WO1997044655A1 - Method and apparatus for sensor and separation in monolayer - Google Patents

Method and apparatus for sensor and separation in monolayer Download PDF

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Publication number
WO1997044655A1
WO1997044655A1 PCT/US1997/008199 US9708199W WO9744655A1 WO 1997044655 A1 WO1997044655 A1 WO 1997044655A1 US 9708199 W US9708199 W US 9708199W WO 9744655 A1 WO9744655 A1 WO 9744655A1
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monolayer
flow
subphase
hydrophobic surface
interface
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PCT/US1997/008199
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French (fr)
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Cary J. Miller
Adam B. Steel
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University Of Maryland
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y10/00—Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/38—Flow patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/64—Electrical detectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/52—Physical parameters
    • G01N2030/524—Physical parameters structural properties
    • G01N2030/525—Physical parameters structural properties surface properties, e.g. porosity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/58—Conditioning of the sorbent material or stationary liquid the sorbent moving as a whole

Definitions

  • the invention relates to a new method and apparatus for measuring physical characteristics of surfaces and interfaces and to sensors and separation using controlled monomolecular and multi-layer systems. Amphiphilic films flowing across a hydrophobic surface in contact with a subphase are used in developing chemical sensors and high resolution separations.
  • Chromatography is a branch of separation science which is particularly sensitive to interfacial phenomenon.
  • the basic mechanism of separation common to all chromatographic techniques is a differential migration of species in a stream of fluid (mobile phase) caused by varying degrees of sorption to a stationary phase (column) .
  • the resolving power of a column is directly dependent on the ability to produce a large differential velocity between all species moving through the column.
  • the resolving power can be increased by increasing the area of the interfacial region between the mobile and stationary phases per unit volume.
  • capillary electrophoresis and high performance liquid chromatography resolution is enhanced by making smaller capillary and using smaller particles in a packed bed, respectively.
  • the present invention provides a new method of chromatography which approaches the fundamental mobile-stationary phase interfacial area limit by employing two distinct monolayer technologies.
  • the bilayer geometry is accomplished using Langmuir films at the air/aqeous interface as the mobile phase and self-assembled monolayers (SAMs) of thiols on gold as the stationary phase.
  • SAMs self-assembled monolayers
  • the new method is referred to as Langmuir film chromatography (LFC) .
  • a continuous interfacial bilayer electrode (“CIBE") described herein can be used in this new method of probing monolayer flow and the molecular properties of interfaces.
  • the CIBE consists of a hydrophobically modified electrode disk oriented perpendicular to the air/subphase interface. The edge of the disk is lowered until it just makes contact with the subphase. By rotating this electrode, fluid
  • Langmuir monolayers are continuously transferred at the instroke of the disk and released at the outstroke.
  • the admittance of this rotating disk electrode is monitored as a function of the molecular area yielding admittance area isotherm to probe the packing and permeability of the transferred monolayers.
  • Measurements of fatty acids of differing chain lengths, lipids and other surface active molecules show the generality of this new technique.
  • the present invention also provides new electrochemical techniques to probe Langmuir monolayers based on changes in the admittance of an electrode in continuous contact with the film.
  • the continuous interfacial bilayer electrode, CIBE allows one to monitor the dielectric properties of transferred monolayer and multilayer assemblies.
  • the bilayer is formed by flowing a monolayer film, such as a Langmuir film, across a hydrophobic surface in contact with a subphase solution.
  • the hydrophobic surface can be fabricated using a number of techniques, such as via the spontaneous self assembly of amphiphilic monolayers onto solid surfaces.
  • Another object of this invention is provide an improved method of chromatography, called Langmuir film chromatography, so that chemical components associated with the flowing monolayer can be separated.
  • This separation is achieved by designing a hydrophobic surface which interacts with the monolayer components in a differential manner resulting in a differential flow rate across the hydrophobically modified surface.
  • a monolayer film having the first and second substances flows over a hydrophobic surface such that the first and second substances flow at different rates.
  • Yet another object of the present invention is to provide an improved sensor capable of detecting small amounts of a substance.
  • a monolayer film flows over a hydrophobic surface in a manner dependent on the presence of the substance.
  • the substance can be located in either the monolayer film or hydrophobic surface via absorption from the subphase or via its introduction into the flowing monolayer.
  • a method of creating a bilayer comprising the steps of: (1) providing a subphase solution; (2) positioning an element with a hydrophobic surface, such as a self assembled monolayer, such that the hydrophobic surface contacts the subphase solution; and (3) introducing an amphiphilic substance to create a flowing monolayer film.
  • the present invention also provides a method of measuring the physical characteristics of a surface or interface, comprising the steps of: (1) providing a subphase solution; (2) positioning an electrode with a hydrophobic surface such that the hydrophobic surface contacts the subphase solution; (3) introducing a amphiphilic mixture to the subphase solution to create a monolayer film that flows across the hydrophobic surface; and (4) measuring the flow rate of the monolayer film.
  • the step of measuring the flow rate is performed by measuring the admittance of the electrode, using fluorescence or monitoring the pressure within an enclosed air/subphase region.
  • the present invention also provides a method of performing chromatography to separate a first substance and a second substance, comprising the steps of: (1) providing a subphase solution; (2) positioning an element with a hydrophobic surface such that the hydrophobic surface contacts the surface of the subphase solution; and (3) introducing the first substance and the second substances to the subphase solution to create a monolayer film such that the first substance and the second substance flow across the hydrophobic surface at different rates.
  • the present invention also provides a method of detecting the presence of a first substance, comprising the steps of: (1) providing a subphase solution; (2) positioning an element with a hydrophobic surface such that the hydrophobic surface contacts the subphase solution; (3) introducing a second substance to the subphase solution to create a monolayer film that flows across the hydrophobic surface in a manner dependent on the presence of the first substance; and (4) measuring the flow rate or surface characteristics of the monolayer film.
  • the present invention also provides an apparatus for measuring the physical characteristics of a surface or interface, comprising: (1) a trough to hold a subphase solution and a monolayer film; (2) a counter electrode; (3) a disc shaped electrode with a hydrophobic surface positioned to contact said monolayer film; and (5) a measuring device to measure the characteristics of said monolayer film.
  • the electrode may also be a rotating disc to maintain a substantially constant contact area with the monolayer.
  • the monolayer films produced and probed by the methods and apparatus of the present invention can be any thin film of an amphiphilic substance formed between a subphase and a hydrophobic substance.
  • the monolayer films include for example, lipids, other biological membrane components, amphiphilic polymers and smaller molecules.
  • the term "subphase" as used herein means any fluid that will support the monolayer film of the invention.
  • the preferred subphase is water or solutions of water and other components such as organic molecules.
  • the subphase may also comprise polar organic molecules or any other fluid, e.g., dimethylformamide or dimethylsulfoxide so long as the amphiphilic substance which forms the monolayer film is insoluble in the subphase.
  • the element with the hydrophobic surface according to the invention may be any solid or liquid material having, or capable of supporting, a hydrophobic surface.
  • Preferred elements include silver or gold supports, and silica or glass, but can comprise various other materials such as a mercury droplet.
  • the hydrophobic surface may be any mono or multi molecular surface that is hydrophobic, i.e. has a measurable contact angle with the subphase substance.
  • the surface may include relatively hydrophilic regions so long as the surface is on the average more hydrophobic than the subphase and thus forms a hydrophobic/hydrophilic interface when contacted with the subphase.
  • the amphiphilic substance which forms the monolayer film in the interface between the hydrophobic surface and subphase according to the invention can be applied directly to the hydrophobic surface to create a monolayer film in the interface, or can be applied to the air/subphase interface adjacent or at a distance from the hydrophobic surface. If applied to the air/subphase interface, the monolayer film flows across the hydrophobic surface to form a monolayer between the hydrophobic surface/subphase interface as if the amphiphilic substance is applied directly to that interface.
  • the rate at which the monolayer film is produced across the hydrophobic surface is referred to herein as the flow rate. This flow rate of the monolayer film can be measured by a variety of methods, preferably by electrochemical methods as described in more detail herein.
  • Physical characteristics of the resulting monolayer, the surface of the element which supports the hydrophobic layer, and the hydrophobic layer itself can also be determined electrochemically and by various other means.
  • the physical characteristics include, for example, mechanical coupling, viscoelastic properties, surface energy and topology, chemical functionality and permeability of the monolayer or surface as the case may be and depending on the particular measuring techniques used, as described in more detail below.
  • the invention provides extremely sensitive systems for detecting or sensing the presence of a component in a biological sample or other mixture suspected of containing the component, and also ultrasensitive methods and apparatus for separating one or more components.
  • the components detected or separated by the invention may be associated with the amphiphilic substance which forms the monolayer film, and may include, for example, cells, inorganic particles, DNA fragments, particles, or proteins dissolved m or simply carried along by the flowing monolayer film.
  • Detectable components may also be part of the subphase which when contacted by the hydrophobic layer interacts with that layer and become part of it.
  • FIG. IA shows a schematic of a Langmuir trough and a
  • FIG. IB shows an expanded view of the rotating electrode geometry at the aqueous interface.
  • FIG. 2A shows pressure-area isotherms for stearic acid in the absence (-) and presence (o) of the rotating electrode.
  • the subphase was 10 nM KC1, pH 2 (HCl), and the temperature was 22 °C.
  • FIG. 2B shows admittance-area isotherm for stearic acid.
  • FIG. 3 shows monolayer capacitance as a function of fatty acid chain length.
  • the monolayer capacitances (o) are calculated from the data for fatty acids given in Table 1.
  • the solid line is monolayer capacitance calculated using a simple capacitor model.
  • FIG. 4 shows a pressure-area and admittance-area isotherms for pentadecanoic acid including admittance data (o) and pressure data (-) .
  • the subphase was 10 mM KC1, pH 2 (HCl), and the temperature was 22 °C.
  • FIG. 6 shows the behavior of fluid Langmuir films, including admittance data (o) and pressure data (-) for arachidic acid on 10 mM KCl, pH 5.5 and temperature of 22 °C. The data was collected one point per two seconds at 30 points per disk revolution.
  • FIGS. 7A to 7D illustrates various stages of a lateral flow.
  • FIG. 8B shows the monolayer capacitance data of FIG. 8A replotted for data analysis.
  • FIGS. 9A to 9C show electrode geometries and positioning at the air/electrolyte interface.
  • FIG. 10 illustrates flow parameter dependence on substrate thickness. The flow parameter was determined for oleic acid on dodecythiol SAM modified gold electrodes of varying thickness. The average flow parameter measured for at least three electrodes per thickness is plotted. The error bars represent 1 standard deviation. The solid horizontal line is the mean flow parameter for all, n > 40, oleic acid on dodecythiol runs. The dash-dot lines
  • FIG. 11 shows flow parameter dependence on piston oil pressure.
  • the solid line is the linear regression best fit line through the data set.
  • FIG. 12 shows flow parameter as a function of SAM chain
  • the error bars represent ⁇ 1 standard deviation.
  • FIG. 13 shows monolayer capacitances for mixed M6, M12 SAMs.
  • the capacitance (o) is plotted versus the mole
  • FIG. 14 shows flow parameter for oleic acid as a function of M12 mole fraction in the SAM.
  • the M12 mole fraction m the SAM is calculated from the capacitance data and parallel capacitor model in FIG. 13.
  • FIG. 15 shows flow parameter for oleic acid as a function of solution mole fraction of thiocholesterol (TC) .
  • the solid line is merely a tie line between the single component SAM values for dipalmitoylphosphatidylthioethanol
  • FIG. 16 shows a diagram of sensing mechanisms available with monolayer flow.
  • FIG. 17A shows the buffer pH effect on the monolayer flow sensor response.
  • the subphase contains 0.1 mM cadmium chloride, 10 mM KCl and ImM total phosphate buffer.
  • FIG. 17B shows the sensor response as a function of cadmium ion concentration.
  • the subphase was lOmM KCl, ImM total phosphate buffer pH 7.5 with the indicated cadmium concentration.
  • FIG. 18 shows the monolayer flow based pH sensor response.
  • the flow parameter for mixed A u /M8 surfaces deposited from a 5 mole percent A n solution shows sensor properties (o) .
  • Control experiments as a single component M8 surface (x) do not exhibit any pH effect.
  • FIG. 19 shows a comparison of mixed alkylthiol and terminating alkylthiol acid pH sensor surfaces.
  • the length of methyl terminated component is increased from M8 to M10.
  • FIG. 20 shows a comparison of sensor responses for M8 and M10 containing surfaces.
  • M8 (o) and M10 (*) The surface concentration of All is not established in either case, but is estimated to be ⁇ 2° by XPS analysis.
  • the lines are present as a guide to the eye only.
  • FIG. 21 shows a flow parameter determination using data obtained in a fluorescence experiment.
  • the fluorescence front location for 1% solutions of DOPE-RLB (o) and DPPE-RLB (x) in oleic acid are depicted.
  • the DOPE-RLB and DPPE-RLB are flourescently labeled lipids used to measure the rate of flow across the hydrophobically modified surface.
  • the flow parameter is equal to the slope of the best fit line through each data set.
  • FIG. 22 shows the ring flow electrode geometry.
  • FIG. 23 is another view of the ring flow electrode geometry of FIG. 22.
  • FIG. 24A shows a ring electrode capacitance-time profile.
  • the capacitance data decays from a value typical of M12 to the M12-oleic acid bilayer in about 800 seconds.
  • FIG. 24B shows a ring electrode pressure-time profile.
  • the pressure inside the ring does not being to rise until 3200 seconds have elapsed.
  • the time dependence of the internal pressure can be used to monitor the monolayer flow rate across the ring.
  • FIG. IA shows a schematic of a Langmuir trough 10 and a continuous interfacial bilayer electrode ("CIBE") monitor according to a preferred embodiment of the present invention.
  • a gold disk 20 derivatized, i.e., coated, with a self-assembled thiol monolayer, is mounted on a shaft parallel to the air-water interface. With the edge 22 of the gold disk 20 touching the air-water interface, the disk 20 is rotated while the admittance of the disk 20 is measured.
  • a Langmuir monolayer at the interface is continuously sampled by the rotating electrode forming a bilayer and changing the electrode admittance.
  • the rotating disk 20 geometry is such that bilayer formation is favored at the m-stroke and the bilayer is released at the i out-stroke.
  • Admittance changes of the rotating disk 20 are measured in tandem with classical surface pressure measurements to give concurrent pressure-area and admittance-area isotherms.
  • Use of the term "Langmuir film” or “Langmuir monolayer” herein means an amphiphilic substance applied to the interface between two phases and which spreads over the interface.
  • the type of information available at the CIBE is qualitatively different from other monolayer techniques.
  • the admittance isotherm monitors changes in the transfer, packing and dielectric properties as the monolayer film is compressed. Phase transitions which modulate the Langmuir monolayer thickness or its permeability to solvent and ions are well characterized by this electrochemical technique.
  • This admittance monitor compared with other monolayer characterization techniques is that it probes the more practically significant properties of the Langmuir monolayer. It is the structure and permeability of Langmuir monolayers which are of primary importance for their uses as photolithographic resists, separation membranes, corrosion inhibitor and biominetic membrane systems.
  • the continuously refreshed bilayer structure on the disk electrode 20 may also be used to probe dynamic interactions between the subphase (i.e., component of liquid phases) species and supported membrane.
  • the new aspect of this disc geometry is that the cylindrical geometry allows a constant contact area facilitating the electrical characterization of the supported membrane.
  • the cylindrical electrode 20 was fabricated from a silver Canadian mapleleaf coin (99.99% purity) .
  • the disk 20 was center tapped and the milled edge removed and polished
  • the polished disk 20 was cleaned by sonicating in ethanol for 10 minutes, exposure to 5% nitric acid for 10 minutes, and sonicating for another 10 minutes in a fresh ethanol solution.
  • the disk 20 Prior to the deposition of a thin layer of gold, the disk 20 was subjected to an argon plasma within the rf sputtering chamber. After coating ca . 30 nm of Cr to promote adhesion followed by ca . 300 nm of gold, the disk 20 was immediately immersed in an ethanolic solution of octadecylmercaptan (ODM) ( ca . 1 mM) and the ODM monolayer was allowed to form overnight. Prior to each use, this derivatized disk 20 was washed extensively with ethanol and deionized water. Although an ODM layer is described, it should be understood that other substances can also be used.
  • ODM octadecylmercaptan
  • FIG. IA shows a schematic drawing of the experimental setup and the sampling geometry. Monolayer surface pressures were recorded by differential weight measurements using filter paper (Whatman, No. 1) and Wilhelmy plate suspended from an analytical balance (Denver Instruments, Model 100A) .
  • the cylindrical electrode 20 was mounted on a rotating spindle 24 connected to a stepper motor 26 via a reducing worm gear allowing 960 steps per revolution, or 1.5 x 10 "3 cm 2 of surface sampled per step.
  • the edge 22 of the disk 20 was positioned to the point of contact with the subphase surface.
  • a saturated calomel electrode (SCE) 30 and a platinum mesh 32 served as the reference and counter electrodes, respectively.
  • SCE saturated calomel electrode
  • the admittance magnitude and phase angle was measured using a lock-in amplifier 40 (Stanford Research Systems, Mo ⁇ el SR530) under computer control at a frequency of 10 Hz with a 10 mV AC excitation.
  • the lock-m amplifier 40 was connected to the cell using a potentiostat 50 (EG&G Princeton, Model 362) which holds the system at 0.0 V versus SCE.
  • Precision control of the stepper motors 26 and timing of the electrochemical and balance measurements was implemented by computer control.
  • the electrolyte used in these experiments was pH 2 HCl, 10 mM potassium chloride unless otherwise noted.
  • the temperature of the subphase was measured and found to be within the range 21 to 24 °C for all the experiments reported here.
  • the entire balance assembly was enclosed in a plexiglass housing (not shown) kept at high humidity.
  • the trough 10 was cleaned and rinsed with deionized water before being filled with the subphase solution 12.
  • the barrier 60 was then swept across the interface to a minimal area and the surface aspirated via a capillary.
  • the CIBE was lowered to initial contact with the interface.
  • the disk 20 was rotated for six revolutions to clean the interface and to test its response. If a steady, cyclic admittance magnitude response was not obtained after the first revolution or if the measured admittance phase angle was below 85° for any point along the revolution, the ODM monolayer was considered incomplete.
  • Disks 20 in such condition were treated in boiling ethanol for several minutes then reimmersed in ODM coating solution before being retested.
  • Test films were spread from a chloroform solution using a microsyringe (Hamilton) . Ten minutes was allowed for solvent evaporation before initiating disk 20 rotation and compression of the film.
  • the typical monolayer compression rate was 0.5 AVmolecule min. Changing the compression rate in the range of 0.25 to 1.5 AVmolecule min did not influence the results significantly.
  • the electrode rotation rate was controlled so that a complete revolution of the disk 20 was achieved for each 1 to 4 AVmolecule of monolayer compression.
  • Rotating a cylindrical electrode 20 with its symmetry axis parallel to the air-subphase interface maintains a relatively constant contact area between the disk edge 22 and the subphase 12 while probing different positions on the disk edge 22.
  • the rotating disk 20 is able to probe the presence and electrical properties of the monolayer by trapping it in a bilayer between the disk and the subphase.
  • the geometry for transfer of the film to the disk is intermediate between two well-established monolayer transfer geometries, vertical dipping, and horizontal lift. This geometry is unique and essential for the continuous nature of this technique.
  • a contact angle greater than 90° is necessary for transfer of Langmuir films to a substrate during an in-stroke and a contact angle of less than 90° is necessary for transfer during removal of the substrate from the subphase.
  • the rotating electrode 20 geometry As shown in the expanded view of the rotating electrode geometry at the aqueous interface of FIG. IB, the hydrophobic ODM layer on the disk displays a contact angle with water of approximately 110°. At the disk/air/subphase triple line associated with the in-stroke, the contact angle favors transfer so that a bilayer is formed; at the triple line associated with the out-stroke, the contact angle does not favor transfer so that the Langmuir film releases from the bilayer.
  • the surface structure of the derivatized electrode is that of a hydrophobic monolayer in contact with the subphase electrolyte.
  • the admittance response in this case is almost purely capacitive with a magnitude consistent with the thickness and dielectric constant of ODM monolayer.
  • the changes in the admittance caused by the transfer of a monolayer are largely capacitive allowing one to relate changes in the electrode capacitance to changes in the "apparent thickness" or "apparent dielectric constant” of the monolayer.
  • the typically smaller changes in the resistive component of the admittance can be used to assess the permeability and structural rigidity of the transferred Langmuir monolayer.
  • a major advantage to this rotating electrode geometry is that the contact area of the electrode is maintained constant during the continuous sampling of the air/subphase interface.
  • the contact area of the electrode and quality of the ODM monolayer vary across the circumference of the disk 20 resulting in a periodic admittance response. Fortunately, this periodic response is quite stable and can be easily removed from the data.
  • FIG. 2A shows typical pressure-area and admittance magnitude-area profiles for an ODM modified gold disk 20 in contact with a stearic acid monolayer.
  • the two pressure-area isotherms 100, 102 shown in FIG. 2A were taken with (o) and without (-) rotation of the Au disk electrode. Measurable deviations between the pressure-area isotherms 100, 102 obtained in the presence or absence of the admittance monitor are only observed for condensed phase films.
  • the isotherms 100, 102 overlap until the stearic acid monolayer is compressed to a solid at a pressure of 25 mN/m at which point the isotherm with disk rotation 100 begins to systematically deviate from the isotherm without disk rotation 102.
  • the linear nature of the pressure deviation with mean molecular area suggests that film collapse is catalyzed at the disk-subphase interface. It is interesting to note that both isotherms 100, 102 extrapolate to give the same limiting area, 19.8
  • the admittance data also indicates that the film at the subphase-disk interface transfers completely on the in-stroke and releases completely on the outstroke.
  • the plateau in the admittance response observed between 24 and 19 AVmolecule strongly suggests a complete and continuous transfer of the stearic acid monolayer onto the ODM surface. Incomplete transfer on the in-stroke would result in an inhomogenous bilayer structure and an irratic admittance profile. Incomplete release of the film on the outstroke would result in multilayer formation and yield an irreversible and decreasing monolayer response.
  • the reversibility in the transfer and release of the monolayer from the ODM disk is further demonstrated by following the reversibility of the admittance response upon compression 106 and expansion 108 of the stearic acid monolayer.
  • the monolayer capacitance can be calculated from the normalized admittance by treating the bilayer as two capacitors in series.
  • the monolayer capacitance for the solid phase of a fatty acid film versus the number of carbons in the acid is plotted in FIG. 3.
  • the solid line 110 in FIG. 3 is the theoretical capacitance values calculated using the following parallel plate capacitor equation:
  • the isotherms for pentadecanoic acid demonstrate this usage.
  • the admittance-area isotherm 114 closely matches the pressure-area isotherm 116 allowing one to track the phase behavior of pentadecanoic acid.
  • the admittance magnitude 114 drops rapidly at the onset of pressure. This admittance magnitude is significantly higher than that characteristic of a solid pentadecanoic acid monolayer.
  • the film passes through the well-studied liquid expanded/liquid condensed first-order phase transition region during which the film pressure 116 remains constant.
  • the admittance signal 114 is nearly constant through this region suggesting that there is little difference in the thickness and packing of these phases.
  • Nonadecanoic Acid 21 15 0.535 + .009 4
  • the surface phase behavior inferred from the pressure-area isotherms is reflected in the admittance-area isotherms in a manner similar to pentadecanoic acid.
  • the reversibility of the transfer and release of the monolayers from the CIBE monitor is typically observed only for fluid monolayer films.
  • For monolayers compressed into a solid phase we typically observed irreversible transfer of the monolayer on the disk. With each rotation of the electrode, a Y-type bilayer is transferred. This difference in the transfer characteristics of fluid and solid monolayers is seen in the admittance-area isotherms for arachidic acid.
  • the transferred monolayer Upon expansion of the monolayer to zero pressure, the transferred monolayer is released and the admittance response 122 returns to its initial value. Pressurizing the monolayer to 16 mN/m into the solid monolayer phase, the CIBE admittance response 122 decreases with each rotation consistent with the continuous transfer of the arachidic acid on the electrode disk.
  • the information accessible by this method is complementary to that obtained by more complex methods such as Brewster angle reflectance and fluorescence microscopy.
  • the CIBE monitor allows one to track the packing and permeability of transferred monolayers in a continuous fashion during the measurement of the pressure-area isotherm.
  • the resultant CIBE admittance-area isotherms can be used to probe the phase behavior of monolayer films, investigate the influence of subphase components on the packing, and permeability of the transferred bilayer or multilayer structure and determine optimal conditions for monolayer transfer.
  • the CIBE is also an ideal tool to monitor the fluidity of potential flowing amphiphilic films.
  • This method relies on a previously unreported property of fluid monolayers, the lateral flow of an amphiphile monolayer along a derivatized surface. It has been observed that piston oil films spontaneously flow into the interface between an aqueous subphase and a hydrophobic surface at the air/subphase interface. Lateral flow rates depend strongly on the chemical functionality and molecular topology of the derivatized surface. As such, this method opens tremendous possibilities m sensor development, surface characterization, and ultrahigh resolution chromatography. Monolayer flow is the pressure-driven movement of a monomolecular film into the interface between a surface and a solution. There are numerous forces present in the flowing monolayer system.
  • FIGS. 7A to 7D illustrate the steps in the monolayer flow experiment.
  • Flow may be observed by positioning a SAM modified electrode 200 at a clean air/water interface, as shown in FIG. 7A, and then introducing a piston oil 204 to the surface.
  • the piston oil 204 spreads to cover the entire interface until the film is self-compressed to its equilibrium spreading pressure.
  • the film 204 then begins to displace the solution at the electrode/solution interface until a complete bilayer is formed between the electrode 200 surface and the solution, as shown in FIG. 7D.
  • FIG. 7C as the monolayer flows into the surface/water interface it dramatically changes the capacitance of the electrode 200.
  • the lateral flow rate is calculated from the capacitance-time curve.
  • the lateral flow system is composed of three distinct regions: the hydrophobic surface, the flowing monolayer, and the subphase. Each of these regions can be varied independently in developing sensors, surface characterization methods, and monolayer-based separation schemes.
  • the interfacial nature of monolayer flow is ideal for developing applications which monitor molecular level surface texture or hydrophobicity.
  • the coupling between the flowing monolayer and the underlying surface structure makes measurements of lateral flow particularly sensitive to chemical topology and a novel surface characterization tool.
  • the flowing monolayer system can also be used as a model system for the study of two-dimensional fluid mechanics and nanotribology which is essential for understanding lubrication processes. The simplicity of this system and the ability to modulate the flow parameter make monolayer flow very attractive for sensor development.
  • the binding of an analyte to the surface from the subphase alters both the surface texture and hydrophobicity of the surface, allowing detection via measurement of the lateral flow rate.
  • the flow rate is quite sensitive as it integrates large numbers of individual chemical recognition events.
  • Lateral flow based sensors offer the potential of greatly simplifying chemical sensor design.
  • FIG. 8A shows the change in capacitance of a dodecylthiol (M12) coated gold electrode as a function of time after the deposition of an excess of oleic acid at the air/electrolyte interface.
  • the electrode capacitance decays to a value consistent with a complete oleic acid/M12 bilayer.
  • the shape of the capacitance profile is well described by a simple pressure driven plug flow model. The lateral flow rate, assuming a linear pressure gradient across the flow path and uniform flow across the gradient, along a surface of length w at time t is
  • the proportionality constant includes a number of factors including the viscosity of the flowing monolayer, the mechanical coupling of the flowing monolayer to the chemically derivatized surface (i.e. molecular friction) the free energy of activation to separate the surface/solution interface, and the viscous drag of solution with the flowing monolayer.
  • k is assumed to be independent of
  • X ⁇ 1/2 w so that a plot of the extent of flowing film incursion into the surface-solution interface, x, versus the square root of time should be linear.
  • the term j2 kA ⁇ is the flow parameter and has units of cm/s 1/2 .
  • Flow parameter determinations are made using chemically modified electrodes. Lateral flow is observed by placing an electrode at a clean air/water interface then introducing a piston oil to the water surface. The piston oil very quickly expands to cover the entire water surface at its equilibrium spreading pressure and in a much slower process flows into the electrode/water interface. Flow of the monolayer into the electrode/water interface has a dramatic effect on the capacitance of that interface. Thus, x is not measured directly, but indirectly by monitoring the capacitance of the electrode. The extent of film flow can be determined from the observed capacitance as a function of time, C ⁇ , by using an equivalent circuit model of two capacitors in parallel. In this model, with the length of the flow path normalized to 1, the observed capacitance is a linear function of x,
  • the flow parameter is determined from the slope of a C ⁇ versus the t' /? plot.
  • FIG. 8A A typical capacitance-time curve is presented in FIG. 8A.
  • the admittance of the SAM modified electrode, C M is measured initially both because its value is needed to calculate the flow parameter and because it provides an indication of the SAMs packing and permeability.
  • the capacitive component of the admittance, calculated from the admittance magnitude and phase angle, is proportional to the monolayer capacitance.
  • the capacitance for the M12 SAM coated electrode is 1.30 ⁇ F/cm ? , in good agreement with a simple capacitor model.
  • the surface pressure increases almost immediately to the equilibrium spreading pressure and the admittance of the electrode then decreases as the oleic acid film flows into the interface.
  • the admittance eventually stabilizes when a complete bilayer is formed at the electrode/solution interface.
  • the capacitance of the bilayer is C M+F .
  • NR normalized response
  • the bilayer capacitance is 0.66 ⁇ F/cm 2 and the normalized response is 0.506 yielding an oleic acid capacitance of 1.33 ⁇ F/cm 2 .
  • the flow parameter is calculated using the slope of the C ⁇ vs t 1/2 plot.
  • the data is linear over more than 85% of the capacitance range, which is typical in our measurements.
  • the flow parameter calculated from the linear fit shown in FIG. 8B is 6.23xl0 "3 cm/s 1/2 .
  • the SAM coated electrode serves as the working electrode in a three electrode cell.
  • a saturated calomel electrode (SCE) and platinum wire, located in a surface-isolated chamber of the Teflon trough serve as the reference and counter, respectively.
  • the admittance magnitude and phase angle were measured using a lock-in amplifier under computer control at a frequency of 10 Hz with a 10 mV ac excitation.
  • the lock-in amplifier was connected to the cell using a potentiostat which holds the system at 0. OV versus the SCE reference.
  • the surface pressure was measured by differential weight measurements using a filter paper Wilhelmy plate suspended from an analytical balance.
  • the electrolyte was 10 mM potassium chloride adjusted to pH 2 with HCl in all cases, except where noted.
  • the temperature of the subphase was found to be within the range 20 to 23°C.
  • the SAM modified gold electrodes were prepared on glass substrates with freshly cleaved smooth edges. Smooth edges were created by scoring a short segment on a plate glass face and carefully snapping the glass to give 2 freshly cleaved edges.
  • the plate glass was then cut into roughly 1x3 inch pieces and cleaned in a chromic acid bath. After rinsing with copious amounts of water the substrates were placed in a radio frequency sputtering chamber with the freshly cleaved edges normal to the metal source. Before coating with the metal layers, the substrates were cleaned with a 50 W argon plasma for 30 seconds. To promote gold adhesion a chromium underlayer of ca . 50 nm was coated on the substrates before the ca . 200 nm gold layer.
  • the coated glass substrates were placed in organic thiol deposition solution ⁇ ca . 1 mM of the organic thiol in absolute ethanol) immediately upon removal from the sputtering chamber and the SAM was allowed to form for at least 12 hours before use.
  • organic thiol deposition solution ⁇ ca . 1 mM of the organic thiol in absolute ethanol
  • a section of a SAM coated slide was cut to give a rectangular glass block with the cleaved edge and the 2 opposing plate faces coated with gold and SAM and 2 uncoated edges of hydrophilic glass. Cut pieces were reimmersed in the appropriate deposition solution for at least 10 minutes prior to use to allow assembly of the SAM on the freshly exposed gold edges.
  • Freshly cleaved glass was preferred to regular microscope slide glass edges because of the large amount of surface roughness on the latters' edge.
  • the variable surface roughness makes reproducible calculation of the SAM capacitance extremely difficult.
  • Lateral flow measurements at microscope slide edges also deviated from the shape predicted by the plug flow model, suggesting that the flow path varied along the length of the substrate.
  • Ring electrodes were created by cutting quartz tubing using a diamond saw. The cut edge was annealed, then cleaned and coated as described for rectangular electrodes. Advantages to using a ring electrode is that there is only one flow front so no compression zone is observed as with a rectangular geometry and that flow can continue after an entire bilayer is formed by flow molecules expanding into the middle of the ring.
  • the electrode was positioned with the SAM coated edge in contact with the water surface in a Teflon trough such that the freshly cut hydrophilic glass edges were wetted as depicted in FIG. 10.
  • the substrate is pulled up from the solution interface to ensure that only the bottom face is wetted.
  • the substrate must also be raised in order to avoid wetting when the piston oil is applied to the liquid interface.
  • the pressure driven flow model adequately describes the observed capacitance-time plots. Several predictions can be made based on the model. We have designed experiments to test these predictions and further validate the model.
  • the model predicts that the measured flow parameter should be independent of the flow path length.
  • electrodes were made using plate glass substrates of varying thickness. All of the electrodes were coated with the same SAM, M12. The observed flow parameter as a function of the flow path length, w, is depicted in FIG. 10. The results presented are from at least 3 different electrodes of each substrate thickness. The average flow parameter determined for each substrate thickness falls within one standard deviation of the average flow parameter for all measurements of oleic acid on a M12 SAM. Therefore, this prediction of the model holds. The model also predicts that the flow parameter should be a linear function of the square root of the piston oil pressure.
  • the derivatives cover a significant pressure range given the subtle differences in molecular structure.
  • the flow parameter for each piston oil was determined at a M12 coated electrode. As predicted, the flow parameter is a linear function of the square root of the equilibrium spreading pressure, FIG. 11. The results presented are from at least 3 different electrodes and 5 individual flow experiments per piston oil.
  • the monolayer capacitance, determined from the normalized response, for each of the piston oils at its equilibrium spreading pressure is also listed in Table 2.
  • Oleyl Acetate 19.30 ⁇ 0.40 5.74 ⁇ 0.25 1.66 ⁇ 0.0 Oleyl Alcohol 36.40 ⁇ 0.50 7.27 ⁇ 0.18 1.33 ⁇ 0.0 Oleyl Butyrate 12.40 ⁇ 0.40 4.39 ⁇ 0.35 1.72 ⁇ 0.0 Oleyl Cyanide 16.80 ⁇ 0.30 4.87 ⁇ 0.20 2.27 ⁇ 0.0 Oleyl Isobutyrate 23.12 ⁇ 0.50 5.63 ⁇ 0.23 1.67 ⁇ 0.0 Oleyl Trifluroacetate 6.30 ⁇ 0.90 2.85 ⁇ 0.26 2.51 ⁇ 0.0
  • the slope of the plot in FIG. 11 is 1.12xl0 "3 cm sec 1 q -1"/2" . According to the model the slope is J2k .
  • k has units of inverse surface poise per square centimeter (sP/cm 2 ) .
  • the value of k is 6.38xl0 "7 cm 2 sec g "1 which corresponds to a surface viscosity for oleic acid flowing at the M12-water interface of 1.57xl0 6 sP/cm 2 , an extremely large value.
  • the surface viscosities of the oleic acid derivatives at the air-water interface are on the order of 0.05 surface millipoise.
  • the large viscosity in the bilayer shows that the rate of monolayer flow is determined primarily by the flowing film/solid surface interaction.
  • the disparate viscosity values validate the assumption that the flowing film-SAM interaction is the dominant term in the flow parameter, providing the basis for applications based on monolayer flow.
  • the flow parameter is observed to decrease with the number of oleyl groups on the same molecule, suggesting that specific chemical interactions between the flowing film and the SAM does influence the flow rate. Differences in the chemical interactions between flowing films can be determined using the simple flow model because the interactions are included in the proportionality constant, k.
  • Interfacial properties of solids are of increasing interest to such diverse fields as polymer formulation, biomaterials development, semiconductors, lubrication, and photography. Understanding lubrication, molecular friction and wetting is essential in many disciplines. The miniaturization of mechanical devices depends on tailoring lubricants for these devices as the ratio of surface to volume increases. Biomedical implants require durable lubricants designed with biocompatibility in mind. Self-assembled monolayers (SAMs) are well-suited for the study and modeling of interfacial phenomenon due to the fine control in forming diverse surfaces. There is a growing interest to produce surfaces with mixed chemical functionality in which the lateral distribution of the components is critical.
  • SAMs Self-assembled monolayers
  • Alkanethiols absorbed on gold provide well characterized model surfaces for the study of lateral flow as a surface characterization tool.
  • SAMs of n-alkanethiols (C n H (2n+1) SH) with 3 ⁇ n ⁇ 20 cover a broad range of surface energy, from highly disordered for small n to highly crystalline structures for n greater than 12, while maintaining the same chemical surface moiety, a methyl group.
  • the flow rate of a monolayer as a function of the n-alkanethiol chain length should primarily indicate surface energy differences as the chemical interactions are identical .
  • One embodiment of this invention relates to probing the dependence of the monolayer properties based on the hydrophobically modified surface.
  • the capacitance of the oleic acid film resulting from lateral flow at the alkylthiol interface can be calculated from the final bilayer capacitance.
  • the capacitance of the oleic acid film is calculated by treating the bilayer as two capacitors in series in an equivalent circuit model.
  • the oleic acid capacitance, C 0A in terms of the bilayer, C Bl , and SAM, C SAM , capacitances is
  • the observed oleic acid monolayer capacitances are depicted in FIG. 15 and the values are independent of the SAM thickness.
  • the average capacitance of the oleic acid film is 1.32 ⁇ 0.07 ⁇ F/cm 2 .
  • a slight decrease in oleic acid film capacitance is observed as the number of carbons in the alkanethiol chain is increased.
  • the smaller capacitance values at longer SAMs are suggestive of a better packed oleic acid monolayer at the longer, more crystalline alkanethiol monolayers.
  • the flow parameter as a function of 1-alkylthiol chain length is depicted in FIG. 12. The maximum flow parameter is observed for the decanethiol SAM.
  • Additional coupling between the flowing monolayer and the surface may be influencing the flow parameter.
  • alkylmercaptans the additional coupling could result from a greater extent of alkyl/oleyl chain interdigitation.
  • alkylmercaptans the additional coupling could be realized at the grain boundaries of crystal domains.
  • the SAM surface designation correspond to the following:
  • Class 1 The first 2 letters designate the surface composition and the number represents the number of carbons in the alkyl chain. Surface designations: BR-terminal bromine; BZ-benzyl mercaptan; CY-cyclohexylmercaptan; EM-methyl ether; PF-perfluoronated; ME-methyl ester; MT-mercaptotoluene; TE-terminal alkene.
  • Class 2 Biomembrane analogs. PC16-dipalmitoyl; PC18-dioleyl; PYT-phytoylmercaptan; TC-thiocholesterol.
  • Class 3 Chromatographic supports.
  • MDS-dodecyl-hexyl disulfide TM12-tert-dodecanethiol.
  • the relative surface concentrations of components in mixed monolayers can be determined by a handful of measurement techniques. When the components have distinct elemental compositions the relative concentrations can be determined by either x-ray photoelectron spectroscopy or surface infrared spectroscopy. For components with the same elemental composition, as is the case with the mixed alkanethiols here, the only method to quantify the relative concentrations is time-of-flight mass spectrometry.
  • the relative surface concentrations can also be determined from the monolayer capacitance by applying the appropriate equivalent circuit model. Two possible equivalent circuit models are based on the film distribution. If the film is ideally phase segregated, then the overall capacitance is modeled by the area-weighted average of the two component capacitances in parallel. If the film is uniformly mixed then the overall capacitance is best modeled by two capacitances in series. The actual behavior of the overall capacitance is bounded by these two models.
  • the observed monolayer capacitance is plotted as a function of mole fraction of M12 in the deposition solution in FIG. 13.
  • the shape of the plot suggests that M12 binds preferentially to M6.
  • An upper bound to the surface concentration of M12 is determined by comparison to the parallel capacitor model (shown as a solid line in FIG. 13) .
  • the deposition solution containing 17% M12 yields a surface with a capacitance equal to that for a 78% M12 surface according the parallel capacitor model.
  • the flow rates of oleic acid on these mixed M6-M12 surfaces are given in FIG. 14.
  • the flow rates are plotted versus the mole fraction of M12 in the self assembled monolayer as determined using the parallel capacitor circuit model discussed above.
  • the observed rates produce a somewhat unexpected profile.
  • the flow parameters are significantly lower than either of the component flow parameters for nearly all mixtures.
  • the mechanism for lowering the flow parameter so dramatically is that the flow parameter is attenuated by increased SAM fluidity for single component alkanethiol
  • Fluidity should cause the flow parameter profile to be the reverse of what is observed because the'-surface fluidity should increase as the M12 mole fraction decreases.
  • An alternate mechanism is that lateral flow is impeded by crossing domain boundaries in phase segregated SAMs. The existence of such a mechanism is suggested by the difference in the flow parameter for the surface equimolar mixed thiols and the M6M12 mixed disulfide. The disulfide can not phase segregate, whereas the mixed thiols can and do. The slow flow parameter observed for the disulfide suggests that the disulfide monolayer geometry allows for greater interaction with the flowing monolayer.
  • the lateral flow profile suggests, in light of the effect of phase segregation, that the number of domains increases steadily until reaching a maximum value at about 80% M12 on the surface. The number of domains then remains constant and only decreases when the mole fraction of M12 on the surface decrease to below 0.5. The number of domains decreases as quickly as it had increased and finally the mixed SAM behaves as if it were a pure M6 monolayer even with 20% of the surface covered with M12.
  • SAM typically an alkanethiol
  • the monolayer capacitance data shows that TC is only slightly preferentially deposited in the monolayer.
  • the lateral flow profile as a function of the solution percent of TC is given in FIG. 15.
  • the flow parameter profile is remarkably different than that observed for the mixed alkanethiol SAMs.
  • the flow parameter is above the tie-line connecting the single component values for all mixtures of PCI6 and TC.
  • cholesterol has a condensing effect on the fluid DPPC. This behavior is seemingly also observed in the mixed SAM films as well.
  • the shape of the flow parameter profile argues against significant phase segregation in the SAM.
  • lateral flow rates are quite sensitive to surface chemical topology and composition. Such dependence is due to the integrating nature of the flowing monolayers movement across a surface.
  • the flow rate depends on the chemical composition of the surface and on the solvent exclusion driving force.
  • the coupling between the flowing monolayer and underlying surface structure is dramatically influenced by changing the chemical composition of the surface to include molecular scale porosity.
  • Studies of mixed monolayer systems highlight the dependence of the lateral flow rate on both the chemical composition of the surface and the spatial distribution of the mixed components.
  • the dependence of the lateral flow rate on the surface chemical composition and film homogeneity make lateral flow a novel method of surface characterization. Sensors are becoming increasingly more commonplace. Issues of sensitivity, selectivity, and expense motivate the investigation and development of new sensor technologies.
  • Monolayer flow technology as described herein provides for a new class of sensors.
  • the appeal of flow-based sensing is due to the versatility of the transduction mechanism in regards to sensor design.
  • Sensing is achieved by modulation of the monolayer flow rate, a mechanical effect or by electrical characteristics of the resulting bilayer film.
  • Most current sensor technologies produce a response in relation to an electrochemical, mass, optical, or conductivity change in the sensing element.
  • Flow rate modulation can be achieved by three distinct mechanisms, depicted in FIG. 16, including: (1) ⁇ binding of a subphase species to the flowing monolayer; (2) permeation into or non-specific binding on the SAM; and (3) specific binding interaction of a subphase species with a minor component of the SAM. Simple sensors based on each of these flow attenuation mechanisms are now described.
  • the simplest means of eliciting a flow-based sensor response is to affect the flowing monolayer.
  • Changing the monolayer viscosity for example, will influence the flow rate.
  • the monolayer viscosity is largely dependent on the film phase state and the extent of mechanical coupling to the subphase.
  • the phase state and subphase coupling can be affected by interaction of a solution species, an analyte, with the flowing monolayer. Additionally, specific interactions can be engineered by the appropriate design of the monolayer head group.
  • the first example of flow-based sensing which we will discuss is that for subphase divalent cations.
  • Interaction of a divalent cation, cadmium for this discussion, with the carboxylic acid head group of oleic acid is the basis for sensor response.
  • the condensing effect of divalent cations on ionized carboxylic acid monolayers is well documented.
  • Each divalent cadmium cation associates with two carboxylates and dramatically increases the monolayer viscosity.
  • the flow rate of the associated monolayer should be slower than for the unassociated monolayer. Because the cation associates with the deprotonated carboxylic acid only, the pH of the subphase is of critical importance.
  • This type of flow-based sensor employs the same experimental procedure for flow parameter determinations as described above.
  • the flowing molecule is oleic acid, with oleyl alcohol serving as a control.
  • the flow surface is M12.
  • the subphase is 10 mM KCl with 1 mM total concentration of phosphate buffer.
  • the relative concentrations of mono- and dibasic phosphate were varied to give the desired pH in the range 3 to 8.5.
  • the observed flow parameter as a function of cadmium ion concentration is given in FIG. 17B. It is apparent that cadmium is only effective at attenuating the flow rate of oleic acid for concentrations greater than 0.01 mM and that the sensor response is limited to cadmium concentrations between 0.05 and 0.2 mM. Control experiments were conducted using oleyl alcohol, which should not associate with subphase cadmium, as the flowing monolayer . The flow parameter was found to be independent of the cadmium ion concentration in the control experiments. Sensor selectivity is quantified by measuring selectivity coefficients for interferences. The selectivity coefficient is defined as the ratio of interferent to analyte concentration for which equivalent sensor responses are produced.
  • the ratio of the formation constants for cadmium and calcium acetate is 4.9, which agrees well with the measured selectivity coefficient.
  • the flow parameter is sensitive to interactions between the flowing monolayer and a subphase solute.
  • the second class of monolayer flow sensors is based on non-specific interactions between a subphase solute and the SAM.
  • the non-specific interaction can be permeation of a hydrophobic solute into the SAM or binding of a solute, e.g. a protein, onto the SAM surface. These two examples of non-specific interactions would produce different mechanisms of attenuating the flow rate.
  • Permeation of a hydrophobic solute into the SAM would produce swelling and thereby increase the surface fluidity.
  • the flow parameter decreases with increased surface fluidity in earlier work, so we would expect the flow parameter to decrease with increasing amounts of permeant. Binding of a protein to the SAM surface is expected to attenuate the flow parameter by increasing the viscosity within the flowing monolayer as it flows around the fixed hydrophilic protein.
  • Non-specific protein adsorption onto hydrophobic surfaces is a critical issue in biosensor design.
  • BSA bovme serum albumin
  • the final, and most preferred, class of monolayer flow sensor is based upon a specific interaction between a subphase solute and a minor SAM component.
  • the sensor is made possible by creating a mixed monolayer flow path.
  • the mixed monolayer consists of an inactive surface (typically a methyl terminated amphiphilic monolayer) with a small percentage of dispersed active surface.
  • the active surface elements may be as simple as a carboxylic acid unit for a pH sensor or as complex as a bound antigen for an antibody sensor.
  • the most difficult part of designing such a sensor is optimizing the percentage of active surface area to give good sensor characteristics. The percentage of active surface area is determined during SAM formation.
  • the competitive adsorption of two distinct thiols results in a dispersed monolayer where the relative surface composition need not correspond to the relative solution concentrations.
  • the component with the lowest relative solubility in the deposition solvent preferentially adsorbs to the gold surface.
  • the distribution of the components in a mixed monolayer is not clear at present, but contact angle and laser desorption mass spectrometry measurements indicate that phase segregated domains must be smaller than 100 nm in diameter.
  • the relative surface concentrations and lateral distribution of the components in a mixed SAM are expected to have a major impact on the sensor characteristics for that surface.
  • the issue of mixed monolayer formation in relation to sensor characteristics is described by way of example, a monolayer flow based pH sensor.
  • a monolayer flow based pH sensor was developed by forming a SAM with mixed methyl and carboxyl terminations.
  • the flow parameter is predicted to depend on the subphase pH due to differences in the extent of carboxyl group ionization. Deprotonation of the acid group results in a much more solvated, charged carboxylate group. The flow parameter will decrease with increased carboxylate density due to an increase in the viscosity within the flowing monolayer.
  • a number of modifications were made to the flow parameter determination procedure. Because the equilibrium spreading pressure, and thereby the flow parameter, of oleic acid are a function of pH, oleyl alcohol whose ESP is independent of pH was selected as the flowing monolayer.
  • the subphase solutions are 10 mM KCl with HCl or KOH added to reach the reported pH values.
  • the mixed methyl and acid terminated SAM sensor surfaces were fabricated by deposition from ethanolic (95%) solutions of octanethiol (M8) and 11-mercapto-l-undecanoic acid (All) .
  • Flow measurements were conducted by positioning cut substrates at the subphase interface as described above. In the sensor experiments, additional time was permitted to establish equilibrium between the sensor surface and subphase before the piston oil was applied to the surface.
  • the capacitance-time curves have the same shape as for single component surfaces, but the slope of the capacitance versus square root of time plot is observed to vary with the subphase pH.
  • the results for the sensor surface deposited from a 5 mole percent solution of All to M8 are summarized in Table 7 and shown in FIG. 18.
  • the control experiment data for the flow parameter of oleyl alcohol on a single component M8 surface is included in FIG. 18 as well.
  • the flow parameter is greatly attenuated at the mixed monolayer surface, even at subphase pH values for which the carboxylic acid groups should be fully protonated.
  • the flow parameter decreases in the region of the All pKa, as predicted.
  • the normalized response a measure of the final bilayer capacitance, also increases with pH. This suggests that areas of the SAM surface are not being covered by a bilayer when the carboxyl groups are deprotonated.
  • the essential element to this class of sensor design is the formation of the mixed SAM surface.
  • the relative solution concentration which provides the optimal sensor response was determined empirically to contain 5 mole percent All to M8.
  • One of the objectives of this invention is to achieve a separation within a monolayer assembly.
  • We accomplish this in one embodiment by marrying two monolayer technologies: Langmuir films and self-assembled monolayers on solid substrates.
  • This Langmuir film chromatography (LFC) was developed using high performance liquid chromatography (HPLC) as a paradigm.
  • HPLC high performance liquid chromatography
  • the components of an HPLC system were re-engineered for the two-dimensional fluid system employed in LFC. Detectors were the first components investigated, resulting in the continuous interfacial bilayer electrode described above.
  • the pump and mobile phase were the next components for consideration.
  • monolayer flow was observed effectively combining the LFC pump and mobile phase.
  • Monolayer flow greatly simplifies the design of LFC experiments and allows us to consider the more difficult question of column design.
  • the use of fluorescent probes greatly simplifies the LFC development process by allowing direct observation of the interactions between the flowing phase and the surface during flow.
  • the experimental procedure for observing flow is altered appreciably to accommodate fluorescence detection.
  • the Teflon trough used in all previously discussed flow measurements is replaced by a glass trough for the fluorescence measurements.
  • a subphase well is-produced in a 1 x 3 inch microscope slide by drilling a hole through the slide using a glass coring bit. The bottom of the well is made by sealing a glass coverslip (ca. 0.15 mm thickness) over the hole using epoxy.
  • the well volume is between 1 and 2 ml. Electrodes are constructed in a similar manner to the method described above using thicker glass, ca. 3 cm, to provide a longer flow path.
  • Fluorescence measurements were performed on a Zeiss Axiovert 135 TV inverted microscope with a Hg lamp source. Fluorescence was observed using an intensifier and a CCD camera and was recorded using a VCR.
  • the label present on all of the fluorescent molecules used for these experiments is Lissamine rhodamine B (LRB) .
  • the excitation and emission wavelengths for this rhodamine are 550 and 590 nm, respectively.
  • the filter sets on the microscope are optimized for use with LRB.
  • Monolayer flow is measured directly as the movement of a fluorescence front in these experiments.
  • the glass trough is filled with subphase electrolyte (50 mM KCl) and the surface aspirated using a capillary.
  • the flow electrode is placed at the liquid interface as described above. After focusing on the electrode edge in the microscope, a flowing mixture of interest is applied to the air/water interface as a chloroform solution. The movement of fluorescently labeled molecules onto the electrode surface is recorded for subsequent analysis.
  • the distance of film flow is measured by the position of the fluorescence front. Distance calibrations were performed using a Neubauer ruling with 0.05 mm line spacings.
  • the flow parameter in this case, is the slope of a distance versus the square root of time plot.
  • the flow data for 1% solutions of dioleylphosphatidylethanolamine-LRB (DOPE-LRB) and dipalmitoylphosphatidyl-ethanolamine-LRB (DPPE-LRB) in oleic acid are given in FIG. 21.
  • the flow parameters for DOPE-LRB and DPPE-LRB determined from the slopes in the FIG.
  • FIGS. 22 and 23 The capacitance flow measurement ring geometry is shown schematically in FIGS. 22 and 23.
  • the counter and reference electrodes 302, 304 and Wilhelmy plate 306 are not present in fluorescence-flow measurements.
  • Capacitance- and pressure-time plots for the flow of oleic acid on a ring electrode modified with M12 are given m FIGS. 24A and 24B.
  • the capacitance profile appears just as would be expected, and the data yields a flow parameter identical to that for a rectangular M12 electrode.
  • the complete bilayer capacitance is reached after 800 seconds of monolayer flow, marked in FIG. 24A by tl.
  • the pressure inside the ring remains constant at zero until 3200 seconds have passed since the oleic acid piston oil was added to the water surface outside of the ring t2 in FIG. 24B.
  • the steady-state molecular flux across the flow surface can be calculated using
  • SA is the surface area inside the ring and A 0 is the area per molecule at which the pressure of oleic acid rises above zero as determined by the pressure-area isotherm (53 AVmolc) .
  • the steady-state molecular flux calculated from the example data set in FIGS. 24A and 24B is 3.24 x 10 n molc/s.
  • the molecular flux can be converted to a effective monolayer velocity, m.v., in the bilayer using
  • m . v. — ⁇ * A ⁇ - inner
  • P ⁇ nner is the inner perimeter of the ring.
  • the monolayer velocity for the example data is 2.3 ⁇ m/s.
  • the ring geometry has several advantages over the rectangular geometry for LFC development. Steady-state flow is much more desirable than the continuously decreasing flow rate profile available in the rectangular geometry. Steady-state flow also permits column conditioning with the mobile phase, given appropriate injection technology. The ability to collect eluants in the ring interior by surface aspiration is a clear advantage. It is clear that the ring geometry is the best geometry for LFC development. Thus, several applications for monolayer flow have been described. It has been demonstrated that the monolayer flow is particularly sensitive to chemical topology and is a novel surface characterization tool. The flowing monolayer system can also be used as a model system for the study of two-dimensional fluid mechanics and molecular friction. The simplicity of this system makes monolayer flow very attractive for sensor development.
  • the flow rate is quite sensitive as it integrates large numbers of individual chemical recognition events.
  • the great chemical sensitivity of flow measurements stems in large part to their similarity to chromatographic separations.
  • Flowing monolayers are an ideal geometry for a chromatographic separation in which the analytes and mobile phase are in continuous contact with the stationary phase.

Abstract

A method and apparatus for creating a bilayer. More particularly, a hydrophobic surface (20) is placed in contact with a subphase (12). When a monolayer film, such as a Langmuir film, is introduced on the subphase (12), the monolayer film will flow across the hydrophobic surface (20) and create a bilayer. The rate of flow of the monolayer film across the hydrophobic surface (20) and the pattern formed by the monolayer film on the hydrophobic surface (20) can be used to determine characteristics of the monolayer film and hydrophobic surface (20). If the hydrophobic surface (20) is formed on a bilayer electrode, the admittance of the bilayer electrode can be used to measure the rate of flow or to infer the pattern of the monolayer film. This method and apparatus can be used to perform chromatography and to prepare extremely sensitive sensors.

Description

METHOD AND APPARATUS FOR SENSOR AND SEPARATION IN MONOLAYER
FIELD OF THE INVENTION The invention relates to a new method and apparatus for measuring physical characteristics of surfaces and interfaces and to sensors and separation using controlled monomolecular and multi-layer systems. Amphiphilic films flowing across a hydrophobic surface in contact with a subphase are used in developing chemical sensors and high resolution separations.
BACKGROUND OF THE ART Understanding and controlling the physical characteristics of surfaces and interfaces is of critical importance to applications in several disciplines. These applications range from electronic devices to medical implants. A commercially significant discipline where interfacial phenomenon are particularly important is separation technology. A significant effort has been expended toward understanding separation processes. Such understanding is essential to the development of more efficient separation technologies which are needed by the biotechnology and chemical separations industries.
Chromatography is a branch of separation science which is particularly sensitive to interfacial phenomenon. The basic mechanism of separation common to all chromatographic techniques is a differential migration of species in a stream of fluid (mobile phase) caused by varying degrees of sorption to a stationary phase (column) . The resolving power of a column, its separation efficiency, is directly dependent on the ability to produce a large differential velocity between all species moving through the column. The resolving power can be increased by increasing the area of the interfacial region between the mobile and stationary phases per unit volume. For two important separation technologies, capillary electrophoresis and high performance liquid chromatography, resolution is enhanced by making smaller capillary and using smaller particles in a packed bed, respectively. Hence, a trend in optimizing chromatographic techniques has been to reduce column packing dimensions as much as possible; however, as the packing dimensions are reduced, the pressure demands on the pumping system increase significantly. The fundamental limit to increasing the interfacial region is to confine the separation process to a continuous bilayer in which one monomolecular layer is the mobile and the other the stationary phase.
The present invention provides a new method of chromatography which approaches the fundamental mobile-stationary phase interfacial area limit by employing two distinct monolayer technologies. In one embodiment of the invention, the bilayer geometry is accomplished using Langmuir films at the air/aqeous interface as the mobile phase and self-assembled monolayers (SAMs) of thiols on gold as the stationary phase. The new method is referred to as Langmuir film chromatography (LFC) .
The possibilities of LFC extend beyond the creation of a new, more powerful, chromatographic technique. Investigation of fluid Langmuir monolayer mechanics is crucial to the development of LFC, and will produce valuable information for the characterization of lubrication processes and biological membranes.
A continuous interfacial bilayer electrode ("CIBE") described herein can be used in this new method of probing monolayer flow and the molecular properties of interfaces. The CIBE consists of a hydrophobically modified electrode disk oriented perpendicular to the air/subphase interface. The edge of the disk is lowered until it just makes contact with the subphase. By rotating this electrode, fluid
Langmuir monolayers are continuously transferred at the instroke of the disk and released at the outstroke. The admittance of this rotating disk electrode is monitored as a function of the molecular area yielding admittance area isotherm to probe the packing and permeability of the transferred monolayers. Measurements of fatty acids of differing chain lengths, lipids and other surface active molecules show the generality of this new technique.
In recent years a number of new experimental techniques have been developed which have further fueled interest in organized monolayers at the air-aqueous interface. A serious challenge in developing new techniques able to characterize these systems is their monomolecular nature. A measuring technique used to study Langmuir monolayers must display impressive sensitivity and surface specificity. Fluorescence and Brewster angle microscopies are notable examples of solutions to this inherent measurement difficulty. Because of their fundamental surface specificity, electrochemical measurements are well suited for characterizing Langmuir films.
The present invention also provides new electrochemical techniques to probe Langmuir monolayers based on changes in the admittance of an electrode in continuous contact with the film. The continuous interfacial bilayer electrode, CIBE, allows one to monitor the dielectric properties of transferred monolayer and multilayer assemblies.
SUMMARY OF THE INVENTION There exists a significant need in the art for an improved method and apparatus for measuring the physical characteristics of surfaces and interfaces.
It is therefore an object of this invention to provide an improved method and apparatus for measuring the physical characteristics of surfaces and interfaces.
It is another object of this invention to provide an improved method of creating a bilayer. The bilayer is formed by flowing a monolayer film, such as a Langmuir film, across a hydrophobic surface in contact with a subphase solution. The hydrophobic surface can be fabricated using a number of techniques, such as via the spontaneous self assembly of amphiphilic monolayers onto solid surfaces.
Another object of this invention is provide an improved method of chromatography, called Langmuir film chromatography, so that chemical components associated with the flowing monolayer can be separated. This separation is achieved by designing a hydrophobic surface which interacts with the monolayer components in a differential manner resulting in a differential flow rate across the hydrophobically modified surface. A monolayer film having the first and second substances flows over a hydrophobic surface such that the first and second substances flow at different rates.
Yet another object of the present invention is to provide an improved sensor capable of detecting small amounts of a substance. A monolayer film flows over a hydrophobic surface in a manner dependent on the presence of the substance. The substance can be located in either the monolayer film or hydrophobic surface via absorption from the subphase or via its introduction into the flowing monolayer.
Briefly described, these and other objects of the invention are accomplished by providing a method of creating a bilayer, comprising the steps of: (1) providing a subphase solution; (2) positioning an element with a hydrophobic surface, such as a self assembled monolayer, such that the hydrophobic surface contacts the subphase solution; and (3) introducing an amphiphilic substance to create a flowing monolayer film.
The present invention also provides a method of measuring the physical characteristics of a surface or interface, comprising the steps of: (1) providing a subphase solution; (2) positioning an electrode with a hydrophobic surface such that the hydrophobic surface contacts the subphase solution; (3) introducing a amphiphilic mixture to the subphase solution to create a monolayer film that flows across the hydrophobic surface; and (4) measuring the flow rate of the monolayer film. The step of measuring the flow rate is performed by measuring the admittance of the electrode, using fluorescence or monitoring the pressure within an enclosed air/subphase region. The present invention also provides a method of performing chromatography to separate a first substance and a second substance, comprising the steps of: (1) providing a subphase solution; (2) positioning an element with a hydrophobic surface such that the hydrophobic surface contacts the surface of the subphase solution; and (3) introducing the first substance and the second substances to the subphase solution to create a monolayer film such that the first substance and the second substance flow across the hydrophobic surface at different rates. The present invention also provides a method of detecting the presence of a first substance, comprising the steps of: (1) providing a subphase solution; (2) positioning an element with a hydrophobic surface such that the hydrophobic surface contacts the subphase solution; (3) introducing a second substance to the subphase solution to create a monolayer film that flows across the hydrophobic surface in a manner dependent on the presence of the first substance; and (4) measuring the flow rate or surface characteristics of the monolayer film.
The present invention also provides an apparatus for measuring the physical characteristics of a surface or interface, comprising: (1) a trough to hold a subphase solution and a monolayer film; (2) a counter electrode; (3) a disc shaped electrode with a hydrophobic surface positioned to contact said monolayer film; and (5) a measuring device to measure the characteristics of said monolayer film. The electrode may also be a rotating disc to maintain a substantially constant contact area with the monolayer.
The monolayer films produced and probed by the methods and apparatus of the present invention can be any thin film of an amphiphilic substance formed between a subphase and a hydrophobic substance. The monolayer films include for example, lipids, other biological membrane components, amphiphilic polymers and smaller molecules. The term "subphase" as used herein means any fluid that will support the monolayer film of the invention. The preferred subphase is water or solutions of water and other components such as organic molecules. The subphase may also comprise polar organic molecules or any other fluid, e.g., dimethylformamide or dimethylsulfoxide so long as the amphiphilic substance which forms the monolayer film is insoluble in the subphase. The element with the hydrophobic surface according to the invention may be any solid or liquid material having, or capable of supporting, a hydrophobic surface. Preferred elements include silver or gold supports, and silica or glass, but can comprise various other materials such as a mercury droplet.
The hydrophobic surface may be any mono or multi molecular surface that is hydrophobic, i.e. has a measurable contact angle with the subphase substance. The surface may include relatively hydrophilic regions so long as the surface is on the average more hydrophobic than the subphase and thus forms a hydrophobic/hydrophilic interface when contacted with the subphase.
The amphiphilic substance which forms the monolayer film in the interface between the hydrophobic surface and subphase according to the invention can be applied directly to the hydrophobic surface to create a monolayer film in the interface, or can be applied to the air/subphase interface adjacent or at a distance from the hydrophobic surface. If applied to the air/subphase interface, the monolayer film flows across the hydrophobic surface to form a monolayer between the hydrophobic surface/subphase interface as if the amphiphilic substance is applied directly to that interface. The rate at which the monolayer film is produced across the hydrophobic surface is referred to herein as the flow rate. This flow rate of the monolayer film can be measured by a variety of methods, preferably by electrochemical methods as described in more detail herein. Physical characteristics of the resulting monolayer, the surface of the element which supports the hydrophobic layer, and the hydrophobic layer itself can also be determined electrochemically and by various other means. The physical characteristics include, for example, mechanical coupling, viscoelastic properties, surface energy and topology, chemical functionality and permeability of the monolayer or surface as the case may be and depending on the particular measuring techniques used, as described in more detail below.
In other embodiments of the invention, rather than probing the physical characteristics of a surface or interface, the invention provides extremely sensitive systems for detecting or sensing the presence of a component in a biological sample or other mixture suspected of containing the component, and also ultrasensitive methods and apparatus for separating one or more components. The components detected or separated by the invention, as described by methods discussed in detail below, may be associated with the amphiphilic substance which forms the monolayer film, and may include, for example, cells, inorganic particles, DNA fragments, particles, or proteins dissolved m or simply carried along by the flowing monolayer film. Detectable components may also be part of the subphase which when contacted by the hydrophobic layer interacts with that layer and become part of it.
With these and other objects, advantages and features of the invention that may become apparent, the nature of the invention may be more readily understood by reference to the following detailed description of the invention, the appended claims and to the several drawings attached herein.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. IA shows a schematic of a Langmuir trough and a
CIBE monitor according to a preferred embodiment of the present invention.
FIG. IB shows an expanded view of the rotating electrode geometry at the aqueous interface.
FIG. 2A shows pressure-area isotherms for stearic acid in the absence (-) and presence (o) of the rotating electrode. The subphase was 10 nM KC1, pH 2 (HCl), and the temperature was 22 °C.
FIG. 2B shows admittance-area isotherm for stearic acid.
FIG. 3 shows monolayer capacitance as a function of fatty acid chain length. The monolayer capacitances (o) are calculated from the data for fatty acids given in Table 1. The solid line is monolayer capacitance calculated using a simple capacitor model. FIG. 4 shows a pressure-area and admittance-area isotherms for pentadecanoic acid including admittance data (o) and pressure data (-) . The subphase was 10 mM KC1, pH 2 (HCl), and the temperature was 22 °C. FIG. 5 shows the behavior of solid Langmuir films, including admittance data (o) and pressure data (-) for arachidic acid, CιgH39COOH, on 10 mM KCl, pH 5.5 and temperature of 22 °C with 0.1 mM CdCl2.
FIG. 6 shows the behavior of fluid Langmuir films, including admittance data (o) and pressure data (-) for arachidic acid on 10 mM KCl, pH 5.5 and temperature of 22 °C. The data was collected one point per two seconds at 30 points per disk revolution.
FIGS. 7A to 7D illustrates various stages of a lateral flow.
FIG. 8A shows a monolayer capacitance during lateral flow for flow of oleic acid on a dodecylthiol coated gold electrode (2w=0.145cm) at the surface of lOmM KCl, pH=2 (HCl. Oleic acid was introduced to the solution surface at t=50 sec.
FIG. 8B shows the monolayer capacitance data of FIG. 8A replotted for data analysis.
FIGS. 9A to 9C show electrode geometries and positioning at the air/electrolyte interface. FIG. 10 illustrates flow parameter dependence on substrate thickness. The flow parameter was determined for oleic acid on dodecythiol SAM modified gold electrodes of varying thickness. The average flow parameter measured for at least three electrodes per thickness is plotted. The error bars represent 1 standard deviation. The solid horizontal line is the mean flow parameter for all, n > 40, oleic acid on dodecythiol runs. The dash-dot lines
represent ±1 standard deviation of the aggregate mean.
FIG. 11 shows flow parameter dependence on piston oil pressure. The solid line is the linear regression best fit line through the data set.
FIG. 12 shows flow parameter as a function of SAM chain
length. The error bars represent ±1 standard deviation. The subphase was 10 mM KCl, pH=2 (HCl), T=21°C.
FIG. 13 shows monolayer capacitances for mixed M6, M12 SAMs. The capacitance (o) is plotted versus the mole
fraction of M12 m the solution. The error bars represent ± standard deviation.
FIG. 14 shows flow parameter for oleic acid as a function of M12 mole fraction in the SAM. The M12 mole fraction m the SAM is calculated from the capacitance data and parallel capacitor model in FIG. 13. The error bars
represent ±1 standard deviation. The flow parameter for oleic acid for the M6M12 disulfide (x) is also included for comparison. FIG. 15 shows flow parameter for oleic acid as a function of solution mole fraction of thiocholesterol (TC) . The solid line is merely a tie line between the single component SAM values for dipalmitoylphosphatidylthioethanol
(PC16) and TC. Error bars represent ±1 standard deviation.
FIG. 16 shows a diagram of sensing mechanisms available with monolayer flow.
FIG. 17A shows the buffer pH effect on the monolayer flow sensor response. The subphase contains 0.1 mM cadmium chloride, 10 mM KCl and ImM total phosphate buffer.
FIG. 17B shows the sensor response as a function of cadmium ion concentration. The subphase was lOmM KCl, ImM total phosphate buffer pH 7.5 with the indicated cadmium concentration.
FIG. 18 shows the monolayer flow based pH sensor response. The flow parameter for mixed Au/M8 surfaces deposited from a 5 mole percent An solution shows sensor properties (o) . Control experiments as a single component M8 surface (x) do not exhibit any pH effect.
FIG. 19 shows a comparison of mixed alkylthiol and terminating alkylthiol acid pH sensor surfaces. The length of methyl terminated component is increased from M8 to M10.
FIG. 20 shows a comparison of sensor responses for M8 and M10 containing surfaces. M8 (o) and M10 (*) . The surface concentration of All is not established in either case, but is estimated to be < 2° by XPS analysis. The lines are present as a guide to the eye only.
FIG. 21 shows a flow parameter determination using data obtained in a fluorescence experiment. The fluorescence front location for 1% solutions of DOPE-RLB (o) and DPPE-RLB (x) in oleic acid are depicted. The DOPE-RLB and DPPE-RLB are flourescently labeled lipids used to measure the rate of flow across the hydrophobically modified surface. The flow parameter is equal to the slope of the best fit line through each data set.
FIG. 22 shows the ring flow electrode geometry. FIG. 23 is another view of the ring flow electrode geometry of FIG. 22.
FIG. 24A shows a ring electrode capacitance-time profile. The capacitance data decays from a value typical of M12 to the M12-oleic acid bilayer in about 800 seconds.
FIG. 24B shows a ring electrode pressure-time profile. The pressure inside the ring does not being to rise until 3200 seconds have elapsed. The time dependence of the internal pressure can be used to monitor the monolayer flow rate across the ring.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Referring now in detail to the drawings wherein like parts are designated by like reference numerals throughout, the following is a detailed description of preferred embodiments of the invention.
Continuous Interfacial Bilayer El ectrode
FIG. IA shows a schematic of a Langmuir trough 10 and a continuous interfacial bilayer electrode ("CIBE") monitor according to a preferred embodiment of the present invention. A gold disk 20 derivatized, i.e., coated, with a self-assembled thiol monolayer, is mounted on a shaft parallel to the air-water interface. With the edge 22 of the gold disk 20 touching the air-water interface, the disk 20 is rotated while the admittance of the disk 20 is measured. A Langmuir monolayer at the interface is continuously sampled by the rotating electrode forming a bilayer and changing the electrode admittance. The rotating disk 20 geometry is such that bilayer formation is favored at the m-stroke and the bilayer is released at the i out-stroke. Admittance changes of the rotating disk 20 are measured in tandem with classical surface pressure measurements to give concurrent pressure-area and admittance-area isotherms. Use of the term "Langmuir film" or "Langmuir monolayer" herein means an amphiphilic substance applied to the interface between two phases and which spreads over the interface.
The type of information available at the CIBE is qualitatively different from other monolayer techniques. The admittance isotherm monitors changes in the transfer, packing and dielectric properties as the monolayer film is compressed. Phase transitions which modulate the Langmuir monolayer thickness or its permeability to solvent and ions are well characterized by this electrochemical technique.
An advantage of this admittance monitor compared with other monolayer characterization techniques is that it probes the more practically significant properties of the Langmuir monolayer. It is the structure and permeability of Langmuir monolayers which are of primary importance for their uses as photolithographic resists, separation membranes, corrosion inhibitor and biominetic membrane systems. The continuously refreshed bilayer structure on the disk electrode 20 may also be used to probe dynamic interactions between the subphase (i.e., component of liquid phases) species and supported membrane. The new aspect of this disc geometry is that the cylindrical geometry allows a constant contact area facilitating the electrical characterization of the supported membrane.
Members of the fatty acid series (CH3 (CH2) n,COOH, (n-2) - 14, 15, 16, 17, 18, 19, 20, 22) were purchased in the highest purity available from Aldrich. For fatty acids which displayed non-ideal pressure/area isotherms, recrystallization from either hexane or ethanol was used to further purify these acids. Octadecylmercaptan (ODM) and cholesterol were purchased from Aldrich and purified by recrystallization from ethanol. Dimyristoyl (DMPC) , dipalmitoyl (DPPC) , distearoyl (DSPC) , dioleoyl (DOPC) phosphatidylcholines, and potassium chloride were purchased from Sigma. Cadmium chloride was purchased from Fisher Scientific. All other chemicals were purchased from Aldrich and used as received. Subphase electrolyte solutions were
made with deionized water (18 MΩ/cm resistivity, Milli-Q Millipore) . The cylindrical electrode 20 was fabricated from a silver Canadian mapleleaf coin (99.99% purity) . The disk 20 was center tapped and the milled edge removed and polished
to 0.05 μ gamma alumina (Beuhler) . The polished disk 20 was cleaned by sonicating in ethanol for 10 minutes, exposure to 5% nitric acid for 10 minutes, and sonicating for another 10 minutes in a fresh ethanol solution. Prior to the deposition of a thin layer of gold, the disk 20 was subjected to an argon plasma within the rf sputtering chamber. After coating ca . 30 nm of Cr to promote adhesion followed by ca . 300 nm of gold, the disk 20 was immediately immersed in an ethanolic solution of octadecylmercaptan (ODM) ( ca . 1 mM) and the ODM monolayer was allowed to form overnight. Prior to each use, this derivatized disk 20 was washed extensively with ethanol and deionized water. Although an ODM layer is described, it should be understood that other substances can also be used.
All experiments were performed in a Langmuir balance with a maximum area of 260 cm/. FIG. IA shows a schematic drawing of the experimental setup and the sampling geometry. Monolayer surface pressures were recorded by differential weight measurements using filter paper (Whatman, No. 1) and Wilhelmy plate suspended from an analytical balance (Denver Instruments, Model 100A) . The cylindrical electrode 20 was mounted on a rotating spindle 24 connected to a stepper motor 26 via a reducing worm gear allowing 960 steps per revolution, or 1.5 x 10"3 cm2 of surface sampled per step. The edge 22 of the disk 20 was positioned to the point of contact with the subphase surface. A saturated calomel electrode (SCE) 30 and a platinum mesh 32 served as the reference and counter electrodes, respectively. The admittance magnitude and phase angle was measured using a lock-in amplifier 40 (Stanford Research Systems, Moαel SR530) under computer control at a frequency of 10 Hz with a 10 mV AC excitation. The lock-m amplifier 40 was connected to the cell using a potentiostat 50 (EG&G Princeton, Model 362) which holds the system at 0.0 V versus SCE. Precision control of the stepper motors 26 and timing of the electrochemical and balance measurements was implemented by computer control. The electrolyte used in these experiments was pH 2 HCl, 10 mM potassium chloride unless otherwise noted. The temperature of the subphase was measured and found to be within the range 21 to 24 °C for all the experiments reported here. The entire balance assembly was enclosed in a plexiglass housing (not shown) kept at high humidity. In a typical experiment the trough 10 was cleaned and rinsed with deionized water before being filled with the subphase solution 12. The barrier 60 was then swept across the interface to a minimal area and the surface aspirated via a capillary. With the Wilhelmy plate 70, reference and counter electrodes 30, 32 m place, the CIBE was lowered to initial contact with the interface. The disk 20 was rotated for six revolutions to clean the interface and to test its response. If a steady, cyclic admittance magnitude response was not obtained after the first revolution or if the measured admittance phase angle was below 85° for any point along the revolution, the ODM monolayer was considered incomplete. Disks 20 in such condition were treated in boiling ethanol for several minutes then reimmersed in ODM coating solution before being retested. Test films were spread from a chloroform solution using a microsyringe (Hamilton) . Ten minutes was allowed for solvent evaporation before initiating disk 20 rotation and compression of the film. The typical monolayer compression rate was 0.5 AVmolecule min. Changing the compression rate in the range of 0.25 to 1.5 AVmolecule min did not influence the results significantly. The electrode rotation rate was controlled so that a complete revolution of the disk 20 was achieved for each 1 to 4 AVmolecule of monolayer compression.
Rotating a cylindrical electrode 20 with its symmetry axis parallel to the air-subphase interface maintains a relatively constant contact area between the disk edge 22 and the subphase 12 while probing different positions on the disk edge 22. For a hydrophobic electrode in contact with a Langmuir film, the rotating disk 20 is able to probe the presence and electrical properties of the monolayer by trapping it in a bilayer between the disk and the subphase. The geometry for transfer of the film to the disk is intermediate between two well-established monolayer transfer geometries, vertical dipping, and horizontal lift. This geometry is unique and essential for the continuous nature of this technique. A contact angle greater than 90° is necessary for transfer of Langmuir films to a substrate during an in-stroke and a contact angle of less than 90° is necessary for transfer during removal of the substrate from the subphase. This is also the case for the rotating electrode 20 geometry. As shown in the expanded view of the rotating electrode geometry at the aqueous interface of FIG. IB, the hydrophobic ODM layer on the disk displays a contact angle with water of approximately 110°. At the disk/air/subphase triple line associated with the in-stroke, the contact angle favors transfer so that a bilayer is formed; at the triple line associated with the out-stroke, the contact angle does not favor transfer so that the Langmuir film releases from the bilayer.
Monitoring the admittance response of the rotating disk 20 allows one to sensitively monitor the transfer and dielectric properties of monolayer films at the air/subphase interface. At a clean interface, the surface structure of the derivatized electrode is that of a hydrophobic monolayer in contact with the subphase electrolyte. The admittance response in this case is almost purely capacitive with a magnitude consistent with the thickness and dielectric constant of ODM monolayer. When a surface active agent is spread at the air/subphase interface, a monolayer of the material is transferred onto the rotating electrode forming a bilayer structure. The admittance of this bilayer is then characteristic of both the underlying ODM monolayer as well as the dielectric properties and packing of the transferred amphiphile. To a good first approximation, the changes in the admittance caused by the transfer of a monolayer are largely capacitive allowing one to relate changes in the electrode capacitance to changes in the "apparent thickness" or "apparent dielectric constant" of the monolayer. The typically smaller changes in the resistive component of the admittance can be used to assess the permeability and structural rigidity of the transferred Langmuir monolayer. A major advantage to this rotating electrode geometry is that the contact area of the electrode is maintained constant during the continuous sampling of the air/subphase interface. The contact area of the electrode and quality of the ODM monolayer vary across the circumference of the disk 20 resulting in a periodic admittance response. Fortunately, this periodic response is quite stable and can be easily removed from the data. These cyclic admittance variations are largely corrected by selecting one period at large molecular area to serve as a reference disk rotation. The data within this period is normalized with respect to the average admittance for the period. The same normalization factor, weighted by the ratio of the average admittance between the reference and subsequent periods, is then used to normalize the entire data set.
FIG. 2A shows typical pressure-area and admittance magnitude-area profiles for an ODM modified gold disk 20 in contact with a stearic acid monolayer. The two pressure-area isotherms 100, 102 shown in FIG. 2A were taken with (o) and without (-) rotation of the Au disk electrode. Measurable deviations between the pressure-area isotherms 100, 102 obtained in the presence or absence of the admittance monitor are only observed for condensed phase films. In FIG. 2A, the isotherms 100, 102 overlap until the stearic acid monolayer is compressed to a solid at a pressure of 25 mN/m at which point the isotherm with disk rotation 100 begins to systematically deviate from the isotherm without disk rotation 102. The linear nature of the pressure deviation with mean molecular area suggests that film collapse is catalyzed at the disk-subphase interface. It is interesting to note that both isotherms 100, 102 extrapolate to give the same limiting area, 19.8
AVmolecule, and the film collapses at the same pressure, 43 mN/m. The normalized admittance data 104 is given in FIG. 2B. At large molecular areas the admittance is unchanged from that observed for ODM layer on a clean interface. We find that there is no detectable transfer of the monolayer to the CIBE unless the monolayer pressure rises above zero. The amphiphiles must be pushed into the electrode/subphase/air phase boundary in order for transfer to take place. At 26 AVmolecule the admittance drops steeply to a value 56% of its original magnitude at less than 2 mN/m of surface pressure. Further compression of this monolayer produces little change in the observed admittance until 19 AVmolecule at a pressure of 26 mN/m when the film is compressed to a solid. Once the film begins to collapse the admittance drops further suggesting that multilayers of the stearic acid are being deposited onto the admittance monitor. The admittance phase angle remains constant at 89 ± 1° until the film is compressed to the collapse point at which the phase angle decreases towards 80 ± 5°. Upon collapse, the increase in the resistive component to the multilayer admittance would be consistent with the introduction of fissures and other imperfections in the transferred layer which allow penetration of solvent and ions within the transferred multilayer.
The admittance data also indicates that the film at the subphase-disk interface transfers completely on the in-stroke and releases completely on the outstroke. In particular, the plateau in the admittance response observed between 24 and 19 AVmolecule strongly suggests a complete and continuous transfer of the stearic acid monolayer onto the ODM surface. Incomplete transfer on the in-stroke would result in an inhomogenous bilayer structure and an irratic admittance profile. Incomplete release of the film on the outstroke would result in multilayer formation and yield an irreversible and decreasing monolayer response. The reversibility in the transfer and release of the monolayer from the ODM disk is further demonstrated by following the reversibility of the admittance response upon compression 106 and expansion 108 of the stearic acid monolayer.
A more quantitative analysis of the admittance response for the fatty acids can be obtained using a simple dielectric model. The monolayer capacitance can be calculated from the normalized admittance by treating the bilayer as two capacitors in series. The monolayer capacitance for the solid phase of a fatty acid film versus the number of carbons in the acid is plotted in FIG. 3. The solid line 110 in FIG. 3 is the theoretical capacitance values calculated using the following parallel plate capacitor equation:
εε0A
C d
where ε is the dielectric constant (ε= 2.1), εo is the
permittivity of free space (εo = 8.85 x 10"14 C2/N cm2), A is the area of the parallel plates (A = 1 cm2) , and d is the thickness of the dielectric medium (d = 1.26 x 10"8 cm/methylene) . The observed monolayer capacitances 112 compare quite well to the calculated values for fatty acids which form condensed films, but we find that such a model tends to underestimate the capacitance for the shorter acids. This suggests that either the effective thickness of the shorter fatty acids is smaller due to greater solvent penetration or the dielectric constants are larger. The admittance-area isotherms can also be used to characterize the different phase transitions of these Langmuir monolayers. The isotherms for pentadecanoic acid, shown m FIG. 4, demonstrate this usage. The admittance-area isotherm 114 closely matches the pressure-area isotherm 116 allowing one to track the phase behavior of pentadecanoic acid. As with all fluid monolayers, the admittance magnitude 114 drops rapidly at the onset of pressure. This admittance magnitude is significantly higher than that characteristic of a solid pentadecanoic acid monolayer. Upon further compression the film passes through the well-studied liquid expanded/liquid condensed first-order phase transition region during which the film pressure 116 remains constant. The admittance signal 114 is nearly constant through this region suggesting that there is little difference in the thickness and packing of these phases. An alternate explanation for the constant admittance value 114 observed in the liquid expanded/liquid condensed phase transition for the pentadecanoic acid would be that the ODM surface is inducing a phase transition upon transfer into a single phase. Given the reported ordering effect of long chain hydrocarbon monolayers, we would expect the more compact liquid condensed phase to be favored at the rotating electrode. The dramatic decrease m the admittance 114 observed upon compression from the liquid condensed to solid phase indicates that a new phase is being deposited onto the ODM surface at these high surface pressures. The range of molecular species amenable to this CIBE technique is by no means limited to simple fatty acids. We have also studied the admittance-area response for a set of more biologically relevant lipids. The results are summarized in Table 1.
Table 1. Admittance Data
Area Pressure Normalized
Amphiphile A2/mole mN/m Resporise n
Saturated Acids:
Myristic Acid 18 25 0.639 ± .012 8
Pentadecanoic Acid 19 28 0.610 + .022 9
Palmitic Acid 20 20 0.583 + .015 5
Heptadecanoic Acid 20 18 0.573 + .014 3
Stearic Acid 20 15 0.548 + .011 18
Nonadecanoic Acid 21 15 0.535 + .009 4
Eicosanoic Acid 21 10 0.521 + .011 4
Docosanoic Acα .d 21 10 0.500 + .010 4
Phosphatidylchol .ines :
Dimyristoyl 50 16 0.78 ± .03 4
Dipalmitoyl 45 22 0.69 ± .07 3
Distearoyl 45 18 0.73 ± .04 3
Dioleoyl 70 12 0.80 ± .06 3
Others:
Octadecanol 18 23 0.523 ± .006 3
Oleic Acid 35 12 0.693 ± .014 6
Cholesterol 44 8 0.62 ± .03 7
Vitamin E 60 5 0.66 ± .02 2
The surface phase behavior inferred from the pressure-area isotherms is reflected in the admittance-area isotherms in a manner similar to pentadecanoic acid. The reversibility of the transfer and release of the monolayers from the CIBE monitor is typically observed only for fluid monolayer films. For monolayers compressed into a solid phase, we typically observed irreversible transfer of the monolayer on the disk. With each rotation of the electrode, a Y-type bilayer is transferred. This difference in the transfer characteristics of fluid and solid monolayers is seen in the admittance-area isotherms for arachidic acid. When Cd?' is present in the subphase, complexation of the carboxylic head groups with the Cd?+ results in solid monolayer aggregates at all pressures. As the monolayer is compressed, these solid aggregates are transferred to the CIBE. In FIG. 5, the CIBE admittance 118 drops to a value consistent with a single monolayer transfer at the onset of pressure. At this point, the surface pressure 120 was held at 8 mN/m for two revolutions resulting in the CIBE admittance 118 decreasing to a level consistent with the transfer of two complete Y-type bilayers. Upon expanding the monolayer to zero pressure, the admittance 118 remains unchanged indicating that no transfers are taking place to or from the electrode disk. When the monolayer is compressed again to 8 mN/m, the cadmium arachidate monolayers continue to transfer as seen from the steady decrease in the CIBE admittance 118. On an acidic subphase in the absence of Cd2+, arachidic acid displays a liquid phase up to ca. 15 mN/m. A similar surface pressure profile 124 on this monolayer results in a distinctly different admittance response 122 of the CIBE monitor shown in FIG. 6. The admittance response 122 drops to a value consistent with a single monolayer coverage at the onset of surface pressure and remains constant during the two revolutions of the CIBE monitor at a pressure of 5 mN/m. Upon expansion of the monolayer to zero pressure, the transferred monolayer is released and the admittance response 122 returns to its initial value. Pressurizing the monolayer to 16 mN/m into the solid monolayer phase, the CIBE admittance response 122 decreases with each rotation consistent with the continuous transfer of the arachidic acid on the electrode disk.
Thus, a simple and inexpensive tool for studying Langmuir films has been described. The information accessible by this method, film thickness and compactness, is complementary to that obtained by more complex methods such as Brewster angle reflectance and fluorescence microscopy. The CIBE monitor allows one to track the packing and permeability of transferred monolayers in a continuous fashion during the measurement of the pressure-area isotherm. The resultant CIBE admittance-area isotherms can be used to probe the phase behavior of monolayer films, investigate the influence of subphase components on the packing, and permeability of the transferred bilayer or multilayer structure and determine optimal conditions for monolayer transfer. The CIBE is also an ideal tool to monitor the fluidity of potential flowing amphiphilic films.
Langmuir Film Chroma tography The principles of chromatography hold that the resolving power of a column increases with the number of eluant-stationary phase interactions. Extension of this premise suggests that the ultimate geometry for a separation would involve continuous contact between the eluant and stationary phase. A bilayer composed of one stationary layer and one flowing layer of molecular thickness would provide the continuous contact necessary for ultimate resolution chromatography. Lateral diffusion within monolayers and bilayers has been reported, but the controlled, directed flow of one monolayer past another necessary for chromatography has proven more difficult. The present invention provides a remarkably simple method to produce the chromatographic bilayer assembly using, for example, self-assembled monolayers (SAMs) and amphiphilic films at the air-water interface. This method relies on a previously unreported property of fluid monolayers, the lateral flow of an amphiphile monolayer along a derivatized surface. It has been observed that piston oil films spontaneously flow into the interface between an aqueous subphase and a hydrophobic surface at the air/subphase interface. Lateral flow rates depend strongly on the chemical functionality and molecular topology of the derivatized surface. As such, this method opens tremendous possibilities m sensor development, surface characterization, and ultrahigh resolution chromatography. Monolayer flow is the pressure-driven movement of a monomolecular film into the interface between a surface and a solution. There are numerous forces present in the flowing monolayer system. Driving forces for monolayer flow are countered by frictional forces between the flowing monolayer and the surface and within the flowing monolayer itself. The hydrodynamic mechanics of monolayer flow at the solid-liquid interface are largely unknown. Monolayer film flow at the air/water interface has been described and substantial coupling of the flowing monolayer to the liquid is observed. The rate of flow is controlled primarily by the hydrophobicity of the surface and the piston oil pressure.
FIGS. 7A to 7D illustrate the steps in the monolayer flow experiment. Flow may be observed by positioning a SAM modified electrode 200 at a clean air/water interface, as shown in FIG. 7A, and then introducing a piston oil 204 to the surface. As shown in FIG. 7B, the piston oil 204 spreads to cover the entire interface until the film is self-compressed to its equilibrium spreading pressure. The film 204 then begins to displace the solution at the electrode/solution interface until a complete bilayer is formed between the electrode 200 surface and the solution, as shown in FIG. 7D. As shown in FIG. 7C, as the monolayer flows into the surface/water interface it dramatically changes the capacitance of the electrode 200. The lateral flow rate is calculated from the capacitance-time curve.
The lateral flow system is composed of three distinct regions: the hydrophobic surface, the flowing monolayer, and the subphase. Each of these regions can be varied independently in developing sensors, surface characterization methods, and monolayer-based separation schemes. The interfacial nature of monolayer flow is ideal for developing applications which monitor molecular level surface texture or hydrophobicity. The coupling between the flowing monolayer and the underlying surface structure makes measurements of lateral flow particularly sensitive to chemical topology and a novel surface characterization tool. The flowing monolayer system can also be used as a model system for the study of two-dimensional fluid mechanics and nanotribology which is essential for understanding lubrication processes. The simplicity of this system and the ability to modulate the flow parameter make monolayer flow very attractive for sensor development. The binding of an analyte to the surface from the subphase alters both the surface texture and hydrophobicity of the surface, allowing detection via measurement of the lateral flow rate. The flow rate is quite sensitive as it integrates large numbers of individual chemical recognition events. Lateral flow based sensors offer the potential of greatly simplifying chemical sensor design.
The great chemical sensitivity of these measurements stems in large part to their similarity to chromatographic separations. Flowing monolayers are an ideal geometry for a chromatographic separation in which the analytes and mobile phase are in continuous contact with the stationary phase. The use of the lateral flow geometry allows ultrahigh resolution separations of membrane components under nearly natural conditions.
The development of surface characterization methods, sensors, and separations based on monolayer flow technology are all dependent on the ability to model and understand the process. With an alkyl thiol SAM derivatized gold electrode positioned at an aqueous electrolyte solution interface, the spreading of a Langmuir monolayer on the interface causes the electrode capacitance to drop as the monolayer flows into the electrode/solution interface. FIG. 8A shows the change in capacitance of a dodecylthiol (M12) coated gold electrode as a function of time after the deposition of an excess of oleic acid at the air/electrolyte interface. The electrode capacitance decays to a value consistent with a complete oleic acid/M12 bilayer. The shape of the capacitance profile is well described by a simple pressure driven plug flow model. The lateral flow rate, assuming a linear pressure gradient across the flow path and uniform flow across the gradient, along a surface of length w at time t is
proportional to the pressure drop along the flow path, ΔI1, and inversely proportional to the length of the flow path, x. Therefore,
dx _ , Δπ dt x
where k is a proportionality constant.
The proportionality constant includes a number of factors including the viscosity of the flowing monolayer, the mechanical coupling of the flowing monolayer to the chemically derivatized surface (i.e. molecular friction) the free energy of activation to separate the surface/solution interface, and the viscous drag of solution with the flowing monolayer. In this model k is assumed to be independent of
the surface pressure, 11. This assumption should be valid so long as the piston oil exists in only one phase state for all pressures across the gradient. Typically, piston oil pressure-area isotherms display liquid-expanded phase behavior for all pressures.
Rearrangement of the above equation and solving for x, as a normalized distance, as a function of t gives
V2ΪcΔπ
X = ■1/2 w so that a plot of the extent of flowing film incursion into the surface-solution interface, x, versus the square root of time should be linear. The term j2 kAπ is the flow parameter and has units of cm/s1/2.
Flow parameter determinations are made using chemically modified electrodes. Lateral flow is observed by placing an electrode at a clean air/water interface then introducing a piston oil to the water surface. The piston oil very quickly expands to cover the entire water surface at its equilibrium spreading pressure and in a much slower process flows into the electrode/water interface. Flow of the monolayer into the electrode/water interface has a dramatic effect on the capacitance of that interface. Thus, x is not measured directly, but indirectly by monitoring the capacitance of the electrode. The extent of film flow can be determined from the observed capacitance as a function of time, Cτ, by using an equivalent circuit model of two capacitors in parallel. In this model, with the length of the flow path normalized to 1, the observed capacitance is a linear function of x,
CT = (l - x)CM + xCmF
where CM is the capacitance of the SAM on the electrode and CM+F is the capacitance of the final bilayer. Solving for x and substituting into the prior equation yields the expression
Figure imgf000037_0001
The flow parameter is determined from the slope of a Cτ versus the t'/? plot.
A typical capacitance-time curve is presented in FIG. 8A. The given data is for the flow of oleic acid on a M12 coated substrate (2w = 0.145 cm) . In all experiments the admittance of the SAM modified electrode, CM, is measured initially both because its value is needed to calculate the flow parameter and because it provides an indication of the SAMs packing and permeability. The capacitive component of the admittance, calculated from the admittance magnitude and phase angle, is proportional to the monolayer capacitance. The capacitance for the M12 SAM coated electrode is 1.30 μF/cm?, in good agreement with a simple capacitor model.
After adequate time has passed to assure that the SAM capacitance is stable, typically 1 minute, the oleic acid piston oil is introduced to the surface (t = 50 sec in FIG. 8A) . The surface pressure increases almost immediately to the equilibrium spreading pressure and the admittance of the electrode then decreases as the oleic acid film flows into the interface. The admittance eventually stabilizes when a complete bilayer is formed at the electrode/solution interface.
The capacitance of the bilayer is CM+F. To assess the completeness of bilayer formation we report a normalized response (NR) parameter for every SAM surface which is the ratio of the bilayer capacitance to the SAM capacitance. The NR is used calculate the capacitance of the oleic acid monolayer, using an equivalent circuit with two capacitors in series.
Figure imgf000038_0001
In the given data the bilayer capacitance is 0.66 μF/cm2 and the normalized response is 0.506 yielding an oleic acid capacitance of 1.33 μF/cm2.
The flow parameter is calculated using the slope of the Cτ vs t1/2 plot. The data set from FIG. 8A is replotted, after setting t = 0 to the introduction of the oleic acid, in FIG. 8B. The data is linear over more than 85% of the capacitance range, which is typical in our measurements. The flow parameter calculated from the linear fit shown in FIG. 8B is 6.23xl0"3 cm/s1/2.
A few comments about the shape of the Cτ vs t1/2 plot are in order. First, that the best fit to the linear region of the curve yields an intercept greater than CM suggests that the initial flow (within the first 3 seconds after introduction of the piston oil) is slow. This is expected as the piston oil does not come up to pressure instantaneously, the driving force for lateral flow has a rise time on the order of 1-3 seconds. Second, the curve becomes non-linear as the capacitance approaches Cmi . This is a result of the flow geometry employed in our experiments. As depicted in FIGS. 7A to 7D, flow occurs from both sides of the substrate. Each flowing film front experiences the entire pressure drop across its length. When the fronts make contact, compression of the film occurs until the entire flowing film is pressurized up to the equilibrium spreading pressure. The capacitance of the flowing film layer is not uniform across the flow path until the entire film is at the same pressure. The capacitance of the oleic acid monolayer at the air/water interface as a function of surface pressure has been measured using the CIBE described above. The monolayer capacitance observed with the CIBE decreases by 11 percent between 0.2 and 32 mN/m, while the capacitance decreases 9 percent from the onset of non-linearity to stabilizing at CM+F in flow experiments. That these two distinct methods of measuring the capacitance of an oleic acid monolayer as a function of pressure/molecular area are in agreement is an indication that the plug flow model corresponds closely to the data. The SAM coated electrode serves as the working electrode in a three electrode cell. A saturated calomel electrode (SCE) and platinum wire, located in a surface-isolated chamber of the Teflon trough serve as the reference and counter, respectively. The admittance magnitude and phase angle were measured using a lock-in amplifier under computer control at a frequency of 10 Hz with a 10 mV ac excitation. The lock-in amplifier was connected to the cell using a potentiostat which holds the system at 0. OV versus the SCE reference. The surface pressure was measured by differential weight measurements using a filter paper Wilhelmy plate suspended from an analytical balance. The electrolyte was 10 mM potassium chloride adjusted to pH 2 with HCl in all cases, except where noted. The temperature of the subphase was found to be within the range 20 to 23°C.
The SAM modified gold electrodes were prepared on glass substrates with freshly cleaved smooth edges. Smooth edges were created by scoring a short segment on a plate glass face and carefully snapping the glass to give 2 freshly cleaved edges. The plate glass was then cut into roughly 1x3 inch pieces and cleaned in a chromic acid bath. After rinsing with copious amounts of water the substrates were placed in a radio frequency sputtering chamber with the freshly cleaved edges normal to the metal source. Before coating with the metal layers, the substrates were cleaned with a 50 W argon plasma for 30 seconds. To promote gold adhesion a chromium underlayer of ca . 50 nm was coated on the substrates before the ca . 200 nm gold layer. The coated glass substrates were placed in organic thiol deposition solution { ca . 1 mM of the organic thiol in absolute ethanol) immediately upon removal from the sputtering chamber and the SAM was allowed to form for at least 12 hours before use. Just prior to use, a section of a SAM coated slide was cut to give a rectangular glass block with the cleaved edge and the 2 opposing plate faces coated with gold and SAM and 2 uncoated edges of hydrophilic glass. Cut pieces were reimmersed in the appropriate deposition solution for at least 10 minutes prior to use to allow assembly of the SAM on the freshly exposed gold edges.
Freshly cleaved glass was preferred to regular microscope slide glass edges because of the large amount of surface roughness on the latters' edge. The variable surface roughness makes reproducible calculation of the SAM capacitance extremely difficult. Lateral flow measurements at microscope slide edges also deviated from the shape predicted by the plug flow model, suggesting that the flow path varied along the length of the substrate.
Experiments were performed with various geometries as shown in FIGS. 9A to 9C. Ring electrodes were created by cutting quartz tubing using a diamond saw. The cut edge was annealed, then cleaned and coated as described for rectangular electrodes. Advantages to using a ring electrode is that there is only one flow front so no compression zone is observed as with a rectangular geometry and that flow can continue after an entire bilayer is formed by flow molecules expanding into the middle of the ring.
The electrode was positioned with the SAM coated edge in contact with the water surface in a Teflon trough such that the freshly cut hydrophilic glass edges were wetted as depicted in FIG. 10. The substrate is pulled up from the solution interface to ensure that only the bottom face is wetted. The substrate must also be raised in order to avoid wetting when the piston oil is applied to the liquid interface.
The pressure driven flow model adequately describes the observed capacitance-time plots. Several predictions can be made based on the model. We have designed experiments to test these predictions and further validate the model.
The model predicts that the measured flow parameter should be independent of the flow path length. To test this supposition, electrodes were made using plate glass substrates of varying thickness. All of the electrodes were coated with the same SAM, M12. The observed flow parameter as a function of the flow path length, w, is depicted in FIG. 10. The results presented are from at least 3 different electrodes of each substrate thickness. The average flow parameter determined for each substrate thickness falls within one standard deviation of the average flow parameter for all measurements of oleic acid on a M12 SAM. Therefore, this prediction of the model holds. The model also predicts that the flow parameter should be a linear function of the square root of the piston oil pressure. This result requires that the same piston oil be applied at different surface pressures so that the same interactions are present between the SAM, flowing film and subphase. That is, the flow model proportionality constant, k, must be constant. A piston oil can hold a pressure below its equilibrium spreading pressure if the monolayer is applied to the air-water interface below complete coverage and pressurized using conventional Langmuir monolayer compression methods. However, if we assume that the interaction between the flowing film and the SAM surface is the dominant interaction in determining the flow parameter and that the interaction between the flowing film and the subphase is minimal, then oleic acid derivatives can be utilized to assess the flow parameter-surface pressure relationship. The equilibrium spreading pressures for the oleic acid derivatives used in this study are listed in
Table 2. The derivatives cover a significant pressure range given the subtle differences in molecular structure. The flow parameter for each piston oil was determined at a M12 coated electrode. As predicted, the flow parameter is a linear function of the square root of the equilibrium spreading pressure, FIG. 11. The results presented are from at least 3 different electrodes and 5 individual flow experiments per piston oil. The monolayer capacitance, determined from the normalized response, for each of the piston oils at its equilibrium spreading pressure is also listed in Table 2.
Table 2. Summary of piston oil parameters Piston Oil ESP FP Cap mN/m cm/s μF/cm
(x 1000)
Ethyl Oleate 15.90 ± 0.70 5.25 ± 0.23 1.85 ± 0.0
Methyl Oleate 19.80 ± 0.30 5.78 ± 0.14 1.79 ± 0.0
Oleic Acid 32.50 ± 0.30 6.31 ± 0.27 1.55 ± 0.0 Table 2. Summary of piston oil parameters
Piston Oil ESP FP Cap mN/m cm/s μF/cm'
(x 1000)
Oleyl Acetate 19.30 ± 0.40 5.74 ± 0.25 1.66 ± 0.0 Oleyl Alcohol 36.40 ± 0.50 7.27 ± 0.18 1.33 ± 0.0 Oleyl Butyrate 12.40 ± 0.40 4.39 ± 0.35 1.72 ± 0.0 Oleyl Cyanide 16.80 ± 0.30 4.87 ± 0.20 2.27 ± 0.0 Oleyl Isobutyrate 23.12 ± 0.50 5.63 ± 0.23 1.67 ± 0.0 Oleyl Trifluroacetate 6.30 ± 0.90 2.85 ± 0.26 2.51 ± 0.0
The slope of the plot in FIG. 11 is 1.12xl0"3 cm sec1 q -1"/2" . According to the model the slope is J2k . Thus, we find that k has units of inverse surface poise per square centimeter (sP/cm2) . The value of k is 6.38xl0"7 cm2sec g"1 which corresponds to a surface viscosity for oleic acid flowing at the M12-water interface of 1.57xl06 sP/cm2, an extremely large value. The surface viscosities of the oleic acid derivatives at the air-water interface are on the order of 0.05 surface millipoise. The large viscosity in the bilayer shows that the rate of monolayer flow is determined primarily by the flowing film/solid surface interaction.
The disparate viscosity values validate the assumption that the flowing film-SAM interaction is the dominant term in the flow parameter, providing the basis for applications based on monolayer flow.
The large surface viscosity characterizing the flowing monolayers suggests that the dominant factor controlling the rate of lateral motion in these monolayers is the mechanical coupling of the piston oil molecules to the SAM. We have performed experiments to probe the influence of piston oil structure on this mechanical coupling. Experiments were also performed to determine the influence of the electrode potential on monolayer flow. The importance of the interaction between the flowing film and the SAM can be probed by linking multiple oleyl groups together. If coupling to the SAM surface is important then tethering oleyl groups together should result in significantly slower flow rates due to increased mechanical coupling per molecule. The results of lateral flow experiments with the listed oleins on a M12 SAM modified electrodes are summarized in Table 3. For easier comparison the flow parameter values have been corrected for the difference in ESP and normalized to the flow parameter for oleic acid.
The flow parameter is observed to decrease with the number of oleyl groups on the same molecule, suggesting that specific chemical interactions between the flowing film and the SAM does influence the flow rate. Differences in the chemical interactions between flowing films can be determined using the simple flow model because the interactions are included in the proportionality constant, k.
Table 3. ESP and flow parameters for olein series
ESP Normalized
Species (mN/m) Flow Parameter
Oleic Acid 32.5 1.00 1, 3-Diolein 30.9 0.90 Table 3. ESP and flow P'arameters for olein series
ESP Normalized
Species (mN/m) Flow Parameter
Oleyl oleate 4.6 0.86
Tπolein 13.1 0.72
The interaction between the flowing film tail group and the SAM is a possible mechanism to produce a separation in monolayer flow. Increasing the coupling between the film and the surface is equivalent to increasing the retention of an eluant in chromatography. The coupling between the piston oil and the surface should have a dramatic effect on the ability to produce separations. The effect of three types of tail group modifications have been investigated:
(1) varying the length of the cis-double bond containing tail; (2) tails containing multiple cis-double bonds; and
(3) exchange of the oleyl group with a saturated tail group. The results are summarized in the following Table 4.
Table 4. Flowing phase tail group influence on the flow parameter
ESP No]rmalized
Piston Oil (mN/m) Flow Parameter
Type 1
Myristoleic Acid 34.7 1.32
Palmitoleic Acid 33.5 1.20
Oleic Acid 32.5 1.00
Vaccemc Acid 33.4 1.16 cιs- 11 -Eιcosanoιc Acid 31.5 1.01 Type 2
Linoleic Acid 30.6 1.26
Lmolenic Acid 29.4 1.33 Type 3 Table 4. Flowing phase tail group influence on the flow parameter
ESP Normalized
Piston Oil (mN/m) Flow Parameter
Phytol 32.2 0.94
Ethyl Myπstate 18.8 1.17
Stearyl Oleate 4.8 0.66
It is apparent that both shortening the cis-double bond containing tail and introducing additional cis-double bonds to the tail increase the monolayer flow rate. Presumably, larger flow parameters are achieved by reducing the coupling between the flowing monolayer and the M12 surface and smaller values by increasing the coupling. Alternatively, the differences in flow parameter could arise from coupling within the flowing monolayer itself, viscosity effects. Piston oil molecules with larger cross-sections than oleic acid, e.g. phytol and stearyl oleate, are observed to flow slower than oleic acid. Again, this is due either to the larger molecular cross-section which permits greater interaction with surface or to viscosity differences.
Thus, a model describing the spontaneous flow of piston oil films into the interface between an aqueous subphase and a hydrophobic surface has been described. Monolayer flow rates depend strongly on the chemical functionality and molecular topology of the derivatized surface. Predictions derived from the model were verified experimentally, providing a more thorough understanding of the lateral flow process. Given this understanding of the factors governing the monolayer flow process, applications based on the technology will now be described.
In the course of developing LFC, the remarkable property of fluid Langmuir monolayers described above was observed. Lateral flow of monolayers provides both the pump and mobile phase for the bilayer separation geometry of LFC. Additionally, it has been realized that lateral flow provides a new means of surface characterization and sensor development. Lateral flow rates are quite sensitive to surface chemical topology. The coupling between the flowing monolayer and underlying surface structure can be dramatically influenced by changing the chemical composition of the surface. The dependence of the lateral flow rate on the surface chemical composition make lateral flow a novel method of surface characterization. The ability to influence the flow rate by modifying the surface structure provides a route toward sensor development. Various separations of components in a mixed flowing monolayer film based on differences in chemical interaction with a SAM stationary phase can easily be performed by one of ordinary skill in the art given the teachings herein.
Interfacial properties of solids are of increasing interest to such diverse fields as polymer formulation, biomaterials development, semiconductors, lubrication, and photography. Understanding lubrication, molecular friction and wetting is essential in many disciplines. The miniaturization of mechanical devices depends on tailoring lubricants for these devices as the ratio of surface to volume increases. Biomedical implants require durable lubricants designed with biocompatibility in mind. Self-assembled monolayers (SAMs) are well-suited for the study and modeling of interfacial phenomenon due to the fine control in forming diverse surfaces. There is a growing interest to produce surfaces with mixed chemical functionality in which the lateral distribution of the components is critical.
The need for convenient analytical techniques to study the diverse interfaces present in these fields is apparent. A number of techniques have been developed to study surfaces, each sensitive to surface structure in a different manner. Average surface structure is probed using electrochemical methods, contact angle geometry, reflection infrared spectroscopy, and ellipsometry. These techniques can be used to ascertain SAM organization, structure, and composition on a global scale, but are very limited in their ability to describe the spatial distribution of SAM components. Local organization and composition can be examined to some extent using scanning microscopies and time-of-flight mass spectrometry; however, global structural information such as the film fluidity is difficult to assess using these methods. Given the strong coupling between the flowing monolayer and a SAM surface in lateral flow the flow rate of a given piston oil should be indicative of the SAM organization, structure, and composition on a microscopic scale.
Alkanethiols absorbed on gold provide well characterized model surfaces for the study of lateral flow as a surface characterization tool. SAMs of n-alkanethiols (CnH(2n+1)SH) with 3<n<20 cover a broad range of surface energy, from highly disordered for small n to highly crystalline structures for n greater than 12, while maintaining the same chemical surface moiety, a methyl group. The flow rate of a monolayer as a function of the n-alkanethiol chain length should primarily indicate surface energy differences as the chemical interactions are identical .
One embodiment of this invention relates to probing the dependence of the monolayer properties based on the hydrophobically modified surface. The capacitance of the oleic acid film resulting from lateral flow at the alkylthiol interface can be calculated from the final bilayer capacitance. The capacitance of the oleic acid film is calculated by treating the bilayer as two capacitors in series in an equivalent circuit model. The oleic acid capacitance, C0A, in terms of the bilayer, CBl, and SAM, CSAM, capacitances is
Figure imgf000050_0001
The observed oleic acid monolayer capacitances are depicted in FIG. 15 and the values are independent of the SAM thickness. The average capacitance of the oleic acid film is 1.32±0.07 μF/cm2. A slight decrease in oleic acid film capacitance is observed as the number of carbons in the alkanethiol chain is increased. The smaller capacitance values at longer SAMs are suggestive of a better packed oleic acid monolayer at the longer, more crystalline alkanethiol monolayers. The flow parameter as a function of 1-alkylthiol chain length is depicted in FIG. 12. The maximum flow parameter is observed for the decanethiol SAM. We believe the flow parameter is tracking the fluidity, or disorder, present in the SAM. The shape of the plot suggests that there exists an optimal SAM phase state for the lateral flow of oleic acid at these surfaces. For very short, extremely disordered, SAMs (n=3-5) the flow parameter is 'significantly lower than for decanethiol. As the crystalline nature of the SAM increases the flow parameter increases until an optimal value is reached for nearly crystalline SAMs, n=10 and 12. The flow parameter then decreases as the SAMs become more crystalline. The flow rate decreases on the more crystalline SAMs because there exists greater area for interaction of the flowing monolayer with the surface. Two explanations for the increased interactions are that the SAM is incompletely ordered or that the number of tilt grain boundaries increases presenting molecular scale pits in the surface and thereby greater area for coupling of the flowing and stationary monolayers.
Table 5. Monolayer data for alkanethiols
Monolayer Contact Angles Flow na Capacitance Water HD Paramter μF/cm2 cm/s°''J
(x 1000)
3 5.2110.23 85 10 2.40±0.12
4 3.77±0.07 93 13 4.74±0.11
5 3.09±0.07 96 16 4.92±0.24
6 2.55±0.14 100 25 5.65±0.44
7 2.2110.09 99 30 5.5410.31
8 2.03±0.11 102 38 5.5210.40
9 1.63±0.12 105 39 5.7310.30
10 1.47±0.04 110 42 6.5510.36
12 1.30±0.05 112 47 6.3110.27
16 0.98±0.04 113 48 6.1310.16
18 0.91±0.03 115 49 6.1010.22
20 0.82±0.03 117 49 5.2510.28 a. The SAM chain length, CH3 (CH2) n.xSH
Additional coupling between the flowing monolayer and the surface may be influencing the flow parameter. For the short, very fluid, alkylmercaptans the additional coupling could result from a greater extent of alkyl/oleyl chain interdigitation. For the longer, more crystalline, alkylmercaptans the additional coupling could be realized at the grain boundaries of crystal domains.
Evidence supporting the grain boundary impedance to flow supposition was obtained in lateral flow measurements performed with a dilute fluorescent probe. In these experiments, which will be described in greater detail later, lateral flow is observed on an inverted fluorescence microscope as the movement of a fluorescent front along the surface. In experiments on M12 SAM modified electrodes the lighted area is homogeneous in intensity; however, on an octadecylthiol SAM modified electrode bright spots develop behind the fluorescence front and remain suggesting that pinning sites have developed on the SAM. The resolution of the microscope limits the site dimensions to less than 10 μm. The existence of "pinhole" defects with .1 - 10 μm diameter in octadecylmercaptan monolayers has been postulated.
The study of n-alkanethiols on gold provides the essential basis for understanding the significance of differences in observed flow parameters. It is clear that the flow parameter is quite dependent on the interfacial surface energy and structure of the SAM at which amphiphile molecules become fixed. The fastest flow rates occur for near-crystalline, homogeneous films. SAM fluidity lowers the flow rate by increasing the flowing monolayer/SAM surface coupling. Lowering the interfacial free energy, thereby increasing the hydrophilicity, lowers the flow rate by decreasing the driving force. Increasing the crystallinity and thereby the number and/or depth of grain boundaries lowers the flow rate by increasing the coupling between the flowing monolayer and the SAM at such pinning sites. To determine the nature of surfaces amenable to sensor development and chromatographic supports, the lateral flow behavior of widely varied SAM surfaces was investigated.
The capacitance, thermodynamic, and lateral flow data for the surfaces investigated in this study are given in
Table 6. There are a number of points which fall well below the alkanethiol data. We suspect that these surfaces have a slower flow rate for oleic acid due to additional coupling, i.e. recognition, with the surface. The surfaces which display this additional coupling are denoted by an asterisk in Table 6. All of these surfaces share a common structural aspect, molecular porosity. These surfaces provide submolecular scale hydrophobic pockets with which the flowing monolayer tail group can have greater interaction. The increased contact area between flowing film molecules and SAM molecules results in a slower flow rate. Such flow rate dependence is evidence that lateral flow based surface characterization is sensitive to molecular topology. The porous SAMs are also reminiscent of traditional chromatographic column packing materials.
Table 6. Monolayer data for terminal functionalized alkanethiols
Monolayer Contact Angles Flow
Figure imgf000054_0001
(X1000)
Class 1 :
BR11 1 . 8610 . 05 89 2.1210.18
BZ 1 5 . 0010 . 18 85 <5 1.4910.12 Table 6. Monolayer data for terminal functionalized alkanethiols
CYO* 4.4310.27 100 <5 1.9910.10
EMU 1.53+0.10 85 37 0.00
PF11 1.7610.10 126 70 5.7810.47
ME11 1.9810.27 73 38 0.00
MTO* 4.7810.27 99 <5 1.7010.15
TEH 1.4910.04 108 38 4.84+0.21
Class 2:
PC16+ 0.9110.07 110 32 4.12+0.29
PC18* 2.2110.29 105 20 4.10+0.25 pyp* 1.2010.06 112 40 3.40+0.18
TC* 1.4210.03 100 10 2.64+0.12
Class 3:
MDS 1.6110.09 112 6 5.07+0.31
TM12* 2.5810.07 103 13 3.29+0.11
a. The SAM surface designation correspond to the following:
Class 1: The first 2 letters designate the surface composition and the number represents the number of carbons in the alkyl chain. Surface designations: BR-terminal bromine; BZ-benzyl mercaptan; CY-cyclohexylmercaptan; EM-methyl ether; PF-perfluoronated; ME-methyl ester; MT-mercaptotoluene; TE-terminal alkene.
Class 2: Biomembrane analogs. PC16-dipalmitoyl; PC18-dioleyl; PYT-phytoylmercaptan; TC-thiocholesterol.
Class 3: Chromatographic supports.
MDS-dodecyl-hexyl disulfide; TM12-tert-dodecanethiol.
The lack of significant chemical recognition for the majority of single component monolayers and the inherent sensitivity of flow to molecular topology naturally leads to the study of lateral flow on mixed monolayers. Practical devices based on lateral flow technology, sensors or separation techniques, will almost certainly rely on mixed monolayer surfaces rather than single component SAMs. While increasing the utility and scope of lateral flow based devices, introducing dual component SAMs adds a great deal of complexity to the interpretation of observed flow rates.
Mixed alkanethiol systems have been thoroughly investigated. Comparison of dual component monolayer properties is complicated because mixed monolayer formation is very dependent on the formation conditions. The relative surface concentrations need not reflect the relative solution concentrations, the surface concentrations being most dependent on the formation solvent. The spatial distribution of the components in the mixture, whether uniformly distributed or phase segregated, is extremely difficult to assess. Both the relative surface concentrations and spatial distribution are important variables in the design of practical devices. For example, the anchoring of nematic liquid crystals at mixed length alkanethiol SAMs is extremely dependent on both of these variables. Lateral flow experiments probe both the composition and distribution of the SAM components on a surface, making them well-suited for the study of mixed SAMs. The relative surface concentrations of components in mixed monolayers can be determined by a handful of measurement techniques. When the components have distinct elemental compositions the relative concentrations can be determined by either x-ray photoelectron spectroscopy or surface infrared spectroscopy. For components with the same elemental composition, as is the case with the mixed alkanethiols here, the only method to quantify the relative concentrations is time-of-flight mass spectrometry. The relative surface concentrations can also be determined from the monolayer capacitance by applying the appropriate equivalent circuit model. Two possible equivalent circuit models are based on the film distribution. If the film is ideally phase segregated, then the overall capacitance is modeled by the area-weighted average of the two component capacitances in parallel. If the film is uniformly mixed then the overall capacitance is best modeled by two capacitances in series. The actual behavior of the overall capacitance is bounded by these two models.
Mixed monolayers of hexylthiol (M6) and dodecanethiol (M12) were made by immersing typical flow electrode substrates in ethanolic solutions containing both thiols.
The observed monolayer capacitance is plotted as a function of mole fraction of M12 in the deposition solution in FIG. 13. The shape of the plot suggests that M12 binds preferentially to M6. An upper bound to the surface concentration of M12 is determined by comparison to the parallel capacitor model (shown as a solid line in FIG. 13) . For example, the deposition solution containing 17% M12 yields a surface with a capacitance equal to that for a 78% M12 surface according the parallel capacitor model.
The flow rates of oleic acid on these mixed M6-M12 surfaces are given in FIG. 14. The flow rates are plotted versus the mole fraction of M12 in the self assembled monolayer as determined using the parallel capacitor circuit model discussed above. The observed rates produce a somewhat unexpected profile. The simplest assumption holds that the flow parameter is a linear combination of the component flow parameters. However, this is clearly not observed. The flow parameters are significantly lower than either of the component flow parameters for nearly all mixtures. The mechanism for lowering the flow parameter so dramatically is that the flow parameter is attenuated by increased SAM fluidity for single component alkanethiol
SAMs. Fluidity should cause the flow parameter profile to be the reverse of what is observed because the'-surface fluidity should increase as the M12 mole fraction decreases. An alternate mechanism is that lateral flow is impeded by crossing domain boundaries in phase segregated SAMs. The existence of such a mechanism is suggested by the difference in the flow parameter for the surface equimolar mixed thiols and the M6M12 mixed disulfide. The disulfide can not phase segregate, whereas the mixed thiols can and do. The slow flow parameter observed for the disulfide suggests that the disulfide monolayer geometry allows for greater interaction with the flowing monolayer. The lateral flow profile suggests, in light of the effect of phase segregation, that the number of domains increases steadily until reaching a maximum value at about 80% M12 on the surface. The number of domains then remains constant and only decreases when the mole fraction of M12 on the surface decrease to below 0.5. The number of domains decreases as quickly as it had increased and finally the mixed SAM behaves as if it were a pure M6 monolayer even with 20% of the surface covered with M12. Among the many claims of potential applications for SAMs, the use as model systems for studying biological membranes has been highly touted. The majority of studies involving biomimetic systems employ the SAM, typically an alkanethiol, as a support for a biomembrane monolayer or bilayer of interest. We have studied the lateral flow behavior of a number of biomembrane components and conducted a detailed study of a mixed system involving thiol derivatives of cholesterol and dipalmitoylphosphatidylglycerol. The capacitance, thermodynamics, and lateral flow values for the single component monolayers are given in Table 6 under the heading class 2. The designations correspond to the following: PCI6 - dipalmitoylphosphatidylthioethanol, PC18 - dioleylphosphatidylthioethanol, PYT - phytoylthiol, TC - thiocholesterol. The flow parameters for all of these monolayers fall within the circle denoting additional flowing film/surface coupling in FIG. 18. The increased coupling presumably arises from the surface fluidity and in the cases of PYT and TC from molecular porosity created by the bulky component structure. The use of alkanethiol SAMs as a support in the study of biomembranes has a serious shortcoming in that the SAM has been observed to have an ordering effect on the supported monolayer. Thus, the use of more fluid biomembrane SAM forming species is preferred as a more natural support. Of particular interest for studies on mammalian systems are mixed monolayers of cholesterol and dipalmitoylphosphatidylcholine (DPPC) . We have studied the lateral flow behavior of mixed SAMs of PC16 and TC.
The monolayer capacitance data shows that TC is only slightly preferentially deposited in the monolayer. The lateral flow profile as a function of the solution percent of TC is given in FIG. 15. The flow parameter profile is remarkably different than that observed for the mixed alkanethiol SAMs. The flow parameter is above the tie-line connecting the single component values for all mixtures of PCI6 and TC. For mixed monolayers on the air-water interface cholesterol and DPPC, cholesterol has a condensing effect on the fluid DPPC. This behavior is seemingly also observed in the mixed SAM films as well. The shape of the flow parameter profile argues against significant phase segregation in the SAM. Though immiscible at low surface pressures (<12 mN/m) on the air-water interface, cholesterol and DPPC are miscible for the lateral pressures common in a membrane bilayer (20-35 mN/m) .14 Therefore, we believe that the mixed PC16-TC monolayer films must be packed in a manner similar to that in a membrane bilayer which is very desirable for applying these SAM systems as supports for model membranes .
We have shown that lateral flow rates are quite sensitive to surface chemical topology and composition. Such dependence is due to the integrating nature of the flowing monolayers movement across a surface. The flow rate depends on the chemical composition of the surface and on the solvent exclusion driving force. The coupling between the flowing monolayer and underlying surface structure is dramatically influenced by changing the chemical composition of the surface to include molecular scale porosity. Studies of mixed monolayer systems highlight the dependence of the lateral flow rate on both the chemical composition of the surface and the spatial distribution of the mixed components. The dependence of the lateral flow rate on the surface chemical composition and film homogeneity make lateral flow a novel method of surface characterization. Sensors are becoming increasingly more commonplace. Issues of sensitivity, selectivity, and expense motivate the investigation and development of new sensor technologies. There are important needs for new analytical sensors in the pharmaceutical, environmental, process control, biomedical, and personal diagnostic analysis fields. Much of the effort in studying SAMs has been motivated by the potential for the development of chemical and biological sensors with increased sensitivity and selectivity. SAM based sensors have already been developed for the detection of ultralow levels of cadmium, ionic surfactants, hydrophobic redox molecules, and biomolecules (proteins, antibodies, etc.) .
Monolayer flow technology as described herein provides for a new class of sensors. The appeal of flow-based sensing is due to the versatility of the transduction mechanism in regards to sensor design. "Sensing" is achieved by modulation of the monolayer flow rate, a mechanical effect or by electrical characteristics of the resulting bilayer film. Most current sensor technologies produce a response in relation to an electrochemical, mass, optical, or conductivity change in the sensing element. Flow rate modulation can be achieved by three distinct mechanisms, depicted in FIG. 16, including: (1)~ binding of a subphase species to the flowing monolayer; (2) permeation into or non-specific binding on the SAM; and (3) specific binding interaction of a subphase species with a minor component of the SAM. Simple sensors based on each of these flow attenuation mechanisms are now described.
Conceptually, the simplest means of eliciting a flow-based sensor response is to affect the flowing monolayer. Changing the monolayer viscosity, for example, will influence the flow rate. The monolayer viscosity is largely dependent on the film phase state and the extent of mechanical coupling to the subphase. The phase state and subphase coupling can be affected by interaction of a solution species, an analyte, with the flowing monolayer. Additionally, specific interactions can be engineered by the appropriate design of the monolayer head group.
The first example of flow-based sensing which we will discuss is that for subphase divalent cations. Interaction of a divalent cation, cadmium for this discussion, with the carboxylic acid head group of oleic acid is the basis for sensor response. The condensing effect of divalent cations on ionized carboxylic acid monolayers is well documented. Each divalent cadmium cation associates with two carboxylates and dramatically increases the monolayer viscosity. The flow rate of the associated monolayer should be slower than for the unassociated monolayer. Because the cation associates with the deprotonated carboxylic acid only, the pH of the subphase is of critical importance.
This type of flow-based sensor employs the same experimental procedure for flow parameter determinations as described above. The flowing molecule is oleic acid, with oleyl alcohol serving as a control. The flow surface is M12. The subphase is 10 mM KCl with 1 mM total concentration of phosphate buffer. The relative concentrations of mono- and dibasic phosphate were varied to give the desired pH in the range 3 to 8.5. Cadmium containing subphases were made using cadmium chloride. Control of the pH is important for two competing reasons. First, the equilibrium spreading pressure and extent of oleic acid ionization, pKa = 6.8, are a function of the pH. Second, the solubility of cadmium ions decreases as the solution becomes more basic, Ksp for Cd(OH)2 = 4.5xl0"15. The effect of the subphase pH on the flow parameter for a subphase containing 0.1 mM cadmium chloride was investigated to determine an appropriate pH value for the sensor subphase solutions. The results are given in FIG. 17A. On acidic subphases the flow parameter is the fastest. The flow parameter decrease rapidly near the pKa for oleic acid, and stabilizes at about 40% of the acidic subphase value by pH 8. We selected a buffer pH of 7.5 for the sensor subphase because it provides nearly complete ionization of the oleic acid film and permits cadmium ion concentrations as large as 25 millimolar.
The observed flow parameter as a function of cadmium ion concentration is given in FIG. 17B. It is apparent that cadmium is only effective at attenuating the flow rate of oleic acid for concentrations greater than 0.01 mM and that the sensor response is limited to cadmium concentrations between 0.05 and 0.2 mM. Control experiments were conducted using oleyl alcohol, which should not associate with subphase cadmium, as the flowing monolayer . The flow parameter was found to be independent of the cadmium ion concentration in the control experiments. Sensor selectivity is quantified by measuring selectivity coefficients for interferences. The selectivity coefficient is defined as the ratio of interferent to analyte concentration for which equivalent sensor responses are produced. The subphase concentration of calcium which produces the same flow parameter as 0.075 mM cadmium, the point in the middle of the linear region, is 0.33 mM. This corresponds to a selectivity coefficient of 4.4. The ratio of the formation constants for cadmium and calcium acetate is 4.9, which agrees well with the measured selectivity coefficient. The flow parameter is sensitive to interactions between the flowing monolayer and a subphase solute.
The second class of monolayer flow sensors is based on non-specific interactions between a subphase solute and the SAM. The non-specific interaction can be permeation of a hydrophobic solute into the SAM or binding of a solute, e.g. a protein, onto the SAM surface. These two examples of non-specific interactions would produce different mechanisms of attenuating the flow rate. Permeation of a hydrophobic solute into the SAM would produce swelling and thereby increase the surface fluidity. The flow parameter decreases with increased surface fluidity in earlier work, so we would expect the flow parameter to decrease with increasing amounts of permeant. Binding of a protein to the SAM surface is expected to attenuate the flow parameter by increasing the viscosity within the flowing monolayer as it flows around the fixed hydrophilic protein.
The influence of subphase octanol on the flow rate of oleyl alcohol at a M12 surface has been investigated. Preliminary results indicate that octanol concentrations m the micromolar to millimolar range (the saturation concentration of octanol m water is 3 millimolar) have a very slight influence on the flow rate.
Non-specific protein adsorption onto hydrophobic surfaces is a critical issue in biosensor design. In experiments using bovme serum albumin (BSA) , exposure of a M12 surface to a 1 μg/ml solution of BSA for 1 minute is enough to effectively stop monolayer flow. Such non-specific adsorption must be minimized m the design of biological sensors.
The final, and most preferred, class of monolayer flow sensor is based upon a specific interaction between a subphase solute and a minor SAM component. The sensor is made possible by creating a mixed monolayer flow path. The mixed monolayer consists of an inactive surface (typically a methyl terminated amphiphilic monolayer) with a small percentage of dispersed active surface. The active surface elements may be as simple as a carboxylic acid unit for a pH sensor or as complex as a bound antigen for an antibody sensor. The most difficult part of designing such a sensor is optimizing the percentage of active surface area to give good sensor characteristics. The percentage of active surface area is determined during SAM formation.
The competitive adsorption of two distinct thiols results in a dispersed monolayer where the relative surface composition need not correspond to the relative solution concentrations. The component with the lowest relative solubility in the deposition solvent preferentially adsorbs to the gold surface. The distribution of the components in a mixed monolayer is not clear at present, but contact angle and laser desorption mass spectrometry measurements indicate that phase segregated domains must be smaller than 100 nm in diameter. The relative surface concentrations and lateral distribution of the components in a mixed SAM are expected to have a major impact on the sensor characteristics for that surface. The issue of mixed monolayer formation in relation to sensor characteristics is described by way of example, a monolayer flow based pH sensor.
A monolayer flow based pH sensor was developed by forming a SAM with mixed methyl and carboxyl terminations.
The flow parameter is predicted to depend on the subphase pH due to differences in the extent of carboxyl group ionization. Deprotonation of the acid group results in a much more solvated, charged carboxylate group. The flow parameter will decrease with increased carboxylate density due to an increase in the viscosity within the flowing monolayer. In developing the flow based pH sensor, a number of modifications were made to the flow parameter determination procedure. Because the equilibrium spreading pressure, and thereby the flow parameter, of oleic acid are a function of pH, oleyl alcohol whose ESP is independent of pH was selected as the flowing monolayer. The subphase solutions are 10 mM KCl with HCl or KOH added to reach the reported pH values. The mixed methyl and acid terminated SAM sensor surfaces were fabricated by deposition from ethanolic (95%) solutions of octanethiol (M8) and 11-mercapto-l-undecanoic acid (All) . Flow measurements were conducted by positioning cut substrates at the subphase interface as described above. In the sensor experiments, additional time was permitted to establish equilibrium between the sensor surface and subphase before the piston oil was applied to the surface. The capacitance-time curves have the same shape as for single component surfaces, but the slope of the capacitance versus square root of time plot is observed to vary with the subphase pH. The results for the sensor surface deposited from a 5 mole percent solution of All to M8 are summarized in Table 7 and shown in FIG. 18. The control experiment data for the flow parameter of oleyl alcohol on a single component M8 surface is included in FIG. 18 as well. The flow parameter is greatly attenuated at the mixed monolayer surface, even at subphase pH values for which the carboxylic acid groups should be fully protonated. The flow parameter decreases in the region of the All pKa, as predicted. Note also that the normalized response, a measure of the final bilayer capacitance, also increases with pH. This suggests that areas of the SAM surface are not being covered by a bilayer when the carboxyl groups are deprotonated.
Table 7. Flow-based pH sensor data
PH SAM Cap Normalized Flow Parameter
Response (x 1000) μF/cm7 cm/s0-5
M8 Control
2.5 2. .17 ± 0.02 0.390 ± 0.003 7, .47 + 0.35
5.8 2. .17 ± 0.03 0.391 ± 0.012 7. .55 + 0.25 11.8 2, .18 ± 0.02 0.387 + 0.030 7. .65 + 0.30 Mixed A11/M8
2.2 2. .34 ± 0.07 0.413 + 0.010 4. .89 + 0.27
5.8 2. .35 ± 0.07 0.419 + 0.003 4, .42 + 0.22
9.0 2. .42 ± 0.02 0.493 + 0.008 3. .10 ± 0.08 10.4 2. .44 ± 0.07 0.504 + 0.038 2. .61 + 0.1-6
11.8 2. .44 + 0.03 0.671 + 0.041 1. .40 + 0.11
The essential element to this class of sensor design is the formation of the mixed SAM surface. The relative solution concentration which provides the optimal sensor response was determined empirically to contain 5 mole percent All to M8.
The data for the 5% All to M8 deposition solution surface has the best sensor characteristics which we have seen to date. Surfaces created from deposition solutions containing greater than 10% All show no monolayer flow activity at all, regardless of pH. Surfaces created from deposition solutions containing 2% All show a similar pH response, but with less attenuation of the flow parameter with respect to the pure M8 surface. We have also observed that the relative surface concentration, as measured by the pH sensor response, is a function of the total thiol concentration in the deposition solution. Increasing the total thiol concentration in the deposition solution results in SAMs which are enriched in the methyl-terminated component .
Increasing the length of the methyl-terminated component should decrease the sensor response of the surface because complete hydration of the deprotonated carboxyl group is more difficult. We have examined a system with mixtures of All and M10. A simplistic picture of the two surfaces is presented in FIG. 19. Interpretation of the methyl-terminated chain length effect is complicated by the difference in relative surface concentrations for the same deposition solution concentrations. Determination of the All surface coverage must be established by a separate technique, such as XPS, for direct comparisons to be made. Higher solution mole percentages of All are required with the M10 system (up to 25%), as is expected given the decrease in solubility of M10, to produce the similar flow parameter attenuation. A comparison of the sensor properties for M8 and M10 surfaces is given in FIG. 20. We are severely limited in our ability to compare the two sensor response curves in FIG. 20 by not knowing the surface concentration of All on the two surfaces. XPS and contact angle data both suggest that the total surface acid concentration is less than 2% in both cases. It is quite remarkable that the flow parameter is so sensitive to such a small surface component. Based on the teachings herein, one of ordinary skill in the art may prepare monolayer flow based biochemical sensors. In particular, sensors for avidin and the dinitrophenol antibody may be prepared by the mixed monolayer approach using the corresponding conjugate. These sensors will rely on the specificity of receptor-substrate pairs in biological systems. Avidin has a strong affinity for biotin (K = 1 x 1015 M-l) . Likewise, antibodies to dinitrophenol have association constants on the order of 107 M-l. Both biotin and dinitrophenol can be conveniently attached to alkylthiols for use in a sensor system like the pH sensor detailed above.
One of the objectives of this invention is to achieve a separation within a monolayer assembly. We accomplish this in one embodiment by marrying two monolayer technologies: Langmuir films and self-assembled monolayers on solid substrates. This Langmuir film chromatography (LFC) was developed using high performance liquid chromatography (HPLC) as a paradigm. The components of an HPLC system were re-engineered for the two-dimensional fluid system employed in LFC. Detectors were the first components investigated, resulting in the continuous interfacial bilayer electrode described above. The pump and mobile phase were the next components for consideration. In the course of investigating piston oil properties, monolayer flow was observed effectively combining the LFC pump and mobile phase. Monolayer flow greatly simplifies the design of LFC experiments and allows us to consider the more difficult question of column design.
Just as the other components of LFC were developed with consideration of their HPLC analogs, the LFC column was developed with chemically bonded stationary phases in mind. The utility of HPLC blossomed with the development of chemically bonded stationary phases in the early 1970s. Chemically bonded phases are constructed by the covalent attachment of an organic thin film on support material, typically silica. Knowledge of the chemistry of the retention process will allow for predictive development of unique stationary-phase materials for targeted separations. Exploration of the retention process has been initiated by investigating the bonded phase environment in situ. However, a complete understanding of the separation mechanism at bonded phases have yet to be made. LFC should be quite useful as a model system for direct observation of substrate structure influence on the interaction with analytes.
While sorption-based separations have not been attempted, groups have attempted to resolve components in a supported bilayer using electrophoretic motion. In these experiments concentration gradients of charged fluorescent probes were generated by applying an electric field. Probe movement is described by competition between random diffusion and electric field-induced drift. The diffusion coefficient of the fluorescent probe within the supported bilayer can be determined from the concentration profile or by fluorescence recovery after photobleaching (FRAP) measurements. We have also used fluorescence microscopy to observe monolayer flow.
The use of fluorescent probes greatly simplifies the LFC development process by allowing direct observation of the interactions between the flowing phase and the surface during flow. The experimental procedure for observing flow is altered appreciably to accommodate fluorescence detection. The Teflon trough used in all previously discussed flow measurements is replaced by a glass trough for the fluorescence measurements. A subphase well is-produced in a 1 x 3 inch microscope slide by drilling a hole through the slide using a glass coring bit. The bottom of the well is made by sealing a glass coverslip (ca. 0.15 mm thickness) over the hole using epoxy. The well volume is between 1 and 2 ml. Electrodes are constructed in a similar manner to the method described above using thicker glass, ca. 3 cm, to provide a longer flow path. Fluorescence measurements were performed on a Zeiss Axiovert 135 TV inverted microscope with a Hg lamp source. Fluorescence was observed using an intensifier and a CCD camera and was recorded using a VCR. The label present on all of the fluorescent molecules used for these experiments is Lissamine rhodamine B (LRB) . The excitation and emission wavelengths for this rhodamine are 550 and 590 nm, respectively. The filter sets on the microscope are optimized for use with LRB.
Monolayer flow is measured directly as the movement of a fluorescence front in these experiments. In a typical experiment the glass trough is filled with subphase electrolyte (50 mM KCl) and the surface aspirated using a capillary. The flow electrode is placed at the liquid interface as described above. After focusing on the electrode edge in the microscope, a flowing mixture of interest is applied to the air/water interface as a chloroform solution. The movement of fluorescently labeled molecules onto the electrode surface is recorded for subsequent analysis.
Determination of the flow parameter in the fluorescence experiments is straightforward. The distance of film flow is measured by the position of the fluorescence front. Distance calibrations were performed using a Neubauer ruling with 0.05 mm line spacings. The flow parameter, in this case, is the slope of a distance versus the square root of time plot. The flow data for 1% solutions of dioleylphosphatidylethanolamine-LRB (DOPE-LRB) and dipalmitoylphosphatidyl-ethanolamine-LRB (DPPE-LRB) in oleic acid are given in FIG. 21. The flow parameters for DOPE-LRB and DPPE-LRB determined from the slopes in the FIG. 21 are 4.38 x 10~3 and 4.11 x 10"' cm/sl/2, respectively. The flow parameter values have been measured for these mixtures at M12 surfaces three times each and the observed values are (4.4 ± 0.3) x 10"' and (4.2 ± 0.3) x 10~3 cm/s,/2 for the dioleoyl and dipalmitoyl containing mixtures, respectively. These values are roughly the same as the flow parameter measured for these mixtures using the capacitance measurement on a larger trough. Why the flow parameters are lower for these mixtures than for pure oleic acid on M12, 6.3 x 10~3 cm/s1/2, is still not understood, but we believe that it is a combination of geometry effects on the glass fluorescence trough, i.e. much smaller solution volumes and thicker, more irregular, flow electrode surfaces. We have observed flow parameters for the fluorescent probe containing mixtures as large as 5.9 x 10"3 cm/s1/2 on the thinner, smoother flow electrodes used in the capacitance measurements. Beyond the obviously slower flow rates, it is apparent that the flow parameter for both of the labeled probes are quite similar, indicating that no resolution of a mixture of these compounds would be expected in a separation experiment.
In order to observe resolution of a mixture in the fluorescence measurements it is necessary to provide for a band injection of material and elute the mixture with a non-fluorescent mobile phase. We have succeeded in injecting a band at the electrode edge by starting flow with the mixed fluorophore solution, aspirating the surface to effectively stop flow by removing the piston oil, and placing the pure piston oil on the surface to continue monolayer flow. As predicted from the individual flow parameters, we do not observe resolution of DOPE-LRB and DPPE-LRB in band injection experiments on M12 surfaces. Additionally, we did not observe resolution of mixtures containing octadecyl-LRB and DOPE-LRB. However, we do observe significant band broadening during the course of band elution experiments. Comparing the length scales for the fluorescence flow rate and the diffusional band broadening plots, we observe that the monolayer flow rate is only ten times that for diffusion. Thus, if LFC is to evolve into a separation technology mobile and stationary phase pairs with faster flow rates must be developed. Faster monolayer flow rates can be achieved by opening up the stationary phase support architecture to include regions of air/water interface, that is, to effectively construct a cross-section of an HPLC column. Such supports can be lithographically produced to yield ideally packed two-dimensional columns.
Practical LFC will also require an improved geometry which addresses issues such as column conditioning and sample collection. To this end, we have utilized ring electrode substrates. The capacitance flow measurement ring geometry is shown schematically in FIGS. 22 and 23. The counter and reference electrodes 302, 304 and Wilhelmy plate 306 are not present in fluorescence-flow measurements. Capacitance- and pressure-time plots for the flow of oleic acid on a ring electrode modified with M12 are given m FIGS. 24A and 24B. The capacitance profile appears just as would be expected, and the data yields a flow parameter identical to that for a rectangular M12 electrode. The complete bilayer capacitance is reached after 800 seconds of monolayer flow, marked in FIG. 24A by tl. The pressure inside the ring remains constant at zero until 3200 seconds have passed since the oleic acid piston oil was added to the water surface outside of the ring t2 in FIG. 24B. The steady-state molecular flux across the flow surface can be calculated using
Φ SA/AC t2~ t;
where SA is the surface area inside the ring and A0 is the area per molecule at which the pressure of oleic acid rises above zero as determined by the pressure-area isotherm (53 AVmolc) . The steady-state molecular flux calculated from the example data set in FIGS. 24A and 24B is 3.24 x 10n molc/s.
The molecular flux can be converted to a effective monolayer velocity, m.v., in the bilayer using
m . v. =— Φ * Aσ- inner where Pιnner is the inner perimeter of the ring. The monolayer velocity for the example data is 2.3 μm/s. We have determined an effective monolayer velocity of oleic acid on a M12 surface of 2.6 ± 0.6 μm/s for six separate ring flow experiments.
The ring geometry has several advantages over the rectangular geometry for LFC development. Steady-state flow is much more desirable than the continuously decreasing flow rate profile available in the rectangular geometry. Steady-state flow also permits column conditioning with the mobile phase, given appropriate injection technology. The ability to collect eluants in the ring interior by surface aspiration is a clear advantage. It is clear that the ring geometry is the best geometry for LFC development. Thus, several applications for monolayer flow have been described. It has been demonstrated that the monolayer flow is particularly sensitive to chemical topology and is a novel surface characterization tool. The flowing monolayer system can also be used as a model system for the study of two-dimensional fluid mechanics and molecular friction. The simplicity of this system makes monolayer flow very attractive for sensor development. The flow rate is quite sensitive as it integrates large numbers of individual chemical recognition events. The great chemical sensitivity of flow measurements stems in large part to their similarity to chromatographic separations. Flowing monolayers are an ideal geometry for a chromatographic separation in which the analytes and mobile phase are in continuous contact with the stationary phase.
Although preferred embodiments are specifically illustrated and described herein, it will be appreciated that modifications and variations of the present invention are covered by the above teachings and within the purview of the appended claims without departing from the spirit and intended scope of the invention.
What is claimed as new and desired to be secured by Letters Patent of the United States is:

Claims

1. A method of creating a monolayer film, comprising the steps of: providing a subphase; positioning an element with a hydrophobic surface such that said hydrophobic surface contacts said subphase; and introducing an amphiphilic substance to said subphase to create a monolayer film in the interface between said hydrophobic surface and said subphase.
2. The method according to claim 1, wherein said hydrophobic surface is a monolayer coating a solid support.
3. The method according to claim 2, wherein said hydrophobic surface is a self assembled monolayer coating a solid support.
4. A method of measuring the physical characteristics of an interface between a hydrophobic surface and a subphase, comprising the steps of: providing a subphase; positioning an element with a hydrophobic surface such that said hydrophobic surface contacts said subphase to form an interface; introducing an amphiphilic substance to create a monolayer film that flows across said interface; measuring the flow rate of said monolayer film across said interface.
5. A method of measuring the physical characteristics of a surface or interface according to claim 4, wherein said element is an electrode and said step of measuring the flow rate is performed by measuring the electrical admittance of the electrode.
6. A method of measuring the physical characteristics of a surface or interface according to claim 4, wherein said step of measuring the flow rate is performed by fluorescent detection of said flowing monolayer.
7. A method of separating a first substance and a second substance dissolved in or in contact with an amphiphilic mixture, comprising the steps of: •- providing a subphase; positioning an element with a hydrophobic surface such that said hydrophobic surface contacts the surface of said subphase to form an interface; introducing said mixture to create a monolayer film in the interface such that said first and second substances flow across said hydrophobic surface at different rates to cause a physical separation between said first and second substances .
8. A method of detecting the presence of a first substance in a material suspected on containing said first substance, comprising the steps of: providing a subphase; positioning an element with a hydrophobic surface such that said hydrophobic surface contacts said subphase for form an interface; introducing an amphiphilic substance to create a monolayer film that flows across said hydrophobic surface in a manner dependent on the presence of said first substance; and measuring characteristics of said monolayer film to determine whether said first substance is present in said material .
9. A method of detecting the presence of a first substance according to claim 8, wherein said monolayer film flows across said hydrophobic surface at a rate dependent on the presence of said first substance and said step of measuring measures the rate of flow of said monolayer film.
10. A method of detecting the presence of a first substance according to claim 8, wherein said step of measuring measures the characteristics of said monolayer film on said hydrophobic surface.
11. A method of detecting the presence of a first substance according to claim 8, wherein said first substance is a component of said hydrophobic surface.
12. A method of detecting the presence of a first substance according to claim 8, wherein said first substance is a component of said amphiphilic substance.
13. An apparatus for measuring the physical characteristics of an interface, comprising: a trough to hold a subphase and a monolayer film; an element with a hydrophobic surface positioned such that said hydrophobic surface contacts said monolayer film; a measuring device to measure the characteristics of said monolayer film; a counter electrode; and wherein said element is a disk-shaped electrode and said measuring device is connected to said counter electrode and said electrode to measure the electrical characteristics of said electrode.
14. An apparatus for measuring the physical characteristics of a surface or interface according to claim 13, wherein said electrode is a rotating disk.
15. The apparatus of claim 14 wherein said rotating disk maintains a substantially constant contact area with said monolayer.
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US5605662A (en) * 1993-11-01 1997-02-25 Nanogen, Inc. Active programmable electronic devices for molecular biological analysis and diagnostics

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