WO2008068678A1 - Biotechnological device including an actuation means for changing the mobility of preselected biomolecules - Google Patents

Biotechnological device including an actuation means for changing the mobility of preselected biomolecules Download PDF

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Publication number
WO2008068678A1
WO2008068678A1 PCT/IB2007/054838 IB2007054838W WO2008068678A1 WO 2008068678 A1 WO2008068678 A1 WO 2008068678A1 IB 2007054838 W IB2007054838 W IB 2007054838W WO 2008068678 A1 WO2008068678 A1 WO 2008068678A1
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Prior art keywords
permeation layer
actuation means
present
changing
ratio
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PCT/IB2007/054838
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French (fr)
Inventor
Ralph Kurt
Murray Fulton Gillies
Roel Penterman
Stefano Cattaneo
Dirk Jan Broer
Emiel Peeters
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Koninklijke Philips Electronics N. V.
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Application filed by Koninklijke Philips Electronics N. V. filed Critical Koninklijke Philips Electronics N. V.
Priority to EP07849282A priority Critical patent/EP2092338A1/en
Priority to JP2009538839A priority patent/JP2010511490A/en
Publication of WO2008068678A1 publication Critical patent/WO2008068678A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/002—Electrode membranes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms

Definitions

  • BIOTECHNOLOGICAL DEVICE INCLUDING AN ACTUATION MEANS FOR CHANGING THE MOBILITY OF PRESELECTED BIOMOLECULES
  • the detection usually occurs in such a way that the fluid to be analyzed is provided on a substrate material, which contains capture sites for the target molecules which are subject to detection.
  • a capture site may comprise a corresponding DNA-strand in case the target molecule is also a DNA-strand or an antibody in the case of a protein assay.
  • the target molecules in the fluid will then bind specifically to the capture site and remain there even after the fluid is removed.
  • the target molecule contains a label and in this way may be detected.
  • the label could be a fluorescent label or a magnetic label depending on the detection means, i.e., the type of bio-sensor, optical or magnetic sensor.
  • the permeation layer provides capture sites for the immobilization of nucleic acids (or other preselected biomolecules or target molecules).
  • nucleic acids or other preselected biomolecules or target molecules.
  • the main function of the permeation layer is to separate the captured target molecules from the highly reactive electrochemical environment generated immediately at the electrode surface.
  • the layer also allows ions and gasses arising from electrochemical reactions at the electrodes to gradually diffuse into the biological solution.
  • the permeation layers which are known in the art are passive hydrogels, i.e., hydrogels that are in wet conditions insensitive to external stimuli. Furthermore, due to the uniform swelling and/or porosity of the permeation layer, the preselected biomolecules or target molecules will in many cases also enter the permeation layer uniformly, i.e., also at positions where no capture sites are present. It is therefore an object of the present invention to provide a device, which is able of at least partially overcoming some of the above-mentioned drawbacks and helps increasing the specif ⁇ ty and the effectivity of the analysis. This object is solved by a composite according to claim 1 of the present invention. Accordingly, a biotechnological device is provided, comprising at least one permeation layer and at least one actuation means which is capable of
  • biotechnological device is to be understood in its widest sense and includes especially one or more of the following devices: - devices for the detection of one or more preselected biomolecules in a fluid sample, especially devices for the detection of biomolecules in aqueous solution. devices for the controlled release of a compound, especially for drug release. - devices for performing amplification reactions such as PCR
  • artificial scaffolds for tissue engineering and (stem) cell therapies including devices for the release of molecules such as growth factors, cytokines etc. to stimulate growth or proliferation of cells and devices which pump nutrients towards cells or accelerate degradation of the scaffold on command.
  • biomolecules as well as “target molecules”, “capture sites” “drugs” according to the present invention are to be understood in the widest sense and especially include and/or mean the product(s) of an amplification reaction, including both target and signal amplification); purified samples, such as purified genomic DNA, RNA, proteins, etc.; raw samples (bacteria, virus, genomic DNA, etc.); biological molecular compounds such as, but not limited to, nucleic acids and related compounds (e.g. DNAs, RNAs, oligonucleotides or analogs thereof, PCR products, genomic DNA, bacterial artificial chromosomes, plasmids and the like), proteins and related compounds (e.g.
  • polypeptides especially means and/or includes the diffusion rate in the parts of the at least one permeation layer affected by the actuation means and/or the average electrophoretic mobility of the preselected biomolecules inside the parts of the at least one permeation layer affected by the actuation means.
  • the transport of biological particles to the capture site is increased and/or influenced by using an electrical field generated by either the same electrodes used for actuating the permeation layer and/or separate electrodes meant specifically for generating an electrical field directed towards the capture site.
  • the transport will be accelerated by charging an electrode near the capture site with a positive voltage.
  • the polarity of charge is dependent on the pH and this should therefore according to a further embodiment of the present invention be taken into account when choosing the polarity of the voltage required for accelerating transport.
  • the dielectrophoretic effect is used to improve the transport of even non-charged bio- particles (e.g. cells).
  • This embodiment is particularly useful when an electric dipole can be induced on the bio-particle.
  • after capturing the frequency of the applied voltage is changed to repel non-specifically bonded bio-particles. This has been shown for a wide range of applications within the present invention to further lower the background signal.
  • distance in connection with mobility in the sense of the present invention especially means and/or includes the average distance from the surface of the permeation layer - especially in the parts of the at least one permeation layer affected by the actuation means - to the capture sites/probes.
  • This distance is according to the present invention especially meant as a macroscopic dimension and is measured in a straight line although the shortest "travel distance" for a molecule flowing from said surface towards said capture sites/probes might be significantly larger in a nanoporous, mesoporous or network like structure.
  • capture site in the sense of the present invention is especially a certain area within the device where one or more (usually identical) capture probes are located.
  • capture probes in the sense of the present invention especially mean and/or include molecules (or assemblies of molecules) which are able to interact specifically with the preselected biomolecules.
  • each of the capture site(s) may comprise only one capture probe (e.g. in case the capture probe is an antibody or a cell) or a high number of capture probes (e.g. in case the capture probe is a DNA-strand).
  • the type of capture probe may vary from site to site on the array.
  • the flow of preselected biomolecules to the capture sites can be increased while in other areas of the . mobility . , , permeation layer the ratio ⁇ p ⁇ is kept constant or even reduced.
  • the design of the device can be made more compact - In many applications of the present invention, a better control over the fluid comprising the preselected biomolecules can be obtained.
  • the present invention provides in one embodiment a device for analyzing one or more samples, especially fluid samples for the presence, amount or identity of one or more preselected biomolecules (which are in this context to be called target molecules) of interest in the samples.
  • the target molecule(s) and the capture site(s) and probe(s) may be, but not limited to, the product(s) of an amplification reaction, including both target and signal amplification; purified samples, such as purified genomic DNA, RNA, proteins, etc.; raw samples (bacteria, virus, genomic DNA, etc.); biological molecular compounds such as, but not limited to, nucleic acids and related compounds (e.g. DNAs, RNAs, oligonucleotides or analogs thereof, PCR products, genomic DNA, bacterial artificial chromosomes, plasmids and the like), proteins and related compounds (e.g.
  • polypeptides polypeptides, peptides, monoclonal or polyclonal antibodies, soluble or bound receptors, transcription factors, and the like), antigens, ligands, haptens, carbohydrates and related compounds (e.g. polysaccharides, oligosaccharides and the like), cellular fragments such as membrane fragments, cellular organelles, intact cells, bacteria, viruses, protozoa, and the like.
  • the actuation e.g. polysaccharides, oligosaccharides and the like
  • cellular fragments such as membrane fragments, cellular organelles, intact cells, bacteria, viruses, protozoa, and the like.
  • means is capable of changing the ratio ⁇ p ⁇ of the preselected biomolecules in at
  • means is capable of changing the ratio ⁇ p ⁇ of the preselected biomolecules in at
  • At least one actuation means is capable or increasing the ratio ⁇ p ⁇ or
  • the actuation means will according to a further embodiment of the present invention be associated with at least one of the capture site(s), which helps to direct the preselected biomolecules to the capture site(s).
  • actuation means is capable of increasing the ratio ⁇ p ⁇ of the preselected
  • biomolecules in at least selected parts of the permeation layer by a factor of >2, preferably by a factor of >5 and most preferred by a factor of >10 (in wet conditions).
  • At least one actuation means is cap 1 able of decreasing ° the ratio " dT ⁇ is—tance of the
  • the actuation means will according to a further embodiment of the present invention be associated with those parts of the permeation layer which are not associated to one of the capture site(s). According to one embodiment of the present invention, at least one
  • actuation means is capable of decreasing the ratio ⁇ p ⁇ of the preselected
  • biomolecules in at least selected parts of the permeation layer by a factor of >2, preferably by a factor of >5 and most preferred by a factor of >10 (in wet conditions).
  • means is capable of changing the ratio ⁇ p ⁇ of the preselected biomolecules in at
  • the swelling of the permeation layer (or parts thereof), the ratio ⁇ p ⁇ can be set
  • the actuation means is capable of changing the swelling of selected parts of the permeation layer by a factor of >1.2 (in wet conditions), preferably by >2, more preferably >5 and most preferably >10 (in wet conditions). According to an embodiment of the present invention, the actuation
  • means is capable of changing the ratio ⁇ p ⁇ of the preselected biomolecules in at
  • the ratio ⁇ p ⁇ can be
  • the actuation means is capable of changing the permeability of selected parts of the permeation layer by a factor of >2, preferably >5 and most preferred >10 (in wet conditions).
  • permeability (commonly symbolized as K, or k) is especially to be understood as a measure of the ability of a porous material to transmit fluids.
  • Ki is the intrinsic permeability [L2]
  • C is a dimensionless constant that is related to the configuration of the flow-paths d is the average, or effective pore diameter [L].
  • the permeability can be measured either directly (e.g. using Darcy's law) or through estimation using empirically derived formulas.
  • Other units are the SI units cm 2 and m 2 .
  • the device comprises at least one capture site, whereby the actuation means is capable of
  • the average size of the at least one capture site is ⁇ lO ⁇ m and ⁇ lOOO ⁇ m, preferably >20 ⁇ m and ⁇ 500 ⁇ m and most preferred >50 ⁇ m and ⁇ 200 ⁇ m.
  • the device comprises >10, preferably >100 and most preferred ⁇ IOOO capture sites.
  • the device comprises at least one active layer part associated with every capture site, whereby the term "active layer part" means the part of the at least one permeation
  • the average ratio of the size of the active layer to the size of the associated capture site is >1 : 1 and ⁇ 10:l, preferably >1,5:1 and ⁇ 3:l.
  • the device comprises at least one transition region between the capture site(s) and/or active layer part(s), which are unaffected by the at least one actuation means.
  • the average diameter of the at least one transition region is >5 ⁇ m and ⁇ 5000 ⁇ m, preferably >10 ⁇ m and ⁇ lOOO ⁇ m, more preferably >20 ⁇ m and ⁇ 500 ⁇ m and most preferred >50 ⁇ m and ⁇ 200 ⁇ m.
  • the at least one permeation layer has an average thickness in wet state (but before changing the permeability and/or swelling of certain regions) in the range of >l- ⁇ 500 ⁇ m, preferably >5- ⁇ 200 ⁇ m, more preferably >10- ⁇ 100 ⁇ m.
  • the actuation in the range of >l- ⁇ 500 ⁇ m, preferably >5- ⁇ 200 ⁇ m, more preferably >10- ⁇ 100 ⁇ m.
  • means is capable of changing the ratio ⁇ p ⁇ of the preselected biomolecules in at
  • means is capable of changing the ratio ⁇ p ⁇ of the preselected biomolecules in at
  • At least selected parts of the permeation layer by changing at least one property/parameter out of the group comprising mobility, distance, permeability and swelling at least of parts of the permeation layer via a change in pH.
  • means is capable of changing the ratio ⁇ p ⁇ of the preselected biomolecules in at
  • the actuation means operates in a pH-range from >2 to ⁇ 12, preferably > 4 to ⁇ 9, more preferably from >5 to ⁇ 8, most preferably >6.5 to ⁇ 7.5 .
  • means is capable of changing the ratio ⁇ p ⁇ of the preselected biomolecules in at
  • means is capable of changing the ratio ⁇ p ⁇ of the preselected biomolecules in at
  • At least selected parts of the permeation layer by changing at least one property/parameter out of the group comprising mobility, distance, permeability and swelling at least of parts of the permeation layer electrically and/or electrochemically.
  • means is capable of changing the ratio ⁇ p ⁇ of the preselected biomolecules in at
  • the voltage applied is chosen so that no gas is generated, which will be for most applications around 2-3V. According to an embodiment of the present invention, the actuation
  • means is capable of changing the ratio ⁇ p ⁇ of the preselected biomolecules in at
  • At least selected parts of the permeation layer by changing at least one property/parameter out of the group comprising mobility, distance, permeability and swelling at least of parts of the permeation layer by electrolysis, i.e. by generating ions electrically or electrochemically.
  • means is capable of changing the ratio ⁇ p ⁇ of the preselected biomolecules in at
  • the actuation by changing at least one property/parameter out of the group comprising mobility, distance, permeability and swelling at least of parts of the permeation layer by applying an electrical current.
  • means is capable of changing the ratio ⁇ p ⁇ of the preselected biomolecules in at
  • the actuation means is capable of changing at least one property/parameter out of the group comprising mobility, distance, permeability and swelling at least of parts of the permeation layer via a change in temperature.
  • the actuation means is capable of changing the permeability and/or swelling by a factor >1.2, preferably >2, more preferably >5 and most preferred >10 per 0.5 0 C.
  • means is capable of changing the ratio ⁇ p ⁇ of the preselected biomolecules in at
  • the actuation means is capable the changing at least one property/parameter out of the group comprising mobility, distance, permeability and swelling at least of parts of the permeation layer via a incident radiation.
  • the actuation means comprises at least one resistive heater element capable of changing the temperature locally in a predefined region of said permeation layer.
  • means is capable of changing the ratio ⁇ p ⁇ of the preselected biomolecules in at
  • the device according to the invention comprises at least one electrodes and/or at least one set of electrodes.
  • preselected biomolecules which may include actuating, accelerating or rejecting preselected biomolecules towards predefined locations by applying an electrical DC or AC field, i.e. by electrophoresis or dielectrophoresis.
  • the permeation layer is provided in close proximity to the at least one electrode and/or the at least one set of electrodes.
  • the actuation means is capable of changing one of the parameters including mobility, distance, permeability and/of swelling of the at least one permeation layer by inducing a phase transition resulting a micro phase separation with a continuous fluid/water phase in the permeation layer.
  • the actuation means is capable of changing one of the parameters including mobility, distance, permeability and/of swelling of the at least one permeation layer by inducing a LCST (lower critical solution temperature) phase transition in the permeation layer.
  • LCST lower critical solution temperature
  • the device comprises a hydrogel material, whereby preferably the permeation layer comprises a hydrogel material.
  • hydrogel in the sense of the present invention especially means that at least a part of the hydrogel material comprises polymers that in water form a water-swollen network and/or a network of polymer chains that are water- soluble.
  • the hydrogel material comprises in swollen state >50% water and/or solvent, more preferably >70% and most preferred >90%, whereby preferred solvents include organic solvents, preferably organic polar solvents and most preferred alkanols such as Ethanol, Methanol and/or (Iso-) Propanol.
  • the hydrogel material comprises a material selected out of the group comprising poly(meth)acrylic materials, subsituted vinyl materials or mixture thereof.
  • the hydrogel material comprises a poly(meth)acrylic material made out of the polymerization of at least one (meth)acrylic monomer and at least one polyfunctional (meth)acrylic monomer.
  • the (meth)acrylic monomer is chosen out of the group comprising (meth)acrylamide, (meth)acrylic acid, hydroxyethyl(meth)acrylate, ethoxyethoxyethyl(meth)acrylate or mixtures thereof.
  • the polyfunctional (meth)acrylic monomer is a bis-(meth)acryl and/or a tri-(meth)acryl and/or a tetra-(meth)acryl and/or a penta-(meth)acryl monomer.
  • the polyfunctional (meth)acrylic monomer is chosen out of the group comprising bis(meth)acrylamide, tripropyleneglycol di(meth)acrylates, pentaerythritol tri(meth)acrylate polyethyleneglycoldi(meth)acrylate, ethoxylated bisphenol-A- di(meth)acrylate , hexanedioldi(meth)acrylate or mixtures thereof.
  • the hydrogel material comprises an anionic poly(meth)acrylic material, preferably selected out of the group comprising (meth)acrylic acids, arylsulfonic acids, especially styrenesulfonic acid, itaconic acid, crotonic acid, sulfonamides or mixtures thereof, and/or a cationic poly(meth)acrylic material, preferably selected out of the group comprising vinyl pyridine, vinyl imidazole, aminoethyl (meth)acrylates or mixtures thereof, co -polymerized with at least one monomer selected out of the group neutral monomers, preferably selected out of the group vinyl acetate, hydroxyethyl (meth)acrylate (meth)acrylamide, ethoxyethoxyethyl(meth)acrylate or mixture thereof, or mixtures thereof.
  • an anionic poly(meth)acrylic material preferably selected out of the group comprising (meth)acrylic acids, arylsulfonic acids, especially st
  • the hydrogel material comprises a substituted vinyl material, preferably vinylcaprolactam and/or substituted vinylcaprolactam.
  • the crosslink density in the poly(meth)acrylic material is ⁇ O.OOOl and ⁇ O.l, preferably ⁇ O.OOl and ⁇ 0.05, or most preferably in the range >0.005 and ⁇ O.Ol.
  • crosslink density means or includes especially the following definition:
  • the crosslink density ⁇ x is
  • ⁇ x 0
  • the hydrogel material comprises a poly(meth)acrylic material co -polymerized with at least one monomer selected out of the group anionic monomers, preferably selected out of the group comprising arylsulfonic acids, especially styrenesulfonic acid, itaconic acid, crotonic acid or mixtures thereof, cationic polymers, preferably selected out of the group comprising vinyl pyridine, aminoethyl (meth)acrylates or mixture thereof, and neutral monomers, preferably selected out of the group vinyl acetate, hydroxy ethyl (meth)acrylate or mixture thereof, or mixtures thereof.
  • group anionic monomers preferably selected out of the group comprising arylsulfonic acids, especially styrenesulfonic acid, itaconic acid, crotonic acid or mixtures thereof
  • cationic polymers preferably selected out of the group comprising vinyl pyridine, aminoethyl (meth)acrylates or mixture thereof
  • neutral monomers preferably selected out
  • the hydrogel material is based on thermo-responsive monomers selected out of the group comprising N-isopropylamide , diethylacrylamide, carboxyisopropylacrylamide, hydroxymethylpropylmethacrylamide, acryloylalkylpiperazine.
  • the hydrogel material is functionalized with reactive side-groups such as amines or active esters, especially to perform in-situ 'cross-linking' of DNA or anti-bodies.
  • a composite, a method and/or device according to the present invention may be of use in a broad variety of systems and/or applications, amongst them one or more of the following: biosensors used for molecular diagnostics rapid and sensitive detection of proteins and nucleic acids in complex biological mixtures such as e.g. blood or saliva high throughput screening devices for chemistry, pharmaceuticals or molecular biology - testing devices e.g. for DNA or proteins e.g. in criminology, for on-site testing (in a hospital), for diagnostics in centralized laboratories or in scientific research tools for DNA or protein diagnostics for cardiology, infectious disease and oncology, food, and environmental diagnostics - tools for combinatorial chemistry tools for amplification of DNA, RNA or peptides analysis devices
  • Fig. 1 shows a very schematic cross-sectional partial view showing a device according to a first embodiment of the present invention with a plurality of capture sites covered by a permeation layer which swelling can be changed electrically
  • Fig. 2 shows the device of Fig. 1 after applying voltage
  • Fig. 3 shows a very schematic cross-sectional partial view showing a device according to a second embodiment of the present invention with a plurality of capture sites covered by a permeation layer which swelling can be changed electrically
  • Fig. 4 shows the device of Fig. 3 after applying voltage
  • Fig. 5 shows a very schematic cross-sectional partial view showing a device according to a third embodiment of the present invention with a plurality of capture sites each covered by a permeation layer whose swelling can be changed electrically Fig.
  • FIG. 6 shows the device of Fig. 5 after applying voltage
  • Fig. 7 shows a very schematic cross-sectional partial view showing a device according to a fourth embodiment of the present invention with a permeation layer comprising two different materials
  • Fig. 8 shows a very schematic cross-sectional partial view showing a device according to a fifth embodiment of the present invention with a permeation layer comprising two different materials
  • Fig. 1 shows a very schematic cross-sectional partial view showing a device 1 according to a first embodiment of the present invention with a plurality of capture sites covered by a permeation layer 20 which swelling can be changed electrically.
  • Fig. 1 is a partial view only and in most applications within the present invention much more capture sites and electrodes will be used.
  • the permeation layer 20 is provided on a substrate 50.
  • the device furthermore comprises a second substrate 60 which carries a counter-electrode to the electrodes 10a-e.
  • a bio liquid in most applications an aqueous solution
  • Fig. 2 shows the device of Fig. 1 after applying a voltage.
  • the components which are identical to Fig. 1 are not explicitly mentioned.
  • Fig. 2 is for explanatory reasons only and does not reflect the actual swelling in most applications. In many applications within the present invention, the amount of swelling is much larger than in Fig.2.
  • This swelling increases the speed with which the preselected biomolecules present in the bioliquid enter the permeation layer 20 and reach the capture sites 10a-e.
  • Fig. 3 shows a very schematic cross-sectional partial view showing a device 1 ' according to a second embodiment of the present invention with a plurality of capture sites covered by a permeation layer whose swelling can be changed electrically.
  • Fig. 3 the design of the device according to Fig. 3 is in some extent similar to that of Fig. 1 and therefore for the sake of brevity the components which are identical to Fig. 1 are not explicitly mentioned.
  • the device of Fig.3 differs from that of Fig. 1 in that that no opposite counter-electrode is present, rather on the substrate 50 first electrodes lOa-c and second electrodes 1 la-b are present. It should be noted that Fig. 3 is a partial view only and in most applications within the present invention much more electrodes will be used. However, only the first electrodes lOa-c have capture sites associated with them. It is also possible to have one shared common electrode. The size ratio of 11 and 10 should be approximately one.
  • Fig. 4 shows the device of Fig. 3 after applying voltage.
  • the first electrodes When applying voltage, the first electrodes will form the anodes and the second electrodes the cathodes (or vice versa, depending on the actual application). This will cause the permeation layer to swell in regions associated with the electrodes lOa-c and to shrink in regions associated with the electrodes 1 la-b.
  • the swelling - as discussed - increases the speed and amount of the preselected biomolecules present in the bioliquid which will then enter the permeation layer 20 and reach the capture sites lOa-c.
  • the preselected biomolecules will less likely enter the permeation layer 20 there, thus furthermore increasing the efficacy of the device for a large number of applications within the present invention.
  • Fig. 5 shows a very schematic cross-sectional partial view showing a device 1 " according to a third embodiment of the present invention with a plurality of capture sites 10a-e each covered by a permeation layer 20a-e whose swelling can be changed electrically.
  • This device has for some applications the advantage that a faster response can be achieved by reducing the dimensions of the hydrogel, as shown in Fig. 5. Furthermore an arrangement like this avoids internal stress and possible adhesion problems between the actuated and non-actuated areas of the hydrogel for a large number of applications within the present invention.
  • the device 1 " includes individually addressable electrodes According to an embodiment of the present invention (not shown in the Figs.) these electrodes and the other suitable components of the device are connected to a large area electronics platform such as amorphous silicon or low temperature polycrystalline silicon (LTPS) on glass or on plastic substrates.
  • amorphous silicon or low temperature polycrystalline silicon (LTPS) on glass or on plastic substrates.
  • Fig. 6 shows the device of Fig. 5 after applying voltage. It can be clearly seen, that - depending on the amount of voltage applied - the different permeation layers will also behave differently.
  • Fig. 7 shows a very schematic cross-sectional partial view showing a device 1 '" according to a fourth embodiment of the present invention with a permeation layer 20 comprising two different materials 22, 24. The device is shown partially only; the overall design of the device will be in analogy to Fig. 5. In accordance, also an electrode 10 and a substrate 50 are present. In Fig. 7, the permeation layer comprises a first material 22, which permeability may be changed when applying voltage and a second material 24, whose permeability does not change or changes only to a small extent.
  • the first material 22 is provided close to the capture site or the capture site is provided within the first material 22.
  • Fig. 8 shows an alternative to Fig. 7.
  • the first material 22 is spatially separated from the substrate .
  • the capture site is also provided within the first material 22.

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Abstract

The present invention relates to a device for analyzing samples including mobility an actuation means for changing the ratio of the preselected biomolecules in the distance sample.

Description

BIOTECHNOLOGICAL DEVICE INCLUDING AN ACTUATION MEANS FOR CHANGING THE MOBILITY OF PRESELECTED BIOMOLECULES
The present invention is directed to the field of biotechno logical devices
In recent years, several biotechnological devices, e.g. analytical biotechnological devices have been described. The detection usually occurs in such a way that the fluid to be analyzed is provided on a substrate material, which contains capture sites for the target molecules which are subject to detection. Such a capture site may comprise a corresponding DNA-strand in case the target molecule is also a DNA-strand or an antibody in the case of a protein assay. The target molecules in the fluid will then bind specifically to the capture site and remain there even after the fluid is removed. The target molecule contains a label and in this way may be detected. The label could be a fluorescent label or a magnetic label depending on the detection means, i.e., the type of bio-sensor, optical or magnetic sensor.
In recent years, several biotechnological devices have been described which use permeation layers. The permeation layer provides capture sites for the immobilization of nucleic acids (or other preselected biomolecules or target molecules). In the case of an electrically assisted assay the main function of the permeation layer is to separate the captured target molecules from the highly reactive electrochemical environment generated immediately at the electrode surface. The layer also allows ions and gasses arising from electrochemical reactions at the electrodes to gradually diffuse into the biological solution.
E.g. from the US 6,960,298 and related patents, which are hereby incorporated by reference, synthetic polymer hydrogel permeation layers are known for use on electronic matrix devices for biological assays.
However, the permeation layers which are known in the art are passive hydrogels, i.e., hydrogels that are in wet conditions insensitive to external stimuli. Furthermore, due to the uniform swelling and/or porosity of the permeation layer, the preselected biomolecules or target molecules will in many cases also enter the permeation layer uniformly, i.e., also at positions where no capture sites are present. It is therefore an object of the present invention to provide a device, which is able of at least partially overcoming some of the above-mentioned drawbacks and helps increasing the specifϊty and the effectivity of the analysis. This object is solved by a composite according to claim 1 of the present invention. Accordingly, a biotechnological device is provided, comprising at least one permeation layer and at least one actuation means which is capable of
changing the ratio ~p~ — of preselected biomolecules in at least selected parts of
the permeation layer.
The term "biotechnological device" is to be understood in its widest sense and includes especially one or more of the following devices: - devices for the detection of one or more preselected biomolecules in a fluid sample, especially devices for the detection of biomolecules in aqueous solution. devices for the controlled release of a compound, especially for drug release. - devices for performing amplification reactions such as PCR
(polymerase chain reaction), QPCR (quantitative PCR), RTPCR (real time PCR). artificial scaffolds for tissue engineering and (stem) cell therapies, including devices for the release of molecules such as growth factors, cytokines etc. to stimulate growth or proliferation of cells and devices which pump nutrients towards cells or accelerate degradation of the scaffold on command.
The terms "biomolecules" as well as "target molecules", "capture sites" "drugs" according to the present invention are to be understood in the widest sense and especially include and/or mean the product(s) of an amplification reaction, including both target and signal amplification); purified samples, such as purified genomic DNA, RNA, proteins, etc.; raw samples (bacteria, virus, genomic DNA, etc.); biological molecular compounds such as, but not limited to, nucleic acids and related compounds (e.g. DNAs, RNAs, oligonucleotides or analogs thereof, PCR products, genomic DNA, bacterial artificial chromosomes, plasmids and the like), proteins and related compounds (e.g. polypeptides, peptides, monoclonal or polyclonal antibodies, soluble or bound receptors, transcription factors, and the like), antigens, ligands, haptens, carbohydrates and related compounds (e.g. polysaccharides, oligosaccharides and the like), cellular fragments such as membrane fragments, cellular organelles, intact cells, bacteria, viruses, protozoa, and the like. The term "mobility" in the sense of the present invention especially means and/or includes the diffusion rate in the parts of the at least one permeation layer affected by the actuation means and/or the average electrophoretic mobility of the preselected biomolecules inside the parts of the at least one permeation layer affected by the actuation means. According to an embodiment of the present invention, the transport of biological particles to the capture site is increased and/or influenced by using an electrical field generated by either the same electrodes used for actuating the permeation layer and/or separate electrodes meant specifically for generating an electrical field directed towards the capture site. Especially in the case of DNA, which carries a negative charge for a wide range of applications within the present invention the transport will be accelerated by charging an electrode near the capture site with a positive voltage. It should be noted that for proteins, the polarity of charge is dependent on the pH and this should therefore according to a further embodiment of the present invention be taken into account when choosing the polarity of the voltage required for accelerating transport. In this context, according to a further embodiment of the present invention, after capturing the polarity of the voltage is reversed, thereby repelling non-specifically bonded bio-particles. This has been shown for a wide range of applications within the present invention to further lower the background signal.
According to an embodiment of the present invention, the dielectrophoretic effect is used to improve the transport of even non-charged bio- particles (e.g. cells). This embodiment is particularly useful when an electric dipole can be induced on the bio-particle. In this context, according to a further embodiment of the present invention, after capturing the frequency of the applied voltage is changed to repel non-specifically bonded bio-particles. This has been shown for a wide range of applications within the present invention to further lower the background signal.
The term "distance" in connection with mobility in the sense of the present invention especially means and/or includes the average distance from the surface of the permeation layer - especially in the parts of the at least one permeation layer affected by the actuation means - to the capture sites/probes.
This distance is according to the present invention especially meant as a macroscopic dimension and is measured in a straight line although the shortest "travel distance" for a molecule flowing from said surface towards said capture sites/probes might be significantly larger in a nanoporous, mesoporous or network like structure.
The term "capture site" in the sense of the present invention is especially a certain area within the device where one or more (usually identical) capture probes are located. The "capture probes" in the sense of the present invention especially mean and/or include molecules (or assemblies of molecules) which are able to interact specifically with the preselected biomolecules.
Depending on the nature of the capture probes, each of the capture site(s) may comprise only one capture probe (e.g. in case the capture probe is an antibody or a cell) or a high number of capture probes (e.g. in case the capture probe is a DNA-strand). The type of capture probe may vary from site to site on the array.
By using such a device at least one or more of the following advantages can be achieved for a wide range of applications within the present invention
Due to the possibility of locally increasing the ratio ~p~
of the preselected biomolecules, the flow of preselected biomolecules to the capture sites can be increased while in other areas of the . mobility . , , permeation layer the ratio ~p~ is kept constant or even reduced.
This allows increasing the spot/background ratio and helps to achieve lower levels of pathogens to be detected.
The design of the device can be made more compact - In many applications of the present invention, a better control over the fluid comprising the preselected biomolecules can be obtained.
Furthermore it is also possible to increase the efficacy of the washing step as compared to an assay with a passive permeation layer. After binding of the preselected biomolecules, in the parts of the permeation layer which are associated with a capture sites the
. mobility . . , . , , , ratio -TT- — is increased, which causes unbound preselected
biomolecules to be removed easier. Actually this is an embodiment of the present invention as will be described in more detail later on. The present invention provides in one embodiment a device for analyzing one or more samples, especially fluid samples for the presence, amount or identity of one or more preselected biomolecules (which are in this context to be called target molecules) of interest in the samples. As will be appreciated by those in the art, the target molecule(s) and the capture site(s) and probe(s) may be, but not limited to, the product(s) of an amplification reaction, including both target and signal amplification; purified samples, such as purified genomic DNA, RNA, proteins, etc.; raw samples (bacteria, virus, genomic DNA, etc.); biological molecular compounds such as, but not limited to, nucleic acids and related compounds (e.g. DNAs, RNAs, oligonucleotides or analogs thereof, PCR products, genomic DNA, bacterial artificial chromosomes, plasmids and the like), proteins and related compounds (e.g. polypeptides, peptides, monoclonal or polyclonal antibodies, soluble or bound receptors, transcription factors, and the like), antigens, ligands, haptens, carbohydrates and related compounds (e.g. polysaccharides, oligosaccharides and the like), cellular fragments such as membrane fragments, cellular organelles, intact cells, bacteria, viruses, protozoa, and the like. According to an embodiment of the present invention, the actuation
means is capable of changing the ratio ~p~ of the preselected biomolecules in at
least selected parts of the permeation layer by a factor of >1.2 (in wet conditions).
It has been shown for a wide range of applications within the present invention that this furthermore helps to increase the specifϊty and the effectivity of the analysis.
According to an embodiment of the present invention, the actuation
means is capable of changing the ratio ~p~ of the preselected biomolecules in at
least selected parts of the permeation layer by a factor of >2, preferably by a factor of >5 and most preferred by a factor of >10 (in wet conditions).
It should be noted that according to one embodiment of the present
, , ^ . . . mobility invention, at least one actuation means is capable or increasing the ratio ~p~ or
the preselected biomolecules in at least selected parts of the permeation layer by a factor of >1.2 (in wet conditions). In this embodiment, the actuation means will according to a further embodiment of the present invention be associated with at least one of the capture site(s), which helps to direct the preselected biomolecules to the capture site(s).
According to one embodiment of the present invention, at least one
actuation means is capable of increasing the ratio ~p~ of the preselected
biomolecules in at least selected parts of the permeation layer by a factor of >2, preferably by a factor of >5 and most preferred by a factor of >10 (in wet conditions).
However, according to another embodiment of the present invention,
at least one actuation means is cap 1 able of decreasing ° the ratio " dT÷is—tance of the
preselected biomolecules in at least selected parts of the permeation layer by a factor of > 1.2 (in wet conditions). In this embodiment, the actuation means will according to a further embodiment of the present invention be associated with those parts of the permeation layer which are not associated to one of the capture site(s). According to one embodiment of the present invention, at least one
actuation means is capable of decreasing the ratio ~p~ of the preselected
biomolecules in at least selected parts of the permeation layer by a factor of >2, preferably by a factor of >5 and most preferred by a factor of >10 (in wet conditions).
It goes without saying that a combination of actuation means which
decrease and actuation means which increase the ratio " dT÷is—tance is also feasible and
another embodiment of the present invention.
According to an embodiment of the present invention, the actuation
means is capable of changing the ratio ~p~ of the preselected biomolecules in at
least selected parts of the permeation layer by changing the swelling of selected parts of the permeation layer.
It has been shown for a wide range of applications that by changing
the swelling of the permeation layer (or parts thereof), the ratio ~p~ can be set
to the desired rate easily and with great efficacy.
According to an embodiment of the present invention, the actuation means is capable of changing the swelling of selected parts of the permeation layer by a factor of >1.2 (in wet conditions), preferably by >2, more preferably >5 and most preferably >10 (in wet conditions). According to an embodiment of the present invention, the actuation
means is capable of changing the ratio ~p~ of the preselected biomolecules in at
least selected parts of the permeation layer by changing the permeability of selected parts of the permeation layer by a factor of >1.2 (in wet conditions).
It has been shown for a wide range of applications that by changing
the permeability of the permeation layer (or parts thereof), the ratio ~p~ can be
set to the desired rate easily and with great efficacy. According to an embodiment of the present invention, the actuation means is capable of changing the permeability of selected parts of the permeation layer by a factor of >2, preferably >5 and most preferred >10 (in wet conditions).
It should be noted that according to the present invention, the term "permeability" (commonly symbolized as K, or k) is especially to be understood as a measure of the ability of a porous material to transmit fluids.
The intrinsic permeability of any porous material is: ^I = ^- " Ω where
Ki is the intrinsic permeability [L2] C is a dimensionless constant that is related to the configuration of the flow-paths d is the average, or effective pore diameter [L].
The permeability can be measured either directly (e.g. using Darcy's law) or through estimation using empirically derived formulas. A common unit for permeability is the darcy (ID = 10 12m2), or more commonly the millidarcy (mD). Other units are the SI units cm2 and m2.
According to an embodiment of the present invention, the device comprises at least one capture site, whereby the actuation means is capable of
• mobility , . . . changing the ratio ~p~ by at least changing one property/parameter out or the
group comprising mobility, distance, permeability and swelling of regions of the permeation layer which are associated with the at least one capture site.
According to an embodiment of the present invention, the average size of the at least one capture site is ≥lOμm and ≤lOOOμm, preferably >20μm and <500μm and most preferred >50μm and <200μm. According to an embodiment of the present invention, the device comprises >10, preferably >100 and most preferred ≥IOOO capture sites.
According to a further embodiment of the present invention, the device comprises at least one active layer part associated with every capture site, whereby the term "active layer part" means the part of the at least one permeation
• mobility . lay Jer where the ratio " dT÷is—tance is chang &ed by J the actuation means. According to a further embodiment, the average ratio of the size of the active layer to the size of the associated capture site (in mm3 to mm3) is >1 : 1 and <10:l, preferably >1,5:1 and ≤3:l.
According to a further embodiment of the present invention, the device comprises at least one transition region between the capture site(s) and/or active layer part(s), which are unaffected by the at least one actuation means.
According to a further embodiment of the present invention, the average diameter of the at least one transition region is >5 μm and <5000μm, preferably >10 μm and ≤lOOOμm, more preferably >20 μm and <500μm and most preferred >50 μm and <200 μm.
According to an embodiment of the present invention, the at least one permeation layer has an average thickness in wet state (but before changing the permeability and/or swelling of certain regions) in the range of >l-≤500μm, preferably >5-<200μm, more preferably >10-<100μm. According to an embodiment of the present invention, the actuation
means is capable of changing the ratio ~p~ of the preselected biomolecules in at
least selected parts of the permeation layer via a change in pH.
According to an embodiment of the present invention, the actuation
means is capable of changing the ratio ~p~ of the preselected biomolecules in at
least selected parts of the permeation layer by changing at least one property/parameter out of the group comprising mobility, distance, permeability and swelling at least of parts of the permeation layer via a change in pH.
According to an embodiment of the present invention, the actuation
means is capable of changing the ratio ~p~ of the preselected biomolecules in at
least selected parts of the permeation layer by a factor of >1.2 per 0.5 pH, preferably by a factor of >2 per 0.5 pH, more preferably by factor > 5 per 0.5 pH, and most preferably by a factor >10 per 0.5 pH. According to an embodiment of the present invention, the actuation means operates in a pH-range from >2 to <12, preferably > 4 to < 9, more preferably from >5 to <8, most preferably >6.5 to < 7.5 .
According to an embodiment of the present invention, the actuation
means is capable of changing the ratio ~p~ of the preselected biomolecules in at
least selected parts of the permeation layer electrically and/or electrochemically.
According to an embodiment of the present invention, the actuation
means is capable of changing the ratio ~p~ of the preselected biomolecules in at
least selected parts of the permeation layer by changing at least one property/parameter out of the group comprising mobility, distance, permeability and swelling at least of parts of the permeation layer electrically and/or electrochemically.
According to an embodiment of the present invention, the actuation
means is capable of changing the ratio ~p~ of the preselected biomolecules in at
least selected parts of the permeation layer by changing at least one property/parameter out of the group comprising mobility, distance, permeability and swelling at least of parts of the permeation layer by applying an electric field . In this context, according to a further embodiment, the voltage applied is chosen so that no gas is generated, which will be for most applications around 2-3V. According to an embodiment of the present invention, the actuation
means is capable of changing the ratio ~p~ of the preselected biomolecules in at
least selected parts of the permeation layer by changing at least one property/parameter out of the group comprising mobility, distance, permeability and swelling at least of parts of the permeation layer by electrolysis, i.e. by generating ions electrically or electrochemically.
According to an embodiment of the present invention, the actuation
means is capable of changing the ratio ~p~ of the preselected biomolecules in at
least selected parts of the permeation layer by changing at least one property/parameter out of the group comprising mobility, distance, permeability and swelling at least of parts of the permeation layer by applying an electrical current. According to an embodiment of the present invention, the actuation
means is capable of changing the ratio ~p~ of the preselected biomolecules in at
least selected parts of the permeation layer via a change in temperature.
According to an embodiment of the present invention, the actuation means is capable of changing at least one property/parameter out of the group comprising mobility, distance, permeability and swelling at least of parts of the permeation layer via a change in temperature. According to an embodiment of the present invention, the actuation means is capable of changing the permeability and/or swelling by a factor >1.2, preferably >2, more preferably >5 and most preferred >10 per 0.50C.
According to an embodiment of the present invention, the actuation
means is capable of changing the ratio ~p~ of the preselected biomolecules in at
least selected parts of the permeation layer via incident radiation.
According to an embodiment of the present invention, the actuation means is capable the changing at least one property/parameter out of the group comprising mobility, distance, permeability and swelling at least of parts of the permeation layer via a incident radiation. According to an embodiment of the present invention, the actuation means comprises at least one resistive heater element capable of changing the temperature locally in a predefined region of said permeation layer.
According to an embodiment of the present invention, the actuation
means is capable of changing the ratio ~p~ of the preselected biomolecules in at
least selected parts of the permeation layer via applying an electrical potential.
To this end, according to an embodiment of the present invention, the device according to the invention comprises at least one electrodes and/or at least one set of electrodes.
By doing so, it is for a wide range of applications within the present invention possible even to control the movement of said preselected biomolecules, which may include actuating, accelerating or rejecting preselected biomolecules towards predefined locations by applying an electrical DC or AC field, i.e. by electrophoresis or dielectrophoresis.
According to an embodiment of the present invention, the permeation layer is provided in close proximity to the at least one electrode and/or the at least one set of electrodes.
According to an embodiment of the present invention, the actuation means is capable of changing one of the parameters including mobility, distance, permeability and/of swelling of the at least one permeation layer by inducing a phase transition resulting a micro phase separation with a continuous fluid/water phase in the permeation layer.
According to an embodiment of the present invention, the actuation means is capable of changing one of the parameters including mobility, distance, permeability and/of swelling of the at least one permeation layer by inducing a LCST (lower critical solution temperature) phase transition in the permeation layer.
According to an embodiment of the present invention, the device comprises a hydrogel material, whereby preferably the permeation layer comprises a hydrogel material.
The term "hydrogel" in the sense of the present invention especially means that at least a part of the hydrogel material comprises polymers that in water form a water-swollen network and/or a network of polymer chains that are water- soluble. Preferably the hydrogel material comprises in swollen state >50% water and/or solvent, more preferably >70% and most preferred >90%, whereby preferred solvents include organic solvents, preferably organic polar solvents and most preferred alkanols such as Ethanol, Methanol and/or (Iso-) Propanol.
According to an embodiment of the present invention, the hydrogel material comprises a material selected out of the group comprising poly(meth)acrylic materials, subsituted vinyl materials or mixture thereof.
According to an embodiment of the present invention, the hydrogel material comprises a poly(meth)acrylic material made out of the polymerization of at least one (meth)acrylic monomer and at least one polyfunctional (meth)acrylic monomer. According to an embodiment of the present invention, the (meth)acrylic monomer is chosen out of the group comprising (meth)acrylamide, (meth)acrylic acid, hydroxyethyl(meth)acrylate, ethoxyethoxyethyl(meth)acrylate or mixtures thereof. According to an embodiment of the present invention, the polyfunctional (meth)acrylic monomer is a bis-(meth)acryl and/or a tri-(meth)acryl and/or a tetra-(meth)acryl and/or a penta-(meth)acryl monomer.
According to an embodiment of the present invention, the polyfunctional (meth)acrylic monomer is chosen out of the group comprising bis(meth)acrylamide, tripropyleneglycol di(meth)acrylates, pentaerythritol tri(meth)acrylate polyethyleneglycoldi(meth)acrylate, ethoxylated bisphenol-A- di(meth)acrylate , hexanedioldi(meth)acrylate or mixtures thereof.
According to an embodiment of the present invention, the hydrogel material comprises an anionic poly(meth)acrylic material, preferably selected out of the group comprising (meth)acrylic acids, arylsulfonic acids, especially styrenesulfonic acid, itaconic acid, crotonic acid, sulfonamides or mixtures thereof, and/or a cationic poly(meth)acrylic material, preferably selected out of the group comprising vinyl pyridine, vinyl imidazole, aminoethyl (meth)acrylates or mixtures thereof, co -polymerized with at least one monomer selected out of the group neutral monomers, preferably selected out of the group vinyl acetate, hydroxyethyl (meth)acrylate (meth)acrylamide, ethoxyethoxyethyl(meth)acrylate or mixture thereof, or mixtures thereof.
According to an embodiment of the present invention, the hydrogel material comprises a substituted vinyl material, preferably vinylcaprolactam and/or substituted vinylcaprolactam.
According to an embodiment of the present invention, the crosslink density in the poly(meth)acrylic material is ≥O.OOOl and ≤O.l, preferably ≥O.OOl and <0.05, or most preferably in the range >0.005 and ≤O.Ol.
In the sense of the present invention, the term "crosslink density" means or includes especially the following definition: The crosslink density δx is
here defined as δ „ = where X is the mole fraction of polyfunctional x L + X monomers and L the mole fraction of linear chain (= non polyfunctional) forming monomers. In a linear polymer δx = 0 , in a fully crosslinked system δx = 1 .
According to an embodiment of the present invention, the hydrogel material comprises a poly(meth)acrylic material co -polymerized with at least one monomer selected out of the group anionic monomers, preferably selected out of the group comprising arylsulfonic acids, especially styrenesulfonic acid, itaconic acid, crotonic acid or mixtures thereof, cationic polymers, preferably selected out of the group comprising vinyl pyridine, aminoethyl (meth)acrylates or mixture thereof, and neutral monomers, preferably selected out of the group vinyl acetate, hydroxy ethyl (meth)acrylate or mixture thereof, or mixtures thereof.
These materials have proven themselves in practice for a wide range of applications within the present invention especially in case they are responsive, especially pH-responsive.
According to an embodiment of the present invention, the hydrogel material is based on thermo-responsive monomers selected out of the group comprising N-isopropylamide , diethylacrylamide, carboxyisopropylacrylamide, hydroxymethylpropylmethacrylamide, acryloylalkylpiperazine. and copolymers thereof with monomers selected out of the group hydrophilic monomers, comprising hydroxyethyl(meth)acrylate, (meth)acrylic acid, acrylamide, polyethyleneglycol(meth)acrylate or mixtures thereof, and/or co -polymerized with monomers selected out of the group hydrophobic monomers, comprising (iso)butyl(meth)acrylate, methylmethacrylate, isobornyl(meth)acrylate or mixtures thereof. These co-polymers are known to be thermo-responsive and therefore may be of use for a wide range of applications within the present invention. According to an embodiment of the present invention, the hydrogel material is functionalized with reactive side-groups such as amines or active esters, especially to perform in-situ 'cross-linking' of DNA or anti-bodies.
A composite, a method and/or device according to the present invention may be of use in a broad variety of systems and/or applications, amongst them one or more of the following: biosensors used for molecular diagnostics rapid and sensitive detection of proteins and nucleic acids in complex biological mixtures such as e.g. blood or saliva high throughput screening devices for chemistry, pharmaceuticals or molecular biology - testing devices e.g. for DNA or proteins e.g. in criminology, for on-site testing (in a hospital), for diagnostics in centralized laboratories or in scientific research tools for DNA or protein diagnostics for cardiology, infectious disease and oncology, food, and environmental diagnostics - tools for combinatorial chemistry tools for amplification of DNA, RNA or peptides analysis devices
The aforementioned components, as well as the claimed components and the components to be used in accordance with the invention in the described embodiments, are not subject to any special exceptions with respect to their size, shape, material selection and technical concept such that the selection criteria known in the pertinent field can be applied without limitations. BRIEF DESCRIPTION OF THE DRAWINGS
Additional details, features, characteristics and advantages of the object of the invention are disclosed in the subclaims, the figures and the following description of the respective figures and examples, which — in an exemplary fashion — show several preferred embodiments of a device according to the invention.
Fig. 1 shows a very schematic cross-sectional partial view showing a device according to a first embodiment of the present invention with a plurality of capture sites covered by a permeation layer which swelling can be changed electrically Fig. 2 shows the device of Fig. 1 after applying voltage Fig. 3 shows a very schematic cross-sectional partial view showing a device according to a second embodiment of the present invention with a plurality of capture sites covered by a permeation layer which swelling can be changed electrically Fig. 4 shows the device of Fig. 3 after applying voltage Fig. 5 shows a very schematic cross-sectional partial view showing a device according to a third embodiment of the present invention with a plurality of capture sites each covered by a permeation layer whose swelling can be changed electrically Fig. 6 shows the device of Fig. 5 after applying voltage Fig. 7 shows a very schematic cross-sectional partial view showing a device according to a fourth embodiment of the present invention with a permeation layer comprising two different materials Fig. 8 shows a very schematic cross-sectional partial view showing a device according to a fifth embodiment of the present invention with a permeation layer comprising two different materials
Fig. 1 shows a very schematic cross-sectional partial view showing a device 1 according to a first embodiment of the present invention with a plurality of capture sites covered by a permeation layer 20 which swelling can be changed electrically.
Associated with each of the capture sites is an electrode 10a-e. The capture sites are not explicitly indicated in the figure for the sake of clearness. It should be noted that Fig. 1 is a partial view only and in most applications within the present invention much more capture sites and electrodes will be used. The permeation layer 20 is provided on a substrate 50. The device furthermore comprises a second substrate 60 which carries a counter-electrode to the electrodes 10a-e. In between the electrode 70 and the layer 20 a bio liquid (in most applications an aqueous solution) is present (which is not explicitly shown). Fig. 2 shows the device of Fig. 1 after applying a voltage. For the sake of brevity the components which are identical to Fig. 1 are not explicitly mentioned. Since the swelling of the permeation layer can be changed electrically, the permeation layer 20 will swell in regions associated with the capture sites. It should be mentioned that Fig. 2 is for explanatory reasons only and does not reflect the actual swelling in most applications. In many applications within the present invention, the amount of swelling is much larger than in Fig.2.
This swelling increases the speed with which the preselected biomolecules present in the bioliquid enter the permeation layer 20 and reach the capture sites 10a-e.
Fig. 3 shows a very schematic cross-sectional partial view showing a device 1 ' according to a second embodiment of the present invention with a plurality of capture sites covered by a permeation layer whose swelling can be changed electrically.
As can be seen, the design of the device according to Fig. 3 is in some extent similar to that of Fig. 1 and therefore for the sake of brevity the components which are identical to Fig. 1 are not explicitly mentioned.
The device of Fig.3 differs from that of Fig. 1 in that that no opposite counter-electrode is present, rather on the substrate 50 first electrodes lOa-c and second electrodes 1 la-b are present. It should be noted that Fig. 3 is a partial view only and in most applications within the present invention much more electrodes will be used. However, only the first electrodes lOa-c have capture sites associated with them. It is also possible to have one shared common electrode. The size ratio of 11 and 10 should be approximately one.
Fig. 4 shows the device of Fig. 3 after applying voltage. When applying voltage, the first electrodes will form the anodes and the second electrodes the cathodes (or vice versa, depending on the actual application). This will cause the permeation layer to swell in regions associated with the electrodes lOa-c and to shrink in regions associated with the electrodes 1 la-b.
The swelling - as discussed - increases the speed and amount of the preselected biomolecules present in the bioliquid which will then enter the permeation layer 20 and reach the capture sites lOa-c. However, due to the fact that the permeation layer shrinks in regions associated with the electrodes 1 la-b, the preselected biomolecules will less likely enter the permeation layer 20 there, thus furthermore increasing the efficacy of the device for a large number of applications within the present invention.
Fig. 5 shows a very schematic cross-sectional partial view showing a device 1 " according to a third embodiment of the present invention with a plurality of capture sites 10a-e each covered by a permeation layer 20a-e whose swelling can be changed electrically.
This device has for some applications the advantage that a faster response can be achieved by reducing the dimensions of the hydrogel, as shown in Fig. 5. Furthermore an arrangement like this avoids internal stress and possible adhesion problems between the actuated and non-actuated areas of the hydrogel for a large number of applications within the present invention.
As can be seen in Fig. 5, the device 1 " includes individually addressable electrodes According to an embodiment of the present invention (not shown in the Figs.) these electrodes and the other suitable components of the device are connected to a large area electronics platform such as amorphous silicon or low temperature polycrystalline silicon (LTPS) on glass or on plastic substrates.
Fig. 6 shows the device of Fig. 5 after applying voltage. It can be clearly seen, that - depending on the amount of voltage applied - the different permeation layers will also behave differently. Fig. 7 shows a very schematic cross-sectional partial view showing a device 1 '" according to a fourth embodiment of the present invention with a permeation layer 20 comprising two different materials 22, 24. The device is shown partially only; the overall design of the device will be in analogy to Fig. 5. In accordance, also an electrode 10 and a substrate 50 are present. In Fig. 7, the permeation layer comprises a first material 22, which permeability may be changed when applying voltage and a second material 24, whose permeability does not change or changes only to a small extent. Preferably the first material 22 is provided close to the capture site or the capture site is provided within the first material 22. Such an embodiment allows for a range of application a better fine- tuning of the materials as well as a more compact set-up of the device. Fig. 8 shows an alternative to Fig. 7. In this device 1 "", the first material 22 is spatially separated from the substrate . Preferably, the capture site is also provided within the first material 22.
The particular combinations of elements and features in the above detailed embodiments are exemplary only; the interchanging and substitution of these teachings with other teachings in this and the patents/applications incorporated by reference are also expressly contemplated. As those skilled in the art will recognize, variations, modifications, and other implementations of what is described herein can occur to those of ordinary skill in the art without departing from the spirit and the scope of the invention as claimed. Accordingly, the foregoing description is by way of example only and is not intended as limiting. The invention's scope is defined in the following claims and the equivalents thereto. Furthermore, reference signs used in the description and claims do not limit the scope of the invention as claimed.

Claims

CLAIMS:
1. A biotechnological device comprising at least one permeation layer and at least
one actuation means which is capable of changing the ratio ~p~ of the
preselected biomolecules in at least selected parts of the permeation layer.
2. The device according to claim 1, whereby the actuation means is capable of
changing the ratio ~p~ — of the preselected biomolecules in at least selected
parts of the permeation layer by a factor of >1.2 (in wet conditions).
3. The device according to claim 1 or 2, whereby the actuation means is capable of changing the permeability and/or swelling of selected parts of the permeation layer.
4. The device according to any of the claims 1 to 3, whereby the actuation means is capable of changing the swelling of selected parts of the permeation layer by a factor of >1.2 (in wet conditions), preferably by >2, more preferably >5 and most preferably >10 (in wet conditions).
5. The device according to any of the claims 1 to 4, whereby the actuation means is
capable of changing the ratio ~p~ of the preselected biomolecules in at least
selected parts of the permeation layer by changing the permeability of selected parts of the permeation layer by a factor of > 1.2 (in wet conditions).
6. The device according to any of the claims 1 to 5, whereby the device comprises at least one active layer part associated with every capture site, whereby the term "active layer part" means the part of the at least one permeation layer where the
• mobility . ratio "TT- is changed by the actuation means. distance ° J
7. The device according to any of the claims 1 to 6, whereby the average ratio of the size of the active layer to the size of the associated capture site (in mm3 to mm3) is ≥l :l and < 10:1, preferably > 1,5:1 and ≤3:l.
8. The device according to any of the claims 1 to 7, whereby the device comprises at least one transition region between the capture site(s) and/or active layer part(s), which are unaffected by the at least one actuation means and preferably the average diameter of the at least one transition region is >5 μm and <5000μm, preferably >10 μm and ≤lOOOμm, more preferably >20 μm and <500μm and most preferred >50 μm and ≤200 μm.
9. The device according to any of the claims 1 to 8, whereby the transport of biological particles to the capture site is increased and/or influenced by using an electrical field generated by either the same electrodes used for actuating the permeation layer and/or separate electrodes meant specifically for generating an electrical field directed towards the capture site.
10. A system incorporating a device according to any of the Claims 1 to 9 and being used in one or more of the following applications:
biosensors used for molecular diagnostics rapid and sensitive detection of proteins and nucleic acids in complex biological mixtures such as e.g. blood or saliva high throughput screening devices for chemistry, pharmaceuticals or molecular biology testing devices e.g. for DNA or proteins e.g. in criminology, for on-site testing (in a hospital), for diagnostics in centralized laboratories or in scientific research tools for DNA or protein diagnostics for cardiology, infectious disease and oncology, food, and environmental diagnostics tools for combinatorial chemistry analysis devices
PCT/IB2007/054838 2006-12-04 2007-11-29 Biotechnological device including an actuation means for changing the mobility of preselected biomolecules WO2008068678A1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010112287A1 (en) * 2009-03-31 2010-10-07 Siemens Aktiengesellschaft Device similar to an electrochemical camera and method for the production and use of the device

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10357251B2 (en) 2015-08-26 2019-07-23 Ethicon Llc Surgical staples comprising hardness variations for improved fastening of tissue

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5534132A (en) * 1995-05-04 1996-07-09 Vreeke; Mark Electrode and method for the detection of an affinity reaction
WO2003049677A2 (en) * 2001-12-10 2003-06-19 Nanogen, Inc. Mesoporous permeation layers for use on active electronic matrix devices
US20060134657A1 (en) * 2004-05-28 2006-06-22 Nanogen, Inc. Nanoscale electronic detection system and methods for their manufacture

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5534132A (en) * 1995-05-04 1996-07-09 Vreeke; Mark Electrode and method for the detection of an affinity reaction
WO2003049677A2 (en) * 2001-12-10 2003-06-19 Nanogen, Inc. Mesoporous permeation layers for use on active electronic matrix devices
US20060134657A1 (en) * 2004-05-28 2006-06-22 Nanogen, Inc. Nanoscale electronic detection system and methods for their manufacture

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KUCKLING D ET AL: "TEMPERATURE SENSITIVE POLYMERS BASED ON 2-(DIMETHYL MALEINIMIDO)-N-ETHYL-ACRYLAMIDE: COPOLYMERS WITH N- ISOPROPYLACRYLAMIDE", POLYMER BULLETIN, SPRINGER, HEIDELBERG, DE, vol. 44, no. 3, April 2000 (2000-04-01), pages 269 - 276, XP000945653, ISSN: 0170-0839 *
MURDAN S: "Electro-responsive drug delivery from hydrogels", JOURNAL OF CONTROLLED RELEASE, ELSEVIER, AMSTERDAM, NL, vol. 92, no. 1-2, 19 September 2003 (2003-09-19), pages 1 - 17, XP004456361, ISSN: 0168-3659 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010112287A1 (en) * 2009-03-31 2010-10-07 Siemens Aktiengesellschaft Device similar to an electrochemical camera and method for the production and use of the device
CN102449467A (en) * 2009-03-31 2012-05-09 西门子公司 Device similar to an electrochemical camera and method for the production and use of the device
US9995703B2 (en) 2009-03-31 2018-06-12 Boehringer Ingelheim Vetmedica Gmbh Device similar to electrochemical camera and method for producing device

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