WO2004097384A1

WO2004097384A1 - Molecular detector arrangement

Info

Publication number: WO2004097384A1
Application number: PCT/GB2004/001788
Authority: WO
Inventors: Brian Philip Allen; Xiao-Feng Zhou; Richard Gilbert
Original assignee: E2V Technologies (Uk) Limited
Priority date: 2003-04-25
Filing date: 2004-04-23
Publication date: 2004-11-11
Also published as: GB0309491D0; GB2400908A

Abstract

An analyte carrier, such as a microtiter plate, microfluidic chip or other suitable arrangements for supporting an analyte has a conducting layer for receipt of an analyte having a non-random surface structure pattern designed to enhance the Raman scattering from molecules in an analysis region. The designed surface structure pattern has regions of high curvature, which increase the electromagnetic field provided from the surface, thereby enhancing the energy transfer from electrons in the metal surface to molecules in a region above the surface. This provides energy to electron bandgap levels in the molecules, thereby enhancing Raman scattering which can be detected by a separate detector.

Description

Molecular Detector Arrangement

Field Of The Invention

The present invention relates to a molecular detector, to a carrier for use in a, molecular detector and in particular to a molecular detector assembly of a carrier and detector, which uses surface, enhanced Raman scattering.

Background Of The Invention

It is known that there are many techniques to detect the action or presence of analyte molecules. One such technique utilises the Raman Scattering (RS) effect. Light incident on a molecule is scattered and, as a result of a transfer of energy, a shift in frequency, and thus wavelength, occurs in the scattered light. The process leading to this inelastic scatter is termed the Raman effect. The shift in frequency is unique to the analyte molecule. The RS effect, however, is very weak, so a technique using colloids is known to be used to enhance the effect . Analyte molecules placed within a few Angstroms of a colloid metal surface, such as silver, gold, copper or other such materials, experience a transfer of energy from the metal surface through various mechanisms. This is known as Surface. Enhanced Raman Scattering (SERS) and the result can be measured using conventional spectroscopic detectors.

We have appreciated the problem that the Raman scattering effect, even using surface enhanced Raman scattering (SERS) , provides a small amount of Raman scattered radiation in comparison to normal scattering (effectively a poor signal to noise ratio) .

Summary Of The Invention

The invention is defined in the claims to which reference is directed. An embodiment of the invention uses surface enhanced Raman scattering (SERS) to detect presence of an analyte in a region near the surface using a laser source incident on the region, but further enhances the SERS effect using a surface having a non-random surface structure pattern.

The non-random surface structure pattern is designed to enhance the electromagnetic field experienced by molecules in the region near the surface, thereby enhancing the transfer of energy from the electrons in the surface to the molecule thus enhancing the SERS effect .

Brief Description Of The Figures

Embodiments of the invention will now be described, by way of example only, and with reference to the accompanying drawings, in which:

Figure 1 shows energy levels of Raman scattering; Figure 2 is a schematic diagram showing a detector using the principle of Surface-Enhanced Raman

Scattering; Figure 3 is a schematic diagram of a first surface structure embodying the invention; Figure 4 is a schematic diagram of a second surface structure embodying the invention; Figure 5 is a schematic diagram of a third surface structure embodying the invention; Figure 6 is a schematic diagram of a fourth surface structure embodying the invention; Figure 7A shows one possible method of forming a surface structure pattern; Figure 7B shows another possible method of forming a surface structure pattern; Figure 8 shows an analyte carrier and detector together forming a detector assembly according to a first, preferred embodiment of the invention; Figure 9 shows an analyte carrier according to a second embodiment of the invention; and Figure 10 shows an analyte carrier according to a third embodiment of the invention.

Description Of Embodiments Of The Invention

The embodiments described use the technique of Surface Enhanced Raman Spectroscopy (SERS) with a surface of known non-random pattern.

The present embodiments comprise two main components -. an analyte carrier which provides an analyte region to support molecules to be analysed; and a detector which provides laser radiation to the analyte region on the carrier and has sensors to detect radiation received from the analyte region. Together the analyte carrier and detector comprise a detector assembly. The analyte carrier can take various forms such as a micro array, microtiter plate or microfluidic chip. These and other sample supports are within the term analyte carrier.

The detector itself can comprise various forms of laser source and sensors as described later. The embodiments of analyte carrier, appropriate to the detector can take various forms . The preferred embodiment is a microfluidic chip, but other embodiments include a suitably modified microtiter plate or a prism arrangement also as described later. The analyte carrier is thus a so-called "lab on chip". Prior to describing the embodiments, the SERS effect will first be described by way of background.

When light is scattered from a molecule, most of the photons are elastically scattered. The majority of the scattered photons have the same energy (and therefore frequency and wavelength) as the incident photons. However, a small fraction of the light (approximately 1 in 10⁷ photons) is scattered at frequencies different from, and usually lower than, the frequency of the incident photons as shown in Figure 1. When the scattered photon loses energy to the molecule, it has a longer wavelength than the incident photon (termed Stokes scatter) . Conversely, when it gains energy, it has a shorter wavelength (termed anti-Stokes scatter) .

The process leading to this inelastic scatter is termed the Raman effect, after Sir C.V.Raman, who discovered it in 1928. It is associated with a change in the vibrational, rotational or electronic energy of the molecule, with the energy transferred from the photon to the molecule usually being dissipated as heat. It is also possible for thermal energy to be transferred to the scattered photon, thus decreasing its wavelength. In classical terms, this interaction can be viewed as a perturbation of the molecule's electric field, which is dependent not just on the specific chemical structure of the molecule, but also on its exact conformation and environment . The energy difference between the incident photon and the Raman scattered photon is equal to the energy of a vibrational state of the scattering molecule, giving rise to scattered photons at quantised energy values. A plot of the intensity of the scattered light versus the energy (wavelength) difference is termed the Raman spectrum [RS] . An explanation of the different energy states is shown in Figure 1. Figure 2 shows how the Raman scattering from a compound or ion within a few tens of nanometres of a metal surface can be 10³ to 10⁶ times greater than in solution. This Surface- Enhanced Raman scattering (SERS) and is strongest on silver, but is readily observable on gold and copper as well. Recent studies have shown that a variety of transition elements may also give useful SERS enhancements. The SERS effect is essentially caused by an energy transfer between the molecules and an electromagnetic field near the surface of a metal. The precise mechanism that leads to the enhancement of Raman scattering using SERS need not be described here and various models such as coupling of an image of an analyte molecule to electrons in the metal are known to the skilled person. In effect, electrons in the metal layer 6 supply energy to the molecule thereby enhancing the Raman effect .

One mechanism for coupling is that the electric vector of an excitation laser beam 2 induces a dipole in the surface of a metal layer 6. The restoring forces from the positive polarisation charge result in an oscillating electromagnetic field at a resonant frequency of this excitation. In the Rayleigh limit, this resonance is determined mainly by the density of free electrons at the surface of the metal layer 6 (the 'plasmons') determining the so-called 'plasma wavelength' , as well as the dielectric constants of the metal and its environment.

The presence of a particular molecule is detected using SERS by detecting the wavelength of scattered radiation shown as scattered beam 4. The scattering is not directional and so the sensor (not shown) could be at any reasonable position to capture scattered radiation to measure the wavelength, and hence energy change, of the scattered radiation. The energy change is related to the band gap of molecular states, and hence the presence of particular molecules can be determined. Typically, a molecule 10 to be analysed is bound to a reporter molecule 8 for analysis .

Molecules in an analyte absorbed on or in close proximity to the surface of the layer 6 experience an exceptionally large electromagnetic field in which vibrational modes normal to the surface are most strongly enhanced. This is the Surface Plasmon Resonance (SPR) effect, which enables through-space energy transfer between the plasmons in the metal layer 6 and the molecules 8 near the surface. Scattered photons may then be measured using conventional spectroscopic detectors (not shown) . The intensity of the SPR is dependent on many factors including the wavelength of the incident light and the morphology of the metal surface, since the wavelength should match the plasma wavelength of the metal .

To perform surface enhanced Raman spectroscopy (SERS) the analyte molecules are adsorbed onto a roughened metal surface and the enhanced Raman scattering is detected. To increase the enhancement further, a dye label may be used to provide an additional molecular resonance contribution to the energy transfer, a technique termed SERRS (surface enhanced resonance Raman spectroscopy) . The huge signal enhancement (10⁹ to 10¹⁴ -fold) means that the SERRS label is selectively detected against the background noise, that a much wider and simpler dye chemistry is available than is the case for fluorescence and that the sharp signals enable the recognition of a specific molecule against a background mixture of labels .

SERS and SERRS are known to be performed using colloidal metal particles or thin metal films. For a 5μm silver particle the plasma wavelength is about 382nm, but it can be as high as 600nm for larger ellipsoidal silver particles. The plasma wavelength is to the red of 650nm for copper and gold particles, the other two metals which show SERS at wavelengths in the 350-lOOOnm region. The best morphology for surface plasmon resonance excitation is a small (<100nm) particle of atomically rough surface on a thin (ca. 50nm) metal film.

It is known that with a surface film, the SERS effect requires activation (i.e. roughening) of the metal surface before the extremely large enhancements in signal strength are achieved. There are two levels at which this roughness is important: atomic scale (crystalline defects, steps and boundaries) , and 'mesoscale' (roughness on the scale of a few tens of nanometres) . It has been reported that the SERS effect is primarily due to a relatively few 'hot spots' on a roughened surface or aggregated colloid.

We have appreciated, though, that the SERS effect can be enhanced by using an engineered or designed surface, rather than a randomly roughened surface and that this can provide both a stronger and also more uniformly consistent effect . The embodiments that will now be described describe a way to selectively enhance the interaction between the surface plasmons and the analyte molecules by rationally engineering specific structures on the surface of a metal film. Mesoscale structures are fabricated on the metal surface in order to selectively enhance the local electromagnetic field in the medium close to the surface of the metal, and therefore to optimise the proportion of the surface able to provide the desired characteristics for a SERS 'hot spot' . Whilst various theoretical models have modelled surfaces as simple geometric shapes, we have appreciated that the SERS effect can be enhanced by actually constructing the surface with such geometry. The surface is thus designed or rationally engineered in the sense that a structure pattern is intentionally created, rather than randomly creating a roughened surface . The designed surface structure pattern is created from "bulk" conductor, in the sense that a layer of the chosen conductor in non-particulate form is rationally engineered to produce the surface structure pattern. This is in contrast to the known methods of using a colloidal metal surface, or roughened metal surface. It is important that the conductor (typically metal) is non-particulate or non- colloidal as the particle size could give an undersized signal enhancement, whereas the set of pattern engineered from "bulk" metal is the only set of features on the appropriate scale to enhance the SERS effect in embodiments of the present invention.

The conducting layer can be engineered so as to provide one area having a surface structure pattern on more than one analysis region, each having a different pattern. The use of differing patterns in areas of the same analyte carrier allows structures appropriate to enhance the SERS signal from different molecules to be placed so as to allow simultaneous testing for different molecules - each having a surface structure designed for optimum enhancement .

Figure 3 shows a first surface structure of a surface 6 embodying the invention. In this embodying structure tetrahedral geometric shapes are formed in a metal surface which may be any one of those described above, such as gold, silver or others. The structure size is on the scale of a few tens of nanometres . The surface morphology includes a repeated series of structures 30 each having an upper area 32 and lower area 34 which forms a junction or node with the next structure in the pattern. Together, the structures form a repeating pattern.

The size s of each structure is typically on the scale of tens of nanometres consistent with the strong SERS signal obtained from 80nm diameter particles observed with colloidal gold suspensions. The size s of the structure is chosen to enhance the local electromagnetic field induced by the plasmon wave. Similarly, the distance d between the upper area 32 of each structure is chosen to enhance the local electromagnetic field, and so is the height h. The thickness t of the metal surface is typically a few tens of nanometers, preferably 50nm for gold.

The rational engineering of the surface morphology of the conductor enhances the signal received by the SERS detector by creating a designed electromagnetic filed close to the surface. The surface morphology may be engineered to include any shape able to induce a suitable evanescent electromagnetic field with the desired properties . The features of the structures which enhance the evanescent field are those which have a high radius of curvature. It is known physics that the E field is proportional to the charge density, which is inversely proportional to the radius of curvature, and hence is the E field also inversely proportional to the radius of curvature .

In the first example of Figure 3, the radius of curvature is high at the peak 32 of each tetrahedron 30 and also at the nodes 34 between the structures, as well as at the conjunction of the faces. In a second example in Figure 4, the upper portion 32 of each hemisphere 30 does not have a high radius of curvature, but the node 34 does have high curvature radius. Similarly, the hemi cylinders 30 of the third example in Figure 5 have high radius curvatures at nodes 34 but not at the upper portion 32.

The surface morphology may be engineered to include any shape able to induce a suitable evanescent electromagnetic field with the desired properties . It can range from an array of simple geometric shapes such as tetrahedra hemispheres, or cylinders described to arbitrarily complex structures such as in Figure 6. The precise dimensions and 2D space group of the array, and indeed whether a regular array or a more complex arrangement is chosen, as well as the precise details of the structures which are formed on the surface are all application-dependent and so other examples are also possible.

The surface morphology of the metal may be engineered in a variety of ways. In a first construction technique shown in Figure 7A, the substrate medium 11 has been shaped so that, when the metal layer 6 is deposited, a series of cylindrical structures are formed. The same surface morphology could be achieved by 'stamping' the cylindrical structure directly onto an originally smooth metal surface as shown in Figure 7B. Although ^'the surface geometries of these two structures may be identical, their electromagnetic properties will differ substantially due to the structural differences in the 'bulk' metal beneath the surfaces: (Figure 7A) contains ridges of substrate material in regions where (Figure 7B) has uniform metal.

In any of the shapes described above, and in either manufacturing technique, the key aspect is that the size and radius of curvature of features of the structures comprising a pattern are chosen so as to enhance the field generated by the Plasmon wave and to increase the evanescent filed created. The layer of conductive material is deposited onto the substrate surface using any known conventional deposition techniques. The morphology of the metal surface may be controlled wither by micro structuring the substrate upon which the conductor is overlaid, or by using a micro structured template as a press to imprint the desired surface features. The desired features of the electromagnetic filed may be engineered using a suitable theoretical model linking surface geometry to field density.

Common to all the surface structures described is that there are regions of high curvature . These regions enhance the electromagnetic field from the surface caused by the surface plasmons . The precise choice of structure will depend upon the molecule to which the SERS effect is to be used, and other factors. The field created is normal to the tangent of the surface at a region of high curvature. Structures can be chosen so that the fields so created intersect within the analysis region, thereby multiplying the field at that intersection point. In such an arrangement, the distances are chosen so that the intersection is at a point, which provides constructive interference.

The surface structure may be engineered so that Surface Plasmon Resonance detection (SPR) can be simultaneously performed using a second laser. In which case, the structure size is such that the surface "appears" smooth to the SPR laser, so that s < 800nm, for example for an optical infrared laser. Otherwise the shapes are key to the benefit of an enhanced field, rather than the size in choosing shapes of high curvature to provide constructive fields .

There is presently a paucity of theoretical modelling for plasmon-induced electromagnetic fields around surface microstructures . However, a theoretical framework is available for ideal colloidal systems, and this may give an indication of the theory relevant to microstructures . The output second harmonic power for spherical particles suspended in a medium (i.e. an ideal colloid) is given by

where ε is the complex dielectric function, given by

and E^!i) is the intensity of the electric field at the surface, ω is the frequency of the exciting radiation, 2ω is the second harmonic frequency, m_e is the effective mass of the electron, j is the particle radius, ε_m is the relative permittivity of the medium, ω_p is the bulk plasma frequency, ε_∞ is the dielectric contribution due to atomic core polarizability, τ_bulk is the electron relaxation time in the .bulk metal, v_f is the electron velocity at the Fermi level, L_eff is the effective electronic mean free path, c is the velocity of light and e is the electronic charge .

For typical aqueous gold colloids used in SERS experiments, R is approximately 80 nm, ε_m is 80, ω_p is 1.36 x 10¹⁶ s^"1, ε_∞ is 9.9, τ_bulk is 9.1 x 10^"15 s, v_f is 1.4 x 10^s ms^"1, and L_eff = 6 nm. The exact value for the dielectric function depends on the particle size (or film thickness) , the crystallographic orientation, lattice imperfections and impurities in the composition of the metal.

The preferred embodiment of the invention is to apply the new technique described above in a so-called lab on chip device. In this arrangement, an analyte carrier is provided (which is disposable) to which a solution containing the molecules to be analysed is added. The carrier is then inserted into a detector comprising a laser and a sensor arrangement to detect the Raman scattered radiation and optionally the SPR radiation.

The embodiments of analyte carrier will now be described, as well as describing the whole carrier and detector assembly.

The preferred embodiment of analyte carrier is shown in Figure 8 and is a form of microfluidic chip. On a substrate 11 of suitable plastic, glass or other appropriate material that is transparent to radiation at the chosen wavelengths, is formed a channel layer 13 having a channel 22. Analyte in solution is introduced to the channel in the direction shown by an arrow. At a region 17 of the channel a conductive or semiconductive layer 6 is formed. This layer is preferably one of copper, aluminium, silver or particularly gold. As previously described, the gold layer is engineered to contain microstructures, the size of which being chosen to provide an appropriate plasmon wavelength as already described. A primary use of the chip is in the detection of proteins . For this use, a reporter dye is provided on the gold surface 6 having a linking molecule to which an antibody or similar receptor attaches, and also a peptide or similar fragment able to mimic a portion of the target protein. The reporter dye is initially held away from the surface by binding to the receptor site on the antibody or receptor molecule. On binding of a target protein, the reporter molecule is displaced and comes within the region 20 of influence of the evanescent field from the metal surface. The reporter dye is chosen depending upon the protein to be analysed. It is the reporter molecule that provides the SERS scattering.

The detector into which the analyte carrier chip is inserted comprises a SERS laser 28 providing a beam 2 to the analyte and reporter molecules at the surface region 17 of the gold layer 16. The SERS laser 28 provides radiation at a wavelength chosen to match a bandgap of the reporter molecule and will vary from molecule to molecule. To provide a flexible detector, therefore, the SERS laser is preferably tunable. As SERS scattering 4 is not directional, the sensor 26 for the scattered radiation could be at any position. However, this sensor is preferably not opposite the SERS laser to avoid direct radiation from the laser reaching the sensor.

A second embodying analyte carrier is shown in Figure 9 and comprises a modified microtiter plate. A microtiter plate is known to the skilled person and comprises a series of wells in a substrate, typically of plastic. Samples of an analyte are introduced to the microtiter plate wells for analysis. In accordance with the embodiment of the invention, the bottom of each well, or side, is modified to include the conductive surface 6 onto which a reporter dye is placed. The analyte in solution is then introduced into each well and the plate inserted into a detector as previously described in relation to Figure 8. The conductive surface is preferably gold of typical thickness 50 to 80 nm as previously described. The detector arrangement can illuminate each well in turn, but preferably has an array of detectors to allow simultaneous illumination and detection from each of a plurality of wells in the plate.

A third embodiment of chip is shown in Figure 10. In this arrangement, a prism is effectively created from a substrate 11 having a reflective surface 15 and a surface 19 on which sensors are mounted for external connection. The gold layer is provided on a side of the prism. The gold layer, laser and sensor are as described in relation to the first embodiment .

For any of the above "lab-on-a-chip" devices, the surface may have any of the patterns of structures described or other structured patterns. There is an additional possibility of controlling the exact composition of the metal layer 16. Modifying the metal surface 16 with a variety of dopant atoms would provide an additional means of modulating the plasma wavelength, maybe even resulting in an electronically controllable SPR field. These are all within the scope of the invention.

Claims

1. An analyte carrier for use in a detector assembly in which laser radiation is used to detect the presence of an analyte by Raman scattering, comprising:

a substrate for supporting the analyte; and a conducting layer on a portion of the substrate having an analysis region for receipt of the analyte, the conducting layer having a non-random surface structure pattern engineered from bulk conductor designed to enhance the Raman scattering from molecules in the analysis region.

2. An analyte carrier according to claim 1, wherein the conducting surface comprises a metal film.

3. An analyte carrier according to claim 2, wherein the metal film is one of aluminium, copper, silver or gold.

4. An analyte carrier according to claims 1, 2 or 3, wherein the conducting surface has a thickness of the order 10-lOOnm.

5. An analyte carrier according to any of claims 1 to 4, wherein the conducting surface has deposited thereon a reporter dye and a binding molecule for selectively binding to an analyte molecule to be analysed.

6. An analyte carrier according to claim 5, wherein the reporter dye is arranged so that, in use, the reporter dye is in the analysis region on binding with a molecule to be analysed, and is otherwise outside this region.

7. An analyte carrier according to any of claims 1 to 6, wherein the analyte carrier comprises a microfluidic chip .

8. An analyte carrier according to claim 7 wherein the microfluidic chip includes at least one channel, a portion of the channel having the conducting surface thereon.

9. An analyte carrier according to claim 7, wherein the the microfluidic chip includes multiple channels, each channel having a portion with a conducting surface thereon, each conducting surface having a different reporter dye deposited thereon.

10. An analyte carrier according to any of claims 1 to 6, wherein the carrier comprises a microtiter plate.

11. An analyte carrier according to claim 10, wherein the microtiter plate has one or more wells, the or each well having the conducting surface at a bottom portion thereof.

12. An analyte carrier according to any of claims 1 to 6, wherein the carrier comprises a prism arrangement, the conducting surface being arranged on one face of the prism.

13. An analyte carrier according to any preceding claim, wherein the non-random surface structure pattern comprises repeated structures .

14. An analyte carrier according to claim 13 , wherein the structures are geometrical shapes .

15. An analyte carrier according to claim 13 , wherein the structures are shapes having portions with a curvature radius that enhances the electromagnetic field in the analysis region.

16. An analyte carrier according to claim 29, wherein the pattern has nodes between the structures, the nodes having a curvature radius that enhances the electromagnetic field in the analysis region.

17. An analyte carrier according to any preceding claim, wherein the surface structure pattern is formed by stamping.

18. An analyte carrier according to any of claims 1 to 16, wherein the substrate has a pattern and the surface structure pattern is formed by depositing the conducting layer on the substrate.

19. An analyte carrier according to any preceding claim, wherein the surface structure pattern is arranged to have portions of higher curvature than others, the portions of higher curvature being designed to produce an enhanced electromagnetic field on ah axis perpendicular to the tangent to the surface at the high curvature portions .

20. An analyte carrier according to claim 19, wherein a plurality of regions of high curvature are arranged so that the fields in a direction perpendicular to the tangent to the curve at the high curvature portions intersect at a point in the analysis region.

21. A detector assembly for detecting the presence of a molecule in an analyte comprising: an analyte carrier according to any preceding claim; a first laser radiation source arranged to provide radiation directed, in use, to the analysis region to cause Raman scattering; and a sensor arranged to detect radiation from the first laser radiation source that has been scattered from the analysis region by Raman scattering to detect the' presence of the molecule.

22. A method of analysing an analyte comprising providing the analyte in an analysis region of a conducting layer, the layer having a non-random surface structure pattern designed to enhance the Raman scattering from molecules in the analysis region, and conducting surface enhanced Raman spectroscopy by detecting laser light scattered from molecules in the analysis region.

23. An analyte carrier according to claim 1, wherein the conducting layer comprises a plurality of areas, each area having a different non-random surface structure pattern.