GB2427022A

GB2427022A - An encoded carrier for analysing a chemical species using surface plasmon resonance

Info

Publication number: GB2427022A
Application number: GB0511672A
Authority: GB
Inventors: Carl Edward Norman; Katja Franziska Kastl
Original assignee: Toshiba Research Europe Ltd
Current assignee: Toshiba Europe Ltd
Priority date: 2005-06-08
Filing date: 2005-06-08
Publication date: 2006-12-13
Anticipated expiration: 2025-06-08
Also published as: GB0511672D0; GB2427022B

Abstract

An encoded carrier comprises a code region having a code and a separate reaction region. The reaction region has a substantially circular diffraction grating on its surface and is configured to support surface plasmons. Incident radiation couples to surface plasmons at a peak angle of incidence. Present on the surface of the reaction region are known probe molecules such as strands of DNA which are exposed to unknown target molecules in a sample. Where hybridisation between probe and target molecules has occurred, the angle of incidence at which surface plasmon resonance occurs shifts, thus indicating the presence of the known molecule in the sample. Use of a circular grating allows multiple carriers to be used easily in a system by simplifying the alignment of the grating with the light source.

Description

1 2427022 An Encoded Carrier The present invention relates to the field of

encoded carriers primarily for use in analysing a particular chemical species or to monitoring molecular reactions.

There has always been a need to be able to quickly and efficiently monitor reactions between different chemicals in order to both identify unknown molecules and to study the reactions of both known and unknown molecules. This need has become all the more acute with the substantial and numerous discoveries being made in the

biotechnology field.

DNA has the capacity to "hybridise", in other words, a single strand of DNA is capable of pairing to its complementary single strand but not pairing to an unrelated sequence.

Using this technique it is possible to identify and monitor the reactions of an unknown strand of DNA by attempting to hybridise it with known DNA strands.

Currently, there are a number of methods for performing this analysis. In one of the known methods, a known molecule or "probe" is reacted with an unknown molecule or "target", to analyse the target molecule. The probe is tethered to a stable material and is generally labelled with a radioactive isotope or fluorophore that can be detected after hybridisation takes place.

W000/1 6893 describes a system where probes are attached to a plurality of different coded carriers. The target molecules are tagged and are introduced to the probes with the coded carriers.

GBP2306484 relates to a technique similar to W000/16893 which uses coded carriers in order to monitor reactions between polymers and the like.

In a further method, a so-called DNA microarray, "spots" of different probe molecules are attached to an inert material such as glass or nylon which are then exposed to labelled target molecules.

Although effective, the above methods are label or tag based methods which require the awkward process of attaching a fluorescent tag molecule to either the probe or the target molecule which can be difficult and time consuming to implement. Interest is now growing in non-tag based methods One envisaged method uses surface plasmon resonance to determine whether or not a reaction has taken place between a probe located in a microarray and a target, see for example, Brockman et al, "Grating Coupled Surface Plasmon Resonance for Rapid Label-Free, Array-based sensing", American Laboratory, June 2001, Pages 37 to 40.

In the above method, the probes are attached to a gold layer which is provided on a plastic optical grating. On performing a surface plasmon resonance measurement, light is reflected off the gold layer to excite the metal surface plasmon at the goldldielectric interface. When light is incident on the grating at a particular angle, light couples to the surface plasmon causing a resonance condition where more of the light is absorbed by the structure causing a decrease of the amplitude of the reflected light. The grating serves to couple radiation with the correct incident angle and properties to the surface plasmon mode.

The above proposed surface plasmon resonance technique still has its drawbacks in that it is difficult to test a large number of different molecules at any one time.

The present invention attempts to address the above problems and in a first aspect provides an encoded carrier comprising a code region having a code and a reaction region separate from said code region, said reaction region having a substantially circular diffraction grating on its surface wherein the reaction region is configured to support surface plasmons and said diffraction grating is configured to couple incident radiation to surface plasmons excited by said radiation.

In practice, a plurality of known molecules or "probes" will be attached to the reaction region of the encoded carrier. A physical property of the reaction region, will then be measured with the probes attached to the reaction region. Examples of physical properties which may be measured is the reflectance or transmission of the reaction region.

The encoded carriers with probes will then be introduced to molecules of a second type or "targets". Depending on the types of molecules used for the probe and the target, a reaction may take place. For example, if the target is a strand of DNA and the probe is a complementary strand of DNA then hybridisation will occur. If the target is a strand of DNA and the probe is a strand of DNA with a sequence unrelated to that of the target then no hybridisation will occur. If a reaction, such as hybridisation takes place, the measured physical property of the reaction region will change, hence it is possible to determine if the reaction has happened.

Attaching probes to the reaction region is a relatively easy process and may be achieved by placing an encoded carrier with a suitable surface into a solution of probes or they may be "spotted" onto the surface of the reaction region. Suitable materials for the reaction region will be discussed later.

Typically, a number of different encoded carriers will be used, each with a different probe. Carriers with different probes will have different codes, so that each probe may be identified by reading the code on the carrier. Thus, different probes may be reacted at once with a target molecule. To determine which of the different probes have reacted with the target, the physical properties of a number of the encoded carriers will be measured, then the codes on the encoded carriers which have reacted will be read to determine exactly which probes reacted with the target.

The above system allows thousands and possibly millions of different probes to be reacted with a target in one experiment, since the different probes may be easily distinguished from one another due to their unique code.

In the present invention, the reaction region is configured to support surface plasmons and the diffraction grating is configured to couple incident radiation to surface plasmons excited by said radiation.

If radiation is incident on the reaction region at a particular angle, the "resonance angle", the incident radiation couples to the surface plasmons and there is a drop in the reflectance of the reaction region at this angle. The resonance angle is determined by a number of factors such as the wavelength of the incident radiation and the composition of the reaction region and general shape and surface topography of the reaction region.

Thus, the resonance angle will vary depending on the molecules (e.g. probe and target molecules) which are attached to the surface of the reaction region.

As previously described, the resonance angle is determined by, in addition to the wavelength of the incident radiation and the composition of the reaction region, the surface topography of the reaction region. Thus, changes in the grating period, pitch and general shape can affect the resonance angle.

It is therefore important that in prior art systems, which utilise either one or two dimensional gratings, the grating is aligned in a certain manner relative to the incident radiation otherwise the effective pitch of the grating (as "seen" by the reader apparatus) may vary from reading to reading. In prior art systems that utilise surface plasmon effects (such as the Brockman et al system described above) the reaction region (and therefore the grating) is located on a microarray. It is thus a relatively simple matter to align the array such that the interrogating radiation source correctly illuminates the grating.

For a system comprising a plurality of encoded carriers however each carrier that is read must first be aligned correctly to ensure accurate readings are obtained. For a system comprising potentially thousands of carriers such an alignment step is laborious and reduces the throughput and efficiency of the system.

In the present invention however the carrier is provided with a circular diffraction grating. Therefore, regardless of the overall rotational orientation of the encoded carrier, one portion of the grating will always be in the correct orientation for a reading to be made.

Conveniently the circular grating can be made to approximate a one dimensional line grating of narrow lateral extent by either only selecting light which reaches the imaging detector from regions along the centre line of the grating or by illuminating only a substantially one dimensional area of the diffraction grating. In the latter case the area illuminated should correspond to a diameter across the grating.

Preferably, the diffraction grating should be a first order or second order diffraction grating.

The grating may comprise a plurality of regularly spaced raised ridges.

The diffraction grating may also comprise a corrugated structure.

Preferably the diffracting grating should have a pitch in the region of 100 nanometres to 2 microns. More preferably, the grating pitch should be in the range 100- 600 nanometres.

Preferably the depth of the diffraction grating should be in the range 2 500 nanometres. More preferably, the grating depth should be 10 - 8Onm.

When viewed in cross-section, the lines of the grating may be sinusoidal, blazed or square in form.

When studying how reflected radiation is affected by the radiation coupling to surface plasmons, the reaction region preferably comprises a dielectric layer and a metal layer.

The metal layer may be selected from Al, Au, Cr, Co, Cu, In, Fe, Pb, Mg, Mn, Mo, Ni, nichrome, Nb, Pd, Pt, Se, Ag, Ta, Te, Sn, Ti, W, Zn, and Zr.

The dielectric layer may be selected from A1203, BaTiO3, CdO, CdSe, CdS, CeO2, Germanium oxide, indium oxide, Fe203, Fe304, MgF2, Si02, SiO, SiO (where O<x<1) Si3N4, tantalum oxide, tin oxide, Ti02, TiO, ZnSe, and ZnS.

When checking for the presence of target molecules by measuring changes in the amplitude of transmitted radiation, the reaction region preferably comprises a metal layer. The metal layer may be any of those described above.

The reaction region may be single sided where the reaction region is planar and the reaction region may only be measured on one side. Alternatively, the reaction region may be two sided such that it may be measured on either side.

Conveniently, for carriers where the reaction region is two sided, the carrier may be provided with probe molecules on one side only such that the carrier can distinguish between the presence of target molecules and external factors such as wholesale changes in the refractive index of the surrounding medium thermal or vibrational effects. For carriers configured in such a manner the presence of target molecules will affect surface plasmon activity only on the side of the carrier provided with the probe molecules. If surface plasmon resonances are affected on both sides then this would be indicative of factors other than the presence of target molecules, be they changes in the overall refractive index of the surrounding medium, system temperature or vibrational effects. In such a case, measuring the plasmon resonance shift on both sides of the carrier will allow a differential measurement to be made, thereby allowing resonance shifts due to target molecules to be separately identified.

Where there is a metal and dielectric layer, which layer is uppermost will mainly be dictated by the bonding characteristics of the probe molecules.

The interface where the plasmons are generated should be close enough to the probe and target molecules such that the presence of target molecules affects the surface plasmons.

Typically, the site at which the target molecules should attach is within 5Omri of the interface where the surface plasmons are generated.

The code located in the code region may be a code such as an alphanumeric code or a barcode. In general, any geometric code may be used.

The carrier may be generally planar and the code may be provided at or close to two or more edges of said carrier.

Preferably said carrier is generally planar and said code is configured such that it can be uniquely identified regardless of which plane of the carrier is uppermost.

The code may extend through the whole of the width of said carrier and is preferably provided at the edge of the carrier where each character of the code is fully open to the edge of the carrier.

Preferably, the encoded carrier is less than 400 jim by 400 jim, more preferably less than 150 jim by 150 jim.

In a second aspect, the present invention provides a method of tracking a chemical reaction between probe molecules of a first type and target molecules of a second type, the method comprising: attaching probe molecules to an encoded carrier, said encoded carrier comprising a code region having a code and a reaction region separate from said code region, said reaction region having a substantially circular diffraction grating on its surface wherein the reaction region is configured to support surface plasmons and said diffraction grating is configured to couple incident radiation to surface plasmons excited by said radiation; introducing the encoded carriers with probe molecules to target molecules; and measuring a physical property of the reaction region to determine if the probe molecules have reacted with the target molecules.

The above method may be extended to a plurality of probes, where: a plurality of different types of probe molecules are attached to the reaction region of a plurality of encoded carriers having different codes, such that each type of probe molecule is attached to carriers having the same code; the plurality of encoded carriers with different codes and probe molecules are introduced to target molecules; a physical property of the reaction region for each carrier is measured and its code is read to determine which of the probe molecules have reacted with the target molecules.

As previously explained measuring a physical property of the reaction region may comprise measuring the reflectance of the reaction region or the transmittance of the reaction region.

The reflectance may be measured as a function of the angle of incidence of radiation reflected from the reaction region, as a function of the wavelength of the reflected radiation.

The reaction kinetics may be monitored since the measured physical property of the reaction region will change dependent on the number of target molecules which have reacted with the probes. Thus, by measuring the physical property of the reaction region at different times after the probes have been mixed with the targets, it is possible to obtain information about the reaction kinetics.

In a third aspect, the present invention provides an apparatus for reading an encoded carrier, said encoded carrier comprising a code region having a code and a reaction region separate from said code region, said reaction region having a substantially circular diffraction grating on its surface wherein the reaction region is configured to support surface plasmons and said diffraction grating is configured to couple incident radiation to surface plasmons excited by said radiation, said apparatus comprising: means to perform a measurement of a physical property of the reaction region; and means to read the code provided on said coding area.

The means to perform a measurement of a physical property may be configured to measure the amplitude of the reflected radiation as a function of angle of incidence of the radiation or as a function of the wavelength of the reflected radiation. Alternatively, the means to perform a measurement of a physical property may be configured to measure the amplitude of the reflected radiation at a predetermined polarisation or the amplitude of the transmitted radiation.

In both the second and third aspects of the present invention the irradiating radiation is preferably polarised. This allows the light to couple efficiently with the grating, thereby enhancing the coupling to the surface plasmon when on resonance.

The incoming light beam is first expanded, then focused onto the reaction region. This allows the detector to investigate a range of angles of incoming light in a single image.

The irradiating radiation may also be conveniently masked such that only the centre line of the grating being read is illuminated.

In the event that polarised light is used to illuminate the carrier then the detection means may also optionally include a polarising filter to reduce stray radiation.

The terms "probe" or "probe molecules" have been used to refer to the molecules which are attached to the encoded carrier. The terms "target" or "target molecules" have been used to refer to the molecule which is to be reacted with the molecules already attached to the encoded carriers. The probes and targets may be chosen from a number of different types of molecules for example, antibodies, antigens, enzymes, toxins, proteins, genes etc. The present invention will now be described with reference to the following non- limiting embodiments in which: Figure Ia is a schematic of an encoded carrier in accordance with an embodiment of the present invention undergoing a surface plasmon resonance measurement prior to reaction of the encoded carrier with a target species, Figure lb is a plot of reflected amplitude against angle of incidence indicating the resonance angle; Figure 2a is a schematic of the encoded carrier of Figure Ia undergoing a surface plasmon resonance measurement after reaction of the encoded carrier with a target species; Figure 2b is a plot of reflected amplitude against angle of incidence indicating the resonance angle; Figure 3 is a schematic of a surface plasmon resonance measurement apparatus; Figure 4 is a known example of an encoded carrier having a planar striped diffraction grating and a bar code; Figure 5 is a known encoded carrier having a planar grid-type diffraction grating and an alphanumeric code; Figure 6 is an example of an encoded carrier in accordance with the present invention; Figure 7 is an encoded carrier in accordance with an embodiment of the present invention having a substantially circular shape and having an alphanumeric code; Figure 8 is an encoded carrier in accordance with an embodiment of the present invention having a an inset alphanumeric code; Figure 9 is an encoded carrier in accordance with an embodiment of the present invention having a substantially circular overall shape, and an alphanumeric code; Figure 10 is a schematic of a cross section through the centre line of the diffraction grating of an encoded carrier in accordance with an embodiment of the present invention, the diffraction grating has two corrugated layers; Figure 11 is a schematic of a cross section through the centre line of the diffraction grating of an encoded carrier in accordance with an embodiment of the present invention, the diffraction grating has three corrugated layers; Figure 12 is a schematic of a cross section through the centre line of the diffraction grating of an encoded carrier in accordance with an embodiment of the present invention, the diffraction grating has five corrugated layers; Figure 13 is a schematic of a cross section through the centre line of the diffraction grating of an encoded carrier in accordance with an embodiment of the present invention, the diffraction grating has three corrugated layers; Figure 14 is a schematic cross section through the centre line of the diffraction grating of an encoded carrier in accordance with an embodiment of the present invention, the diffraction grating has multiple periodic corrugated layers; Figure 15 is a schematic of a reading apparatus for the encoded carriers of the present invention; Figure 16 shows how polarised light from a known direction interacts with a circular grating; Figure 17 is a schematic of a cross section through the centre line of the diffraction grating of an encoded carrier in accordance with an embodiment of the present invention, the diffraction grating has three corrugated layers, and; Figure 18 shows the carrier of Figure 17 following reaction with a target molecule.

Figures 1 and 2 explain how surface plasmon modes can be used to monitor molecular reactions. As such therefore they are applicable both to prior art systems that utilise surface plasmon resonance and also the embodiments of the present invention.

Figure 1 a is a schematic cross-section of an encoded carrier 1. The encoded carrier 1 comprises a first dielectric layer 5 of Si02 and a second metal layer 7 of Au. The order of these two layers may be reversed.

Region 3 is the reaction region of the encoded carrier 1, in this region, the first dielectric layer 5 and second metal layer 7 are corrugated to form a diffraction grating.

A beam of radiation 10 is incident on said carrier 1 at angle 0. Radiation 10 enters the first layer 5 and the second layer 7. When the radiation has the appropriate wavelength, polarisation and angle of incidence, the energy from the incident radiation couples to the electrons of the metal layer 7 to excite surface plasmons at the interface 9 between the first dielectric layer 5 and the second metal layer 7. At this "resonance" condition, there is a steep fall in radiation reflected from the reaction region since this radiation is now absorbed by coupling to the surface plasmon mode.

The surface plasmon mode travels along the plane of the interface and extends for approximately SOnm on either side of the interface 9. The characteristics of the surface plasmon mode can thus be governed by physical properties of layers which are within the range of the surface plasmon. Thus, providing that the interface is close enough to a surface of the reaction region, chemical species 11 adhering to the surface of the reaction region may affect the surface plasmon characteristics.

The present invention is primarily intended for determining if molecules of a first type or "probes", 11, react with molecules of a second type or "targets", 13.

In figure 2a, the probes 11 have adhered to the targets 13 and thus both chemical species are attached to metal layer 7. The presence of the targets 13 will further affect the angle at which incident radiation couples to the surface plasmons, or the "resonance angle".

In figure 1 a, where just the probes 11 adhere to the surface of the reaction region, the resonance angle is 0 to the surface normal. In figure 2b, where both the probes 11 and targets 13 adhere to the surface of the reaction region, the resonance angle is 0' to the surface normal. This is illustrated in figures lb and 2b respectively which show a plot of the reflected amplitude of radiation against the incident angle of radiation.

Thus, since the reaction between the probes 11 with the targets 13 causes a variation in resonance angle, measurement of the "resonance" angle, can be used to determine if the second type of molecules 13 has reacted to the first type of molecules 11.

Figure 3 schematically illustrates a known measurement apparatus. Here, an encoded carrier 21 is provided on a substrate 23. The substrate 23 may be a glass or other type of substrate. The encoded carrier 21 may be the same encoded carrier described with reference to Figures Ia and 2a.

Laser 25 is rotatably mounted about a rotation axis which is roughly in the plane of the interface between the first and second layers of the encoded carrier 21 (the first and second layers are layers 5 and 7 respectively described with reference to Figures 1 a and 2a). Laser 25 is rotatably mounted so that the angle of incidence 0, may be varied.

Similarly, the detector 31 may be moved in order to detect the reflected radiation.

Laser 25 emits a beam of radiation which impinges on beam splitter 27. Beam splittter 27 sends part of the beam to a reference detector 29. Reference detector 29 ensures that the laser 25 is producing a known output. The remainder of the light is transmitted through beam splitter 27 and impinges on encoded carrier 21 at an angle 0 to the surface normal. Light reflected from encoded carrier 21 is then reflected at an angle 0 to the surface normal away from laser 25 and towards detector 31. Detector 31 is also rotatably mounted about a rotation axis which is roughly in the plain between the first and second layers of encoded carrier 21.

When the angle of incidence 0 is such that the incident radiation couples to the surface plasmon mode, there is a sharp decrease in the amount of radiation reflected and thus detected by detector 31. As this angle 0 is dependent on the chemical species attached to the surface of reaction region 3 (Figure 1), it is possible to determine whether or not the probes have reacted with the targets.

Figure 4 illustrates a known (i.e. prior art) encoded carrier. The encoded carrier has a reaction region 43 and a coding region 45 comprising a code 47.

The reaction region 43 comprises a diffraction grating 49. In this example, the diffraction grating comprises elongate rectangular holes 51 which are oriented parallel to one another and extend through the whole of the encoded carrier 41. Thus, the diffraction grating 49 comprises a plurality of stripes. Although in this particular example, the holes 51 extend through the whole of the encoded carrier, they may also extend just part of the way into the reaction region, such that the diffraction grating 49 comprises alternating stripes of different thickness in the direction into the plane of the encoded carrier 41.

The code 47 is a bar code provided at the edge of the encoded carrier 41 and extends over the edge of the carrier 41. The code has been designed so that if the encoded carrier 41 is read from the opposite side, i.e. from underneath the plain of paper, each numeral can still be uniquely identified.

Corner 53 of encoded carrier 41 has been removed so that the orientation of the carrier can be determined. Confirmation of the orientation of the carrier 41 is necessary in order to correctly measure the reaction area since the orientation of the diffraction grating 49 with respect to the laser should be the same for all encoded carriers 41. The orientation of the carrier 41 may also be useful for reading the code 47.

Typically, the encoded carrier 41 will be 50 to 100 im square. Each of the characters of the bar code will typically have a length between 5 and 10 m.

In use, probes will be attached to reaction region 43. The molecules may be attached using a number of methods, but will preferably be attached by placing the encoded carriers in a solution of probes.

The encoded carriers 41 will then be introduced to molecules of a different type, "target molecules". Typically, this will again be performed by placing the encoded carriers with the probes in a solution or suspension of the target molecules.

If the probes attached to reaction area 43 react with the targets, the surface plasmon resonance characteristics of the reaction area will change as described with reference to Figures ito 3.

In practice, a plurality of encoded carriers 41 will be prepared, by attaching a first type of probes to a first plurality of encoded carriers having the same "first" code, a second type of probes (different to the first type of probes) to a second plurality of encoded carriers having the same "second" code. The second code is different to the first code.

Further encoded carriers with third, fourth, fifth etc codes and types of probes will be prepared. The reaction region 43 for each different type of encoded carrier having a different probe will be analysed as explained with reference to Figure 3 to determine the "resonance angle" prior to reaction.

The different types of encoded carriers 41 will then be introduced into asolution containing the target molecules. The encoded carriers may then be removed from the solution and measured. Alternatively, the carriers may be read while in the solution.

Each type of encoded carrier may be determined from its code. An SPR measurement can then be performed to determine if the probes on the carrier have reacted with the target molecule.

The encoded carriers 41 may also be used to monitor the progress of a reaction between a molecule attached to the carrier and a target molecule. At the start of the reaction, only a few of the probes attached to the encoded carrier will react with target molecules.

As the reaction progresses, more and more of the probes will react with the target molecules allowing the reaction kinetics to be studied.

Figure 5 schematically illustrates a further known encoded carrier 61. As for figure 4, the carrier 61 has a reaction region 63 and a code region 65.

The reaction region 63 comprises a diffraction grating 67. The diffraction grating 67 comprises a gird of holes 69 which extend through the whole of the encoded carrier 61.

However, alternatively, the holes 69 may only extend part of the way through the encoded carrier to form a grid of indents.

The code region 65 comprises a code 71. The code 71 is an alphanumeric code which is designed so that each character can be uniquely identified regardless of whether the code 71 is read from above or below the plane of the encoded carrier 61. The code 71 is provided right at the edge of the carrier 61 and each character of the code is open to the edge of carrier 61. By forming the code at the edge of the carrier, there are no small pieces of debris which could become lodged in other characters of the code or at other positions on the carrier or there is a very small chance of a character not being fully formed.

Indents 73 are provided on either side of the code 71 and are used to indicate the central line of the code 71.

As in the carrier of figure 4, corner 75 has been removed to allow the orientation of the carrier 61 to be determined.

Figure 6 illustrates an encoded carrier 76 in accordance with an embodiment of the present invention. The carrier 76 has a reaction region 77 and a code region 78.

The reaction region 76 comprises a circular diffraction grating 79 formed from laminated corrugated layers. This diffraction grating allows incident radiation to couple to the surface plasmon as described with reference to figures 1 and 2. The code region 78 comprises a code 80.

Figure 7 is a schematic representation of a further encoded carrier in accordance with the present invention. The carrier 81 comprises a reaction region 83 and a code region 85. The reaction region 83 comprises a circular diffraction grating as shown in Figures 6 and 7. The carrier further comprises an arrow shaped notch 87 which indicates to a reader apparatus which way up the particle is lying - e.g. if it points clockwise, the particle is one way up, if it points anti- clockwise, the particle is the other way up.

Figure 8 is a schematic representation of a further encoded carrier in accordance with the present invention. The carrier 91 comprises a reaction region 93 and a code region 95. The reaction region 93 comprises a circular diffraction grating as shown in Figures 6 and 7.

The code region 95 comprises a code 97. The code 97 is an alphanumeric code which is readable from either side of the plane of the carrier 91. The code 97 is inset from the edge of the carrier 91 so that each character of the code is fully bounded. Elongate holes 99 are provided on either side of the code defining the middle line of the code.

Figure 9 schematically illustrates a further type of encoded carrier 101 in accordance with the present invention. As for the encoded carrier described with reference to Figures 6, 7 and 8, the encoded carrier 101 comprises a reaction region 103 and a code region 105.

The reaction region 103 comprises a circular diffraction grating as described previously with reference to figures 6 and 7.

In this particular embodiment, the reaction area 103 is the central region and the coding region 105 extends around the edge of the encoded carrier 101. Specifically, carrier 101 has a square shape and the code region 105 extends along all four sides of the square. A barcode 107 is provided on each side of the square. The same code is provided on each side of the square so that the code can more easily be read. The code must also be designed so that it can be uniquely determined regardless of whether the code is read from above or below the plane of the carrier 101.

Figure 10 is a schematic cross section of an example of the reaction region of the carrier of any of figures 6 to 9. The reaction region 121 comprises two corrugated layers 123 and 125. In this particular example, the upper corrugated layer 123 is a dielectric layer and the lower corrugated layer 125 is a metal layer.

Radiation 127 impinges on the upper corrugated layer 123 and travels through this layer 123 to the interface 129 between the upper 123 and lower 125 corrugated layers. Here if the radiation is at the "resonance angle", surface plasmons will be excited and will travel along the plane of the interface as shown by arrow 130.

Upper corrugated later 123 is thin enough such that chemicals attached to the upper surface of the upper corrugated layer 123 affect the resonance conditions for surface plasmon resonance.

Although the dielectric layer 123 is shown as the upper layer, either the dielectric or the metal layer can be used as the uppermost layer in the structure. Typically, the layer which will adhere to the "first type of molecule" will be chosen at the uppermost layer.

Figure 11 schematically illustrates a cross section of a further example of the reaction region of any of figures 6 to 9. The reaction region 131 comprises three corrugated layers, an upper metal corrugated layer 133, a middle dielectric corrugated layer 135 and a lower metal corrugated layer 137.

Incoming radiation 139 impinges on the upper corrugated layer 133 and is transmitted through to the interface 141 between the upper corrugated layer 133 and the middle corrugated later 135. If the incident radiation satisfies the resonance condition, it couples to surface plasmon 143 which travels in the metal layer along the plane of the interface.

The upper corrugated layer 133 is thin enough such that chemicals adhering to the upper surface of this layer 133 will affect the surface plasmon resonance condition. The lower corrugated layer 137 is too thick for chemicals adhering to its surface to affect the surface plasmon resonance condition. Thus, the reaction region 131 is "one-sided".

Figure 12 is a schematic cross section of a further example of a reaction region for the encoded carrier of any of figures 6 to 9. The reaction region 151 is "double sided".

Here, upper corrugated dielectric layer 153 is provided overlying and in contact with upper metal layer 155. A thick middle dielectric layer 157 is then provided underneath metal layer 155.

A lower metal layer 159 is then provided on the opposing side of middle dielectric layer 157 to upper metal layer 155. The structure is then finished with lower dielectric layer 161. All layers are corrugated layers.

The middle thick dielectric layer is thick enough to ensure that plasmons generated at the upper interface 163 between the upper dielectric layer 153 and the upper metal layer do not interfere with plasmons generated at the lower interface 165 between the lower metal layer 159 and the lower dielectric layer 161.

The reaction region works in a similar manner to that described with reference to Figure 11. However, the reaction region can cope with light impinging on either side and thus the reaction region may be analysed regardless of which plane of the encoded carrier is uppermost during the analysis process.

Figure 13, is a schematic cross section of a further example of a reaction region for the encoded carrier of any of figures 6 to 9. The reaction region 171 is "double sided".

Here, upper corrugated metal layer 173 is provided overlying and in contact with middle dielectric layer 175. Lower metal layer 177 is provided on the opposite side of said middle dielectric layer 175 to said upper metal layer 173.

The reaction region works in a similar manner to that described with reference to Figure 11. However, the reaction region 171 can cope with light impinging on either side and thus the reaction region may be analysed regardless of which plane of the encoded carrier is uppermost during the analysis process.

The middle thick dielectric layer 175 is thick enough to ensure that plasmons generated at the upper interface 179 between the upper metal layer 173 and the middle dielectric layer 175 do not interfere with plasmons generated at the lower interface 181 between the lower metal layer 177 and the middle dielectric layer 175.

Figure 14, is a schematic cross section of a further example of a reaction region for the encoded carrier of any of figures 6 to 9. The reaction region 191 is again "double sided". The reaction region comprises alternating metal and dielectric corrugated layers, the structure being terminated with an upper dielectric layer 193 and a lower dielectric layer 195. The structure is periodic.

The particular structure of figure 14 has 9 layers and hence 8 interfaces at which surface plasmons may be generated. When light impinges on the upper dielectric surface 197 it travels through the first dielectric layer 193 to the first interface 199 between the first dielectric layer 193 and the first metal layer 201. This light will be reflected along path 202. The amount of radiation reflected will depend on whether or the conditions for surface plasmon resonance are satisfied.

Some of the incident radiation will pass through the first metal layer 201 to second interface 203 between first metal layer 201 and second dielectric layer 205 and hence the radiation may also couple to a surface plasmon mode at the second interface 203.

Radiation may penetrate further into the structure and excite surface plasmons at a plurality of interfaces thus outputting radiation which has reflected from a number of different interfaces within the structure. As the structure is symmetric, it is double sided.

Figure 15 schematically illustrates a reading apparatus in accordance with an embodiment of the present invention. The reading apparatus comprises a stage 220 which is provided on anti-vibration table (not shown). The stage is capable of moving a sample placed on the stage in either the x or y directions.

The stage 220 is configured to move an encoded carrier 222 in accordance with the present invention placed on the stage such that the code on the carrier can be read and the reaction region analysed. In the Figure the carrier 222 is enclosed in a fluid cell 224.

The code is read by a digital camera 226. Analysis software is also provided to be able to read the bar code and possibly, an optical character recognition package will be provided if the code is an alphanumeric code. These techniques are well established and will not be discussed further here. The code can be read from any orientation.

The reaction region analysis is configured to perform surface plasmon resonance measurements in order to determine whether or not the target molecule has reacted with molecules on the encoded carrier. It is noted that the presence of a circular diffraction grating on the carrier means that it is not necessary to orientate the carrier prior to analysis.

The analysis apparatus which analyse the carrier in the analysis area comprise a radiation source 228 (in this instance a laser) and a detector 230 (in this case a CCD).

A proportion of the light emitted by the laser 228 is directed into a reference diode 232 by a beam splitter 234. The diode 232 measures any fluctuations in the output of the laser and allows the reader apparatus to compensate accordingly.

The reader apparatus of Figure 15 further comprises a polariser 236, beam expander 238, focussing lens 240 and slit 242 located between the beam expander and the carrier 222 on the stage 220.

The laser light that passes through the beam splitter 234 is then (linearly) polarised by the polariser 236. This polarised light is then directed onto the carrier 222 by means of the beam expander 238 and lens 240.

The carrier 222 in Figure 15 generally resembles the carrier depicted in Figure 6. The grating in this case comprises a series of circular corrugations. It is noted that the purpose of linearly polarising the incoming light is to permit the light to couple to selected areas of the grating.

Where the direction of with the incoming light is perpendicular to the direction of the grating corrugations then photons of incoming light encounter a spatially modulated surface and "see" the grating. When the direction of the incoming light is parallel to the direction of the grating corrugations then the incoming light does not "see" a grating.

This is shown schematically in Figures 1 6a and 1 6b.

In Figures 1 6a and 1 6b the incident light is arriving and departing obliquely along the direction 300 (shown as a vertical arrow in the Figures). In Figure 1 6a the sections where the incident light direction 300 and corrugation directions 302 are at right angles is highlighted. In Figure 16b the sections where the polarisation direction 300 and corrugation directions 304 are essentially parallel are highlighted. The light will couple to the grating (and thence to surface plasmons) more readily in the situation shown in Figure 16a.

Using polarised light helps to select a substantially one dimensional section of the carrier's grating.

Optionally the reader apparatus of Figure 15 may also include masking means, e.g. a slit 242, between the focussing lens 240 and the carrier 222 in order to direct the illuminating radiation onto the desired area of the carrier.

An optional second polariser 244 is shown in Figure 15 between the CCD detector 230 and the carrier. This element may be included in order to remove stray light reaching the detector and also to monitor whether any rotation of the polarisation of the incident light beam is taking place.

Figure 17 shows a schematic cross section of a further example of a reaction region for the encoded carrier of Figures 6-9. The carrier in this figure is similar to the carrier depicted in Figure 13, however although the carrier comprises reaction regions on both sides, only one of the two sides is functionalised such that it will be affected by the presence of a target molecule.

In Figure 17, upper corrugated metal layer 405 is provided overlying and in contact with middle dielectric layer 407. Lower metal layer 409 is provided on the opposite side of said middle dielectric layer 407 to said upper metal layer 405. A barrier 411 can optionally be included in the dielectric layer 407 to prevent cross-talk between the upper and lower metal layers.

A reaction region 413 is provided on the upper layer 405. The region depicted comprises a plurality of chemical species 415 (similar to those described in relation to Figure 1). The reaction region 414 of the lower layer 409 does not comprise the chemical species.

A beam of radiation 41 7a incident on the upper surface 405 will generate plasmons at the interface 406 between the upper metal layer 405 and the dielectric layer 407. A beam of radiation 419a incident on the lower surface 409 will generate plasmons at the interface 410 between the lower metal layer 409 and the dielectric layer 407.

As described with reference to Figure 1 at the appropriate resonance condition there will be a steep fall in radiation reflected from the surface of the carrier since this radiation is now absorbed by coupling to the surface plasmon mode. Radiation 417b reflecting from the upper surface has a resonance angle of 0'. Radiation 41 9b reflecting from the lower surface 409 has a resonance angle of 0.

Figure 18 shows the carrier of Figure 17 following reaction with molecules 421 (like features are denoted by like numerals). As shown the molecules 421 have attached to the chemical species 415 and the resonant angle with respect to the upper layer 405 has changed from 0' to 0".

The reaction region 414 on the lower surface 409 however has not been altered by the presence of the molecules 421 and therefore its resonance angle is unchanged at 0.

The carrier shown in Figures 17 and 18 therefore comprises a double sided structure wherein one side is functionalised such that the presence of a certain molecule will affect the surface plasmon characteristics of that side and the second side is not functionalised such that the surface plasmon characteristics of the second side are unaffected by the presence of the target molecules 421. This carrier structure provides a means of preventing false measurements resulting from external factors such as thermal or vibrational conditions. If both sides exhibit resonance angle changes then this is likely due to external factors. However, if only one side exhibits a resonance angle change then this is likely to be due to the presence of target molecules.

Where the two sides exhibit a dissimilar change in resonance angle, the net change due to the presence of target molecules can be obtained from the difference between the two observed changes

Claims

CLAIMS: 1. An encoded carrier comprising a code region having a code and a

reaction region separate from said code region, said reaction region having a substantially circular diffraction grating on its surface wherein the reaction region is configured to support surface plasmons and said diffraction grating is configured to couple incident radiation to surface plasmons excited by said radiation.
2. A carrier as claimed in claim 1 wherein said diffraction grating has a pitch of 100 nm to 2 tm.
3. A carrier as claimed in claim 1 or 2 wherein said diffraction grating has a depth of 2- 500nm.
4. A carrier according to any preceding claim wherein said diffraction grating comprises a corrugated structure.
5. A carrier according to any preceding claim, wherein said reaction region comprises a dielectric layer and a metal layer.
6. A carrier according to any preceding claim, wherein molecules of a first type are attached to said reaction area.
7. A carrier according to any preceding claim wherein i) the carrier is generally planar such that it has a first and second side; ii) a reaction region is present on both the first and second side, and; iii) molecules of a first type are attached to the reaction area on the first side of the carrier.
8. A method of tracking a chemical reaction between probe molecules of a first type and target molecules of a second type, the method comprising: attaching probe molecules to an encoded carrier, said encoded carrier comprising a code region having a code and a reaction region separate from said code region, said reaction region having a substantially circular diffraction grating on its surface wherein the reaction region is configured to support surface plasmons and said diffraction grating is configured to couple incident radiation to surface plasmons excited by said radiation; introducing the encoded carriers with probe molecules to target molecules; and measuring a physical property of the reaction region to determine if the probe molecules have reacted with the target molecules.
9. A method according to claim 8, wherein: a plurality of different types of probe molecules are attached to the reaction region of a plurality of encoded carriers having different codes, such that each type of probe molecules are attached to carriers having the same code; the plurality of encoded carriers with different codes and probe molecules are introduced to target molecules; a physical property of the reaction region for each carrier is measured and its code is read to determine which of the probe molecules have reacted with the target molecules.
10. A method according to either of claims 8 or 9, wherein measuring a physical property of the reaction region comprises measuring the reflectance of the reaction region.
11. A method according to claim 10, comprising measuring the reflectance of the reaction region as a function of the angle of incidence of radiation reflected from the reaction region.
12. A method according to any of claims 8 to 11 wherein the incident radiation is polarised.
13. A method according to any of claims 8 to 12 wherein the incident radiation is directed along a diameter of the grating of each encoded carrier that is read.
14. A method according to any of claims 8 to 13 wherein the incident radiation is masked such that it only illuminates a substantially one dimensional line on the surface of each grating of each encoded carrier that is read, said one dimensional line being substantially aligned with a diameter of the grating.
15. An apparatus for reading an encoded carrier, said encoded carrier comprising a code region having a code and a reaction region separate from said code region, said reaction region having a substantially circular diffraction grating on its surface wherein the reaction region is configured to support surface plasmons and said diffraction grating is configured to couple incident radiation to surface plasmons excited by said radiation, said apparatus comprising: means to perform a measurement of a physical property of the reaction region; and means to read the code provided on said coding area.
16. An apparatus according to claim 15, wherein the means to perform a measurement of a physical property is configured to measure the amplitude of the reflected radiation as a function of angle of incidence of the radiation.
17. An apparatus according to claim 15, wherein the means to perform a measurement of a physical property is configured to measure the amplitude of the reflected radiation at a predetermined polarisation.
18. An apparatus according to any of claims 15 to 17 wherein the incident radiation is polarised.
19. An apparatus according to any of claims 15 to 18 wherein the incident radiation is directed along a diameter of the grating of the encoded carrier that is read.
20. An apparatus according to any of claims 15 to 19 wherein the apparatus comprises means to mask the radiation incident on the carrier such that it only illuminates a substantially one dimensional line on the surface of the grating of the encoded carrier that is read, said one dimensional line being substantially aligned with a diameter of the