WO2008152367A1

WO2008152367A1 - An optical sensing device

Info

Publication number: WO2008152367A1
Application number: PCT/GB2008/001966
Authority: WO
Inventors: Paola Borri; Wolfgang Langbein; Julie Lutti
Original assignee: UK Secretary of State for Defence
Current assignee: UK Secretary of State for Defence
Priority date: 2007-06-15
Filing date: 2008-06-11
Publication date: 2008-12-18
Anticipated expiration: 2009-12-15
Also published as: GB0711600D0

Abstract

An optical sensing device comprising an optical resonator; a planar surface, near field coupler for coupling evanescent light to the resonator; and fixing means for positioning the resonator relative to the coupler, in which the resonator comprises a microsphere having refractive index 1.50 to 1.95 and diameter 5 to 100 μm and is positioned adjacent the planar surface of the coupler with a separation gap of 200 to 2000 nm.

Description

AN OPTICAL SENSING DEVICE

The present invention is directed to an optical sensing device comprising a microsphere optical resonator and to a sensing chip comprising the same.

The recent increase in the prevalence of antibiotic resistant bacteria and the escalated risk of biological warfare or terrorism have emphasised the need for rapid and cost effective determination of the presence of pathogens in both the civilian and military environment.

Optical biosensors provide a superior method for the detection of these pathogens in that they allow real time monitoring of an environment according to changes in an optical property associated with a biological sample.

Such sensors are commonly based on optical structures in which an evanescent wave associated with an optical mode existing in the structure extends into a sensing layer comprising the biological sample. A change in the refractive index, for example, of the sample by interaction with or binding to a pathogen, leads to a change in an optical property of the mode which can be readily detected.

Examples of such evanescent sensors include surface plasmon resonance (SPR) sensors, resonant mirror (RM) sensors and metal- or dye-clad leaky waveguide (M/DCLW) sensors.

The sensitivity of these biosensors for detection of particles such as bacteria or viruses is, however, limited by poor particle penetration of the sensing layer and/or poor particle overlap with the evanescent field.

A new biosensor based on near field (evanescent) coupling of light from an eroded optical fibre to an optical resonator comprising a dielectric microsphere has recently been reported (Vollmer, F. et al., Appl. Phys. Lett., 2002, 80, 4057-4059). The resonant optical modes (whispering gallery modes, WGM) generated in the microsphere are confined close to its surface by repeated total internal reflection and, in turn, lead to an associated evanescent field. The binding of a target analyte to the microsphere is detected by a change in the resonance wavelength of the biosensor.

The new biosensor is much more sensitive than the planar surface biosensors mentioned above because the light within the microsphere can orbit many thousands of times before coupling back into the fibre.

Indeed the sensitivity of the technique can be related to the quality of the resonance (the "Q- factor") of the microsphere, a measure common to all resonators which expresses the rate of loss of power (damping).

Particular applications of the biosensor include detection of nucleic acids (Vollmer, F. et al., Biophys. J., 2003, 85, 1974-1979), proteins (Arnold, S. et al., Optics Letters, 2003, 28(4), 272-274 and Noto, M. et al., Appl. Phys. Lett., 2005, 87, 223901) and characterisation of nanolayer formation (Noto, M, et al., Optics Letters, 2005, 30(5), 510-512) and photo- induced molecular transformations (Topolancik, J. and Vollmer, F., Biophys. J., 2007, 92, 2223-2229).

Theoretical studies treat the resonance shift of WGM in terms of perturbation theory (Teraoka, I. et al., J. Opt. Soc. Am. B, 2003, 20(9), 1937-1946; Teraoka, I. and Arnold, S., ibid, 2006, 23(7), 1381-1389). Another theoretical study examines the conditions necessary for optimal coupling from optical fibre and planar surface couplers to high Q factor microspheres (Gorodetsky, M.L. and Ilchenko, V.S., ibid, 1999, 16(1), 147-154). The effect of the dielectric substrate of planar surface couplers is reported to lead to line broadening in the scattering spectrum from small dielectric microspheres (Le Thomas, N. et al., ibid, 2006, 23(11), 2361-2365).

The present inventors have now determined that high intrinsic Q factors, not limited by evanescent field coupling back to the substrate, are obtainable from WGMs of microspheres held in aqueous buffer at predetermined distances from planar surface couplers.

Accordingly, the present invention provides an optical sensing device comprising an optical resonator; a planar surface, near field coupler for coupling evanescent light to the resonator; and fixing means for positioning the resonator relative to the coupler, in which the resonator comprises a microsphere having refractive index 1.50 to 1.95 and diameter 5 to 100 μm and is positioned adjacent the planar surface of the coupler with a separation gap of 200 to 2000 run.

As used herein, the term planar surface, near field coupler refers to a coupler in which the frustrated total internal of light is incident at a planar surface.

It will be understood that in order that the device function as a high Q factor optical resonator the microsphere must comprise a dielectric material having low extinction of light.

For sensitive detection of refractive index changes a microsphere with a high responsivity and sharp resonances, i.e. small linewidths, is required. Suitable materials include, but are not limited to, polystyrene and glass, the latter being preferred because it can have lowest extinction of light. Suitable diameters and separation (gap) distances will depend on the refractive index of the material as well as that of the surrounding medium.

For example, suitable diameters for microspheres comprising polystyrene (refractive index 1.58) for use in aqueous solution may be 25 to 45 μm, 20 to 30 μm, 25 to 30 μm, or 30 to 40 μm. Suitable diameters for microspheres comprising glass (refractive index 1.52) for use in aqueous solution may be 50 to 60 μm.

Suitable separation distances for use with polystyrene microspheres are, for example, 200 to 600 run, more particularly, 300 to 400 nm or 400 to 600 nm. Suitable separation distances for glass microspheres are 600 to 800 nm and preferably 700 to 800 nm.

The near field coupler may, in particular, comprise a prism, lens or waveguide coupler but other planar surface couplers may also be suitable. In one embodiment, the near field coupler comprises a half ball lens.

The use of the device for aqueous samples requires that the refractive index of the half ball lens is greater than that of water. In one embodiment, it is 1.825 but other values above 1.33 will also be suitable, provided that an efficient coupling exciting WGMs is obtainable at convenient angles above the critical angle.

The near field coupler includes a monochromatic light source, which can be mechanically swept across a range of angles above the critical angle for total internal reflection.

Advantageously, the light source is a laser (wavelength 700 to 900 nm) which can be tuned to provide a suitable wavelength for measuring the reflectivity spectrum.

It will be appreciated that the reflectivity spectrum is measured by tuning the wavelength of the laser and that the minimum detectable line width in the reflectivity spectrum is dictated by the line width of the laser.

In one embodiment, the light source comprises a tuneable laser of line width about one twenty fifth of the WGM resonance line width found in the reflectivity spectrum, hi this embodiment, the laser may comprise a tuneable DFB laser emitting at a wavelength of ~ 770 nm (corresponding to photon energy of ~ 1.60 eV).

The fixing means may comprise any means for positioning the microsphere above, but not in contact with, the planar surface of the coupler. Suitable fixing means known to those skilled in the art include optical tweezers or an x-y-z mechanical stage.

In the case of the mechanical stage, the microsphere is formed with an integral stem portion by, for example, burning an optical fibre end in a butane/nitrous oxide flame.

An aspect of the present invention provides for a novel optical tweezers arrangement. The tweezers enable the microsphere to be held in aqueous solution at a chosen distance above the upper surface of the coupler.

The tweezers arrangement uses a cone of light at large angle obtained by focusing an annular beam, created from a TEM_0O mode of a Nd: YAG continuous wave laser source at 1064 nm by a pair of axicons, with a high numerical aperture (NA = 1.3) microscope objective.

It is noted that the optical trapping force is (according to geometric optics) increased in this arrangement by a factor of two compared with a full cone illumination having the same maximum aperture angle and power.

Advantageously, the ring illumination facilitates the use of a standard oil immersion objective for trapping in water by reducing the impact of the spherical aberrations created by the refractive index mismatch (between water and the oil in which the objective is immersed).

In one embodiment, a cone angle of 64° and a trap optical power of 250 mW holds a polystyrene microsphere of diameter 15 to 40 μm through 80 μm distance in water against the action of gravity (see Figure 1). Thermal position fluctuations along the axial trapping direction are small compared with the WGM evanescent field decay length (20 to 25 run; RMS) and the trap can, therefore, precisely control the distance of the microsphere from the planar surface coupler.

Preferably, the microsphere is positioned in relation to the near field coupler so that the whole or at least a major portion thereof overlaps with the planar surface.

In a further aspect, the present invention provides for fixing means comprising a transparent separation layer of refractive index matched to a sensing medium, which is provided to the planar surface of the coupler and to which the microsphere is adhered.

As used herein, the term "sensing medium" refers to the medium which is to be interrogated by the device. In a preferred embodiment, the sensing medium is water, or an aqueous solution, and the separation layer has a refractive index about 1.33.

It will be appreciated that requirement that the refractive index of the separation layer is similar to that of the sensing medium is so that the layer does not interfere with WGMs of the microsphere in the medium. Matching of the refractive index of the separation layer and the refractive index of the surrounding medium maximises the Q-factor that can be achieved. It has been found, however, that the refractive index need not be identical and that small deviations from the actual value of solution are acceptable.

In one embodiment, the separation layer comprises a cured polymer since the (thermal or optical) curing process enables direct adherence of the microsphere.

A particularly, suitable polymer comprises a co-polymer of perfluoro alkenyl vinyl ethers, such as CYTOP® (for example, CT-SOLV 180, CTL-809A, CT-PlO; Ashahi Glass Co. Ltd.) which has a refractive index of 1.34. The CYTOP® separation layer can be spin coated to the lens, prism or waveguide or to a glass plate which is or can be arranged for optical matching with the lens, prism or waveguide.

In another embodiment, the separation layer comprises an inorganic material such as magnesium fluoride (MgF₂, refractive index 1.36). Glass slides coated with this material are commercially available.

In that case, the separation layer includes a layer or spot of a transparent adhesive to which the microsphere is adhered. The refractive index of the transparent adhesive may be similarly matched to the target medium.

The adhesive may be a UV-curable adhesive so as to enable spot adhesion of a microsphere positioned by optical tweezers by focusing light with a microscope or by illumination parallel to the separation layer. The uncured adhesive is subsequently washed away with an appropriate solvent.

Suitable transparent adhesives for this purpose comprise CYTOP® and a polyacrylate adhesive, which is available under the proprietary name, DeIo Photobond 4436.

The microsphere may alternatively be adhered by thermal curing of a layer or spot of adhesive comprising or consisting the material in the separation layer, for example CYTOP®. For example, 50 μm glass microspheres may be adhered to a cured CYTOP® separation layer through further thermal curing, thus softening the layer, and allowing the microspheres to penetrate/sink into the layer to the desired depth. Polystyrene microspheres may be adhered to a surface by spin coating a second layer onto the surface, prior to deposition of the microspheres, followed by a curing step. The thickness of the separation layer is chosen for a particular microsphere so as to meet the aforementioned requirements for sharp resonances in the reflectivity spectrum.

The separation layer offers a practical chip for use with planar surface near field couplers (see later) and avoids the problem of fluctuation in the amplitude of resonances over time (so called "jitter") in the reflectivity spectrum found for microspheres in optical tweezers.

This jitter is attributed to rotation of the microsphere in the optical tweezers and the fact that the microsphere is imperfectly spherical - leading to changing coupling strength to WGMs with time.

In embodiments of the present invention in which the microsphere comprises polystyrene (refractive index 1.58) and has diameter 20 to 45 μm, the thickness of the separation layer may be 400 to 600 nm.

Alternatively, the separation layer may have thickness 600 to 800 nm and adhere a glass microsphere (refractive index 1.52) having diameter 40 to 50 μm. hi another example, the separation (and adhesive) layer has thickness 250 to 350 nm and adheres a glass microsphere (refractive index 1.92) having diameter 5 to 15 μm.

hi a preferred embodiment, the microsphere is coated with a specific recognition element for a target analyte. The specific recognition element may, for example, comprise an antibody, hapten or a nucleic acid.

The specific recognition element may be attached to the microsphere by any suitable covalent and/or electrostatic chemistry. For example, glass microspheres may be derivatised by silanisation and subsequent functionalisation permitting the attachment of a coating of antibody, hapten or nucleic acid. In general, the microsphere will be coated following its adherence to the separation layer but it is envisaged that provided the coating survives, for example, the thermal curing of the separation layer and/or adhesive, it may be coated prior to its adherence.

The glass microspheres may, for example, bear a carboxylic or amine functionality which can be derivatised following adherence to the separation layer by conventional chemical methods so as to permit attachment of the specific recognition element.

Suitable methods and/or chemistry permitting selective coating of the microsphere will be apparent to those skilled in the art.

In a preferred embodiment, the device is associated with a flow cell and pump means providing for the passage of a sensing medium, for example aqueous buffer, over the planar surface or separation layer of the device.

In a further aspect, the present invention provides for a sensing chip for use with a near field coupler, comprising a surface planar, glass or waveguide substrate provided with a separation layer having a refractive index matched to that of a sensing medium and thickness 200 to 2000 nm, to which an optical resonator comprising a microsphere having refractive index 1.50 to 1.95 and diameter 5 to 100 μm is adhered.

Embodiments of the sensing chip will be apparent from the foregoing description of the device and the following claims.

The present invention also envisages a device comprising a near field coupler including a single chip provided with a plurality of waveguides each having a microsphere coated with a specific recognition element which is different to any other. In particular, the chip may comprise a plurality of waveguides providing coupling of an evanescent field to the microspheres at different wavelengths of incident light.

Alternatively, the chip may comprise a single waveguide provided with a plurality of microspheres each coated with a specific recognition element different to any other, which is associated with a linear photodectector array.

Preferably, the chip includes an uncoated microsphere as a reference function to allow fluctuations in the refractive index not due to target analyte to be taken into account.

The device may be used for multiplex detection in conjugation with a tuneable (wavelength) laser by, for example, measurement of the intensity coupled to the WGMs as a function of wavelength or of the intensity of scattering in the upper hemisphere of the microsphere.

The target analyte is not limited by the present invention, but preferably comprises a protein, nucleic acid, bacterium, spore or virus.

The present invention will now be described with reference to the following examples and drawings in which

Figure 1 is a scheme showing a device according to one embodiment of the present invention;

Figure 2 is a graph showing the reflectivity spectrum obtained with the device of Figure 1 on a polystyrene microsphere of diameter 30 μm;

Figure 3 is a graph showing the variation in line width of a resonance peak in the reflectivity spectrum obtained with the device of Figure 1 on a polystyrene microsphere of diameter 28 μm with its distance from the coupler; Figure 4 is a graph showing fine structure in a resonance peak of the reflectivity spectrum of Figure 2;

Figure 5 is a schematic representation of a substrate or chip provided with a polymer separation layer and an adhesive layer fixing a polystyrene microsphere;

Figure 6 is graph showing the change in the reflectivity spectrum of the device of

Figure 1 over time in response to a 5% (v/v) solution of glycerol;

Figure 7 is a graph showing the change in the reflectivity spectrum of a device over time according to Figure 1 in response to a solution of streptavidin (0.1 mg/L);

Figure 8 is a graph showing part of the reflectivity spectrum of a device according to Figure 1 incorporating a substrate provided with a separation layer;

Figure 9 is a graph showing the change in the reflectivity spectrum of Figure 8 to fluctuations in the refractive index of an aqueous sample;

Figure 10 is a graph showing the change in the reflectivity spectrum over time of a device similar to that of Figure 1 incorporating a substrate according to Figure 5; and

Figure 11 is a graph showing relating the graph of Figure 10 to the centre peak energy of a particular resonance peak.

Figure 12 a) and b) are graphs showing the theoretical relationship of responsivity to line width for a variety of microsphere refractive index detected through polarisations coupled to a) the TE mode of the microsphere and b) the TM mode of the microsphere. The diameter of the beads are, from left to right 12 to 6 μm (1.9), 25 to 10 μm (1.68), 45 to 20 μm

(1.58), 65 to 30 μm (1.51) and 75 to 40 μm (1.48). Figure 13 is a graph showing TE and TM polarised reflectivity spectra for an embodiment of the invention comprising a polystyrene bead (44 μm) glued to a Cytop® layer.

Figure 14 is a graph showing TE polarised reflectivity spectra, with TM polarisation used as reference, for an embodiment of the invention comprising a polystyrene bead (44 μm) glued to a Cytop® layer.

Figure 15 is a graph showing the change in resonance energy against glycerol concentration for one embodiment of the present application.

Referring now to Figure 1, there is shown a schematic representation of a device according to one embodiment of the present invention.

The device, generally designated 10, includes a transparent flow chamber 11 which together with the upper surface of the coupler defines a conduit of width 80 μm and pumps for controlled delivery of aqueous sample (not shown).

The optical tweezers, generally designated 1OA use a cone of light at large angle obtained by focusing an annular beam 12 with a high numerical aperture (NA = 1.3) standard oil immersion microscope objective 13. The annular beam profile is created from a TEMo₀ mode of Nd: YAG laser 14 of wavelength 1064 nm by a pair of axicons 15. The optical tweezers use a cone angle of 64° and an optical trap power of 250 mW to trap a polystyrene bead 16 of diameter 15 to 40 μm in water within the flow chamber.

The position of the high NA objective is controlled by a precision mechanical stage (not shown). The near field coupler, generally designated 1OB, comprises a tuneable DFB laser 17 emitting at a wavelength of ~ 770 nm, corresponding to photon energy ~ 1.60 eV, a high refractive index (1.83) half ball lens 18 and a photodiode detector array and spectrograph 19. The coupler also includes means for selecting the polarisation of light 20 in the incident and reflected light beam 21.

The spot Gaussian beam diameter of ~ 5 μm and the excitation angle θ ~ 55° are adjusted for good field overlap with the WGMs at the surface. The WGM resonances are detected as dips in the measured reflectivity spectrum.

Horizontal and vertical (z) polarisations are detected simultaneously, which couple respectively to the transverse magnetic (TM) and transverse electric (TE) modes of the microsphere.

Operating the DFB laser under threshold provides for a spectrally broad amplified spontaneous emission which is used for the excitation of WGMs over a spectral range of up to 25 meV. In that case the reflection is dispersed by a high resolution (~ 5 μeV) grating spectrometer and detected by a cooled CCD camera.

Above threshold, the DFB laser emission is ultra-narrow (0.02 μeV) and can be temperature tuned over a range of 5 meV as well as rapidly scanned over a range of about 100 μeV using a triangular current modulation at about 50 Hz. In this case, the detection uses two photodiodes and an oscilloscope to measure the difference between the vertically and horizontally polarised reflected intensities.

A combination of λ/2 and λ/4 plates can be used to selectively excite and detect TE and TM polarised WGMs. Exciting both polarisations simultaneously and using a balanced detection scheme enables the resonant transmission to be measured in both intensity and phase. This interferometric detection provides a background free dispersive spectral line shape of half the mode line width.

The inset image of Figure 1 shows a trapped microsphere coupled to the excitation beam.

Example 1

Referring now to Figure 2, there is shown a graph of the reflectivity spectrum measured on the polystyrene microsphere (Polysciences Inc.; diameter 24 to 31 μm) held in aqueous buffer (purified water; Triton X-100 at 1 x 10^"4 v/v) at a distance of about 300 run above the substrate. The TM spectrum is displaced vertically by 0.5 for clarity.

The mode order n and mode number / of the WGM resonances (as well as the diameter of the sphere) are determined by comparison to the explicit asymptotic formulae of Mie theory (see Lam CC, et al., in J. Opt. Soc. Am. B, 1992, 9, 1585).

Measurement of line width against amplitude in the TEl WGM resonance in the reflectivity spectrum at various distances from the substrate and excitation angles θ gave an optimum excitation angle of θ = 55° (+/-3). This corresponds to an effective WGM index (n_eff) ~ 1.50.

Example 2

The graph of Figure 3 shows the effect of distance from the substrate on amplitude and line width of the TEl WGM resonance (θ = 55°) for a microsphere of 28 μm diameter.

As may be seen, the signal broadens (and deepens) as the microsphere is brought closer to the substrate - indicating stronger coupling to the excitation beam and other substrate optical modes. The resonances are approximately Lorentzian for distances from 0 to about 400 nm and the line width decreases exponentially with increasing distance to the substrate with characteristic length zo = 168 nm.

For distances larger than 400 nm, the effect of coupling back the substrate is no longer dominant in the WGM line width - sharp resonances are observed. The line widths are not Lorentzian but instead consist of an ensemble of dips - attributed to lifting of the degeneracy of the 2/ + 1 azimuthal modes due to imperfectly spherical microsphere.

Jitter

The high resolution spectrum of Figure 4 shows this lifting of degeneracy for a TEl mode in the microsphere of Example 1 (distance from substrate 700 nm). While the microsphere rotates freely in the optical trap the spatially localised excitation couples for different rotation angles to different sub-ensembles of modes which are solidal to the microsphere so that the weight of different modes in the reflectivity spectrum changes with time.

This effect, referred to previously as jitter, is clearly seen in the inset figure, which is a graph of the reflectivity spectrum as a function of time on a grey scale from 1 (white) to 0.96 (black). The observed oscillatory structure is attributed to a cyclic rotation dynamics of the sphere induced by residual flow of the surrounding sample.

The fact that the spectral position of individually resolved resonances is unchanged with time, whilst their amplitude varies, rules out a change in the dielectric properties of the surrounding medium as causative.

The total spectral width of the measure mode ensemble of individual TE_n; or TM_n/ resonances was typically less than 200 μeV which corresponds to an asphericity of the microsphere in the 1 x 10^"4 range. Intrinsic O-Factor

The strongest peak in Figure 4 has line width (full width at half maximum) of 0.4 μeV (0.2 pm) and thus has a Q-f actor (E/δE) of 4 x 10⁶ - suggesting a photon storage time in the microsphere of τ ( = λQ/2πc) 1.6 ns.

As mentioned above, this Q-factor is largely attributable to intrinsic Q-factor because high Q- loading by the coupler at this distance means a very small line width attributable to the substrate.

The intrinsic Q-Factor of a 30 μm diameter polystyrene microsphere in water in the Mie theoretical limit is 1.5 x 10⁸. The difference in this value over the determined value can be attributed to absorption losses in that the related calculated absorption co-efficient of the microsphere α ( = ηp/cτ) of 0.032 cm^"1 is consistent with that reported for copolymers of pentafluoro-styrene at 770 nm (see Pitois C, et al., J. Opt. Soc. Am. B. 2001, 18, 908).

Minimum detection limit

The high intrinsic Q-factor of a 30 μm diameter polystyrene microsphere held in aqueous buffer by optical tweezers suggests sensitivity to a change in the refractive index of the surrounding medium of as little as 4 x 10^" (line width of DFB laser is 1.6 x 10^" eV).

For biosensing applications it is possible to infer (see Arnold, S. et al., J. Opt. Soc. Am. B, 2003, 20, 1937) a protein detection sensitivity of 0.25 pg/mm² in terms of surface mass loading and 0.7 fg in terms of minimum detectable total mass loading - a result that at least 40 times better than in conventional SPR methods.

Example 3 Referring now to Figure 6, there is shown a graph of the reflectivity spectrum as a function of time measured on a polystyrene microsphere of diameter 25 μm held by optical tweezers at a distance 300 nm above the substrate.

The graph shows a shift in the resonance spectrum for TM mode (n = 1) of 356 μeV when a 5% (v/v) solution of glycerol in water is progressively introduced to the flow cell (flow rate 50 μl/sec).

The difference in refractive index of the glycerol solution over aqueous buffer is 0.00743.

Example 4

Referring now to Figure 7, there is shown a similar graph measured on a polystyrene microsphere of diameter 25 μm coated with biotin-labelled bovine serum albumin (BSA) held by optical tweezers a distance nm above the substrate.

The graph shows a shift in the resonance spectrum for TM mode (n = 1) of 245 μeV when a solution of streptavidin (0.1 mg/L) is introduced into the flow cell (flow rate 50 μl/sec).

Separation layer

Referring now to Figure 5 one embodiment of the present invention comprises a device in which a separation layer generally designated 22 of CYTOP® 23 is provided to the half ball lens 18 substrate of the coupler. The separation layer includes an adhesive overlay 24 to which the microsphere 16 is adhered.

Example 5

Referring now to Figure 8, there is shown a graph of the reflectivity spectrum measured on the polystyrene microsphere (diameter 43 μm) held in aqueous buffer in contact with a CYTOP® separation layer of thickness 575 nm by optical tweezers. It may be seen that WGMs (TE mode, n = 1) are not significantly broadened by contact with the separation layer. The line widths are between 0.7 and 1 μeV - corresponding to Q-factors of 2.3 x 10⁶ and 1.6 x 10⁶.

Figure 9 reports a shift of 66 μeV in the reflectivity spectrum over a time interval of 34 minutes which is attributed to temperature drift and/or binding of surfactant. The shift suggests the sensitivity of the device to be similar to that mentioned above.

Example 6

An 800 nm layer of CYTOP® was spin-coated on the half ball lens of the coupler and cured at 18O⁰C for 20 minutes. After cooling, a 400 nm layer of CYTOP®, as an adhesive layer, was spin-coated onto the cured layer. To this additional layer 50 μm diameter soda lime glass beads (refractive index 1.51) were added via a mechanical funnel (across a surface area of 1 mm²) and the whole cured at 180⁰C for one hour. Use of Cytop® as both the separation layer and a glue layer enabled accurate RI matching and thus avoided degradation of the Q factor.

The coated half ball lens was used in a device similar to that described in relation to Figure 1, but without optical tweezers.

The soda lime glass beads were able to withstand a flow of liquid at 125 mm s^"1 over the surface of the sensing device, and did not show mode jumping, thus overcoming a major limiting factor of microspheres held by optical tweezers which are free to rotate.

Example 7

Referring now to Figure 10, there is shown a graph of the reflectivity spectrum (TM mode, n = 1) over time measured on a device including the substrate obtained by Example 6. The measured line width of this resonance is ~ 70 μeV, which corresponds to a Q-factor of ~ 2.3 x 10⁴. This corresponds to an ability to detect changes in refractive index of the surrounding medium of as little as about 3 x 10^"5.

The graph shows a shift in the resonance peak of 183 μeV (see Figure 11), when a 5% solution of glycerol in water is progressively introduced in to the flow cell (flow rate 50 μl/sec).

As may be seen from the level values over time, the fixing of the microsphere without rotation in optical tweezers or due to sample flow, substantially removes jitter.

The present inventors have shown sharp WGMs resonances in the reflectivity spectrum of microspheres held in aqueous sample which are attributable to high intrinsic Q.

These sharp resonances are unaffected by the presence of a separation layer fixing the distance of the microspheres from the upper surface of a near field coupler and are shifted by, for example, specific binding of proteins (streptavidin) to the microspheres.

However, it was noted that temperature curing often resulted in softening of the separation layer and subsequent seeking of the microspheres, with the consequence that control over the effective thickness of the separation layer and the microsphere area covered by Cytop® was lost.

Example 8

Referring now to Figure 12 a) and b), for sensitive detection of refractive index changes a microsphere should have a high responsivity and produce sharp resonances (small linewidths). Soda lime glass beads (refractive index 1.51) and polystyrene (refractive index

1.58) both have high responsivity and sharp resonances, though soda lime glass beads have a slightly higher responsivity than polystyrene at a particular linewidth for TE polarisation. Soda lime glass beads also offer the advantage of a known chemistry for surface functionalisation. Figure 12 b) also shows that TM polarisation corresponds to higher responsivity than TE polarisation.

Example 9

An alternative method for attaching soda lime glass beads to the separation layer investigated removal of the adhesive or glue layer and placing the bead directly onto the cured separation layer of Cytop®, followed by a re-curing step at a lower temperature. Bead attachment was studied as a function of time and temperature, and the strength of attachment tested against a controlled liquid flow. The depth of attachment was tested by measuring the 'footprint' using a light microscope. Optimum re-curing conditions were found to be at a temperature of between 15O⁰C and 175°C for a period of about 10 min. Strong attachment and sinking of the bead by less than 200 nm was achieved.

However, due to the surface roughness, distorted shape, and/or surface damage of commercially available soda lime glass beads, even with the thickness of the separation layer and sinking under control, Q factors higher than 10⁴ were not detected, even when mode strength in reflectivity (~ 10%) indicated only a small effect of microsphere coupling to the substrate on the Q factor.

Example 10

Polystyrene beads have sufficient surface quality to achieve improved Q factors to about 4 x 10⁶. However, since polystyrene beads have a similar glass transition temperature to materials in the separation layer, i.e. Cytop® (glass transition temperature of Cytop® is 108⁰C and of polystyrene is approximately 100⁰C) adherence of polystyrene beads to the separation layer through temperature curing is limited. Adherence of dried polystyrene beads through curing onto a cured Cytop® layer at 125⁰C for 13 min resulted in the beads often not attaching or partial melting of polystyrene and/or intermixing with Cytop®. The optimised procedure, as a compromise between adherence strength and preservation of bead optical quality was spin- coating and high temperature curing (18O⁰C) of Cytop® separation layer (thickness between 200 nm and 800 nm), spin-coating of Cytop® 'glue' layer (thickness between 100 nm and 200 nm), addition of beads in water onto the soft 'glue' layer, and temperature curing at 115⁰C for lO mins.

Microscopy of the sample immersed in water showed no sign of index discontinuity at the attachment region. This indicates that the beads keep their shape and are not significantly intermixing with Cytop® during curing.

Referring now to Figure 13, whispering gallery mode detection through TE and TM polarised reflectivity spectra is illustrated for this embodiment of the invention (polystyrene bead of diameter 44 μm adhered to the Cytop® separation layer, and penetrating the Cytop® layer of thickness of 550 to 650 nm by approximately 165 nm). The orbital coverage of the bead in the layer is approximately 4%.

Referring now to Figure 14, R/Ro for the glued polystyrene beads (44 μm) on the Cytop® separation layer is dependent on the angle of incidence of the coupling angle and on the mode order (n) of the bead, with the higher angles selectively exciting the smaller n order modes.

Example 11

Referring now to Figure 15, the resonance position of the bead in the sensor of example 10 was monitored whilst flowing glycerol solutions of various concentrations through the sensor, at an acquisition rate of 1 Hz. The refractive index change with glycerol concentration is 1.38E^"3 ± 2E⁴ RIU per %vol. From the measured variation of the resonance position of 3.0785E^"5 ± 2E^"8 eV per %vol, we obtain a responsivity of -22 ± 2 meV per RIU. The precision of the peak energy from fitting a single spectrum acquired in 18 ms is 1 neV, corresponding to a sensitivity of 5 x 10^"8 RIU (for comparison, the sensitivity of a Biacore instrument was calculated from measured data to be 1.5 x 10^~7 RIU at an acquisition rate of 1 Hz). At a detection rate of 1 Hz, the standard deviation of the fitted resonance peak energy is 30 neV. This value is limited by the jitter of the optical excitation wavelength which is caused by instability of the DFB laser diode temperature and fast fluctuations in the laser diode current. This precision results in 1.4 x 10^~6 in terms of minimum detectable refractive index change, and 0.7 pg/mm or 4 fg in terms of molecular mass coverage.

The sensitivity of the sensor may be improved by increasing the responsivity of the sensor, which could be achieved by reducing the diameter of the polystyrene beads and by using TM polarisation, as shown in Figure 12 a) and b). This could improve the responsivity by a factor of 2. The jitter could be reduced by optimising the DFB laser diode temperature control. Jitter could also be reduced by using a reference bead having resonances sufficiently close in energy to be scanned by the DFB laser in a single scan. However, fast fluctuations in the DFB laser diode current, also responsible for the jitter, cannot be cancelled by referencing. Solutions to this problem would be using a laser current driver optimised for low fluctuations or an external cavity tuneable laser which can have a linewidth -1000 sharper than a DFB laser. Other improvements could be provided by suppression of digitisation noise in the data, reduction of the shot noise by increasing the optical power, though this is probably limited to one order of magnitude because of optical non-linearities in the beads which would appear at higher power, and production of sharper resonance lines. A sensitivity of 3 x 10^"10 RIU at a rate of 54 Hz is believed to be achievable. Embodiments of the present invention in accordance with the following claims will,

therefore, be apparent to those skilled in the art.

Claims

1. An optical sensing device comprising an optical resonator; a planar surface, near field coupler for coupling evanescent light to the resonator; and fixing means for positioning the resonator relative to the coupler, in which the resonator comprises a microsphere having refractive index 1.50 to 1.95 and diameter 5 to 100 μm and is positioned adjacent the planar surface of the coupler with a separation gap of 200 to 2000 run.

2. A device according to Claim 1, in which the coupler is a prism, lens or a waveguide coupler.

3. A device according to Claim 1 or Claim 2, in which the fixing means comprise an optical tweezers or x-y-z mechanical stages.

4. A device according to Claim 1 or Claim 2, in which the fixing means comprise a transparent separation layer of refractive index matched to a target medium, provided to the upper surface of the coupler, and to which the microsphere is adhered.

5. A device according to Claim 4, in which the separation layer comprises a cured polymer of refractive index of about 1.33.

6. A device according to Claim 5, in which the separation layer comprises a co-polymer of perfluoro alkenyl vinyl ethers.

7. A device according to any preceding Claim, in which the microsphere comprises a polystyrene or glass bead.

8. A device according to any preceding Claim, in which the microsphere comprises a polystyrene bead of diameter 25 to 45 μm and is positioned 400 to 600 nm above the planar surface of the coupler.

9. A device according to any one of Claims 1 to 8, in which the microsphere comprises a glass bead having refractive index 1.52 and diameter 40 to 50 μm and is positioned 600 to 800 run above the planar surface of the coupler.

10. A device according to any one of Claims 1 to 8, in the microsphere comprises a glass bead having refractive index 1.92 and diameter 5 to 15 μm and is positioned 250 to 350 nm above the planar surface of the coupler.

11. A device according to any preceding Claim, in which the coupler has tuneable laser emitting at a wavelength of light of wavelength 600 to 800 nm.

12. A device according to any preceding Claim, in which the microsphere is coated with a specific recognition element for a target analyte.

13. A device according to Claim 12, when dependent on Claim 4, comprising an array of microspheres adhered to the separation layer in which each microsphere is coated with a specific recognition element for a different target analyte.

14. A device according to any preceding Claim, further comprising a flow cell for the passage of an aqueous sample.

15. A device according to Claim 14, in which the flow cell defines a conduit of about 80 micron width.

16. A sensing chip for use with a near field coupler, comprising a glass or waveguide substrate having a planar surface provided with a transparent separation layer of refractive index matched to a sensing medium, which has thickness 200 to 2000 nm and to which an optical resonator comprising a microsphere having refractive index 1.50 to 1.95 and diameter 5 to 100 μm is adhered.

17. A chip according to Claim 16, in which the separation layer comprises a cured polymer of refractive index about 1.33.

18. A chip according to Claim 17, in which the separation layer comprises a co-polymer of perfluro alkenyl vinyl ethers.

19. A chip according to any one of Claims 16 to 18, in which the separation layer has thickness 400 to 600 nm and the microsphere comprises a polystyrene bead having diameter 20 to 45 μm.

20. A chip according to any one of Claims 16 to 18, in which the separation layer has thickness 600 to 800 nm and the microsphere comprises a glass bead having refractive index 1.52 and diameter 40 to 50 μm.

21. A chip according to any one of Claims 16 to 18, in which the separation layer has thickness 250 to 350 nm and the microsphere comprises a glass bead having refractive index 1.92 and diameter 5 to 15 μm.

22. A chip according to any one of Claims 16 to 21, in which the microsphere is coated with a specific recognition element for a target analyte.

23. Use of the chip of Claim 22 or Claim 23, for detection of one or more target analyte comprising a protein, nucleic acid, bacterium, spore or virus.