The present invention broadly relates to a sensor and a method for characterising a dielectric material and relates particularly, though not exclusively, to an evanescent field sensor and a method of characterising the dielectric material using the evanescent waves.
Surface plasmons are coherent oscillations of electrons along an interface between two materials at which the real part of the dielectric function changes sign. The energy of the surface plasmons depends on properties of the materials. Consequently, the detection of surface plasmons can be used to detect the materials.
In the past optical sensors have been proposed and typically comprise a prism having a thin metal coating, such as a thin silver or gold coating on a surface. The thin metal coating is in contact with an external sample dielectric material, such as a biological suspension. The surface plasmons at the interface between the coating and the sample dielectric material can be excited when the propagation constant of the incident light is equal to the propagation constant of the surface plasmon, and the wavelength at which this occurs depends on the refractive index of the sample dielectric material and the wavelength of the light. The generation of surface plasmons resonances results in spectral minimum of transmitted or reflected light intensity at the metal/dielectric interface. Consequently, it is possible to characterise the sample dielectric material by characterising a property of the transmitted light.
More recent optical sensors comprise optical waveguides that replace the prism and the metallic coating is applied onto the optical waveguide.
However, the detection limit of existing waveguide-based method is unsatisfactory for some applications. There is a need for technological advancement.
The present invention provides in a first aspect a method of characterising a dielectric material, the method comprising the steps of:
Throughout this specification the term “dielectric material” is used for any type of material that has dielectric properties and including for example suitable gaseous, solid, liquid materials. In one example the dielectric material is a solution or suspension of a material, such as a biological material.
The sensor typically comprises an optical waveguide, such as an optical fibre or any other suitable type of optical waveguide. A film formed from a material suitable for generation of surface plasmons may be positioned at a surface of the optical waveguide. The surface of the sensor may be a surface of the film and the film may be arranged such that the evanescent field of light guided through the optical waveguide penetrates through the surface of the film.
The step of collecting an intensity of light from the interface typically comprises collecting light that penetrates through at least a portion of the dielectric material.
Embodiments of the present invention have significant practical advantages. A dependency of a limit of detection on the thickness of the film typically is reduced compared with conventional transmission methods. Further, a detection limit that is achievable with the above-defined method may be improved compared with conventional waveguide based transmission methods.
The spectral parameter of the light typically is a wavelength of the light, but may alternatively also be a frequency or an energy of the light or any other parameter that is either directly or indirectly related to the wavelength.
In one example the apparatus comprises a first and a second optical waveguide and the step of guiding light along at least a portion of the sensor comprises guiding light through the first waveguide. In this case the step of collecting an intensity of light from the interface may comprise coupling the intensity of light from the interface into the second waveguide. A film formed from a material suitable for generation of surface plasmons may be positioned at a surface of the first optical waveguide. Alternatively, a film formed from a material suitable for generation of surface plasmons may be positioned at a surface of the second optical waveguide.
The step of guiding light along at least a portion of the sensor may comprise absorbing light from the light source and emitting suitable fluorescence light to generate the surface plasmons.
In one example the method comprises collecting an intensity of light from at least one interface with at least one sample dielectric material and from at least one interface with at least one reference dielectric material. Alternatively, the method may for example comprise collecting an intensity of light only from an interface with a sample dielectric material.
The sensor may be one of at least two sensors and the method may comprise exposing at least one sensor to the sample dielectric material and at least one sensor to a reference dielectric material. In this case the method typically comprises collecting light from interfaces at the at least two sensors using respective collector elements of the collector. In one specific embodiment the at least two sensors comprise respective portions of an optical waveguide and are positioned in sequence along that optical waveguide.
In a specific example the sample dielectric material contains a solute, such as a biological solute, in a solvent and the reference dielectric material comprises the solvent only. Alternatively, the dielectric material may for example be provided in the form of a suspension, such as a suspension of a virus or any other suitable biological sample, and the reference dielectric material may comprise only the liquid that suspends the biological sample.
The step of exposing the surface of the sensor to the dielectric material may also comprise functionalising the surface and thereby providing a surface specificity such that predominantly a predetermined biological species, such as a virus, adsorbs at the surface when the surface is exposed to a suitable dielectric material. In this case the step of collecting an intensity of light from the interface may comprise detecting a change of a property of the light as a function of adsorbed dielectric material and thereby characterising the dielectric material.
Alternatively, the step of exposing the surface of the sensor to the dielectric material may also comprise coating the surface with a coating material that is selected so that the dielectric material, for example a suitable chemical such as molecule that is capable of selectively cleaving spacer molecules (for example an enzyme), will remove molecules of the coating material from the surface when the surface is exposed to the dielectric material. In this case the step of collecting an intensity of light from the interface may comprise detecting a change of a property of the light as a function of removal of coating material and thereby indirectly characterising the dielectric material.
The step of collecting an intensity of light typically comprises generating electronic data and the method typically also comprises the step of processing the electronic data, which may for example comprise comparing collected light intensities for the sample dielectric material with those for the reference dielectric material.
In one specific embodiment the step of processing the electronic data comprises identifying a spectral maximum of the light intensity data for the sample dielectric material compared with light intensity data for the dielectric reference material.
The present invention provides in a second aspect an apparatus for characterising a dielectric material, the apparatus comprising:
The structured surface of the film typically is structured so that the surface has a roughness, but may also have a corrugation, such as a corrugation on a micro-scale, or may be otherwise structured in a regular or irregular manner.
The spectral parameter of the light typically is a wavelength of the light, but may alternatively also be a frequency or an energy of the light or any other parameter that is either directly or indirectly related to the wavelength.
In one example the light source is arranged for emitting light having a continuous wavelength range, such as a suitable “white” light source. In this case the collector typically comprises a spectrometer for detecting the intensity of the light from the at least one interface as a function of the spectral parameter. Alternatively, the light source may be a tunable light source or may comprise one or more monochromatic light sources.
The apparatus may comprise any suitable type of optical waveguide, such as an optical fibre.
In one specific example a sample dielectric material contains a biological solute in a solvent and a reference dielectric material comprises the solvent only.
Alternatively, the dielectric material may be provided in the form of a suspension of a biological sample, such as a suspension of a virus, and the reference dielectric material may comprise only the liquid that suspends the biological sample or a reference biological suspension.
In one specific embodiment the sensor is one of at least two sensors and the collector comprises at least two collector elements for collecting light from respective sensors. For example, the at least two sensors may each comprise respective regions of the optical waveguide and may be positioned in succession along that optical waveguide. At least one sensor may be arranged for contact with a sample dielectric material and at least one sensor may be arranged for contact with a reference material. In this case the apparatus has the significant practical advantage that sample and reference measurements can be performed substantially in parallel and light originating from the interfaces may also be multiplexed. For example, an effect of a change in temperature or other environmental fluctuation on a measurement result typically can be corrected in a relatively simple manner or even neglected if the first and the second sensing regions experience the same or a similar change in temperature.
In one embodiment the apparatus comprises a fluorescent material for absorption of light from the light source and emission of fluorescence radiation. The fluorescent material typically is arranged such that at least a portion of emitted fluorescence light is used for generation of surface plasmons. The film may be positioned over the fluorescent material. Alternatively or additionally the fluorescent material may be positioned within the optical waveguide or in any other suitable area on the waveguide.
The fluorescent material typically is selected to supplement a light intensity and/or a wavelength range of the light source.
In one specific embodiment the apparatus comprises a first optical waveguide for guiding the light from the light source and a second optical waveguide into which in use light from the interface is coupled. The film formed from a material suitable for generation of surface plasmons typically is positioned at a surface of the first optical waveguide.
In one example the apparatus comprises an array of sensors and is arranged so that a distribution of a property of the dielectric material can be detected. The apparatus may comprise an array of m×n sensors and m first optical waveguides and n second optical waveguides, each first optical waveguide having n sensing regions and each second optical waveguide being arranged to receive light form m interfaces, wherein the apparatus is arranged so that a distribution of a property of the dielectric material can be detected.
The film typically comprises Ag, Au, Al or Cu or any other material that is suitable for generation of surface plasmons. The film typically has a thickness within the range of 20-150 nm, such as approximately 50 nm. The film may be fabricated using any suitable deposition technique that results in a film having a surface roughness, such as a film having a granular structure. Suitable film deposition techniques include chemical and physical vapour deposition techniques or using suitable chemical reactions, such as a Tollens reaction or suitable chemical or physical adsorption of metallic nanoparticles.
The present invention provides in a third aspect method of characterising a dielectric material, the method comprising the steps of:
The second intensity of light may be associated with light emitted by label molecules.
The steps of collecting the first and second intensities of light typically comprise collecting the first and second intensities of light from the interface. The first and second intensities of light may be collected sequentially or simultaneously.
The property of the dielectric material typically is indicative of an immobilisation of a biological species at the interface and the label molecules typically emitting fluorescence radiation having a spectral distribution that is also indicative of immobilisation of the specific biological species with the label molecules at the interface.
In one specific example the label molecules are Qdots. The dielectric material may comprise a biological suspension and the method may comprise functionalising the surface at the interface thereby providing a surface specificity such that predominantly a predetermined biological species adsorbs and the label molecules adsorb at the biological species whereby both the first and second light intensities are independently indicative of immobilisation of the biological species at the interface.
The method may also comprise exposing the surface to spacer molecules that are arranged for adsorption at the surface of the interface and are also arranged for coupling to label molecules, such as fluorescent labels that may also locally increase the refractive at the surface. In this case the method typically comprises adsorption of the spacer molecules on a surface of the interface and coupling of the label molecules to the spacer molecules.
Throughout this specification the term “spacer molecules” is used for any type of molecules that is suitable for adsorption at the surface of the interface, coupling to the label molecules and are arranged for cleaving by a predetermined type of molecule.
In one specific embodiment the spacer molecules are arranged for cleaving by a chemical or a biological species of the dielectric material (for example an enzyme). In this case the method typically comprises detecting a spectrally dependent change in the intensity, which is indicative of cleaving of the spacer molecules and adsorption of the chemical or a biological species at the cleaved spacer molecule on the surface of the interface. Alternatively, the method may comprise detecting a correlated spectral change in the first and second intensities, which is indicative of cleaving of the spacer molecules and adsorption of the chemical or biological species at the cleaved spacer molecule on the surface of the interface.
The method may also comprise the step of re-attaching cleaved portions to respective cleaved spacer molecules at the interface such that the spacer molecules are again arranged for coupling to the label molecules.
The method in accordance with the third aspect of the present invention typically comprises the method in accordance with the first aspect of the present invention.
Alternatively or additionally, the second intensity of light may relate to second harmonic generation (SHG) associated with a surface plasmon excitation at the interface. In this case the method typically comprises directing suitable light to the interface, the suitable light having a wavelength in the range of a fundamental resonance wavelength of the plasmonic excitation. The method typically comprises the step of analysing the second intensity to obtain information concerning an orientation or change thereof and/or a comformation or change thereof of biological species at the interface.
The present invention provides in a fourth aspect an apparatus for characterising a dielectric material, the apparatus comprising:
The second light intensity may include an intensity of fluorescence light and the label molecules may comprise Qdots that emit the fluorescence light having a spectral distribution that is indicative of immobilisation of the label molecules at the interface.
The apparatus typically comprises the apparatus in accordance with the second aspect of the present invention.
The apparatus according to the fourth aspect of the present invention has significant practical advantages. The apparatus enables (for the first time) performing surface plasmon resonance studies together with another sensing techniques, such as fluorescence spectroscopy, using the same apparatus. Consequently, the apparatus combines key advantages of both sensing techniques within a single platform, which is not possible using existing platforms. Further, it is possible to provide independent confirmation of a diagnostic using the other sensing technique, which also increases the detection specificity. The invention will be more fully understood from the following description of specific embodiments of the invention. The description is provided with reference to the accompanying drawings.
a) and (b) are graphs showing spectral characteristics of light transmitted through a sensor and light emitted from a side portion of the sensor, respectively, using the apparatus of
a) and (b) are graphs showing spectral characteristics of light transmitted through a sensor and light emitted from a side portion of the sensor, respectively, using the apparatus of
a) and (b) are graphs showing spectral characteristics of light transmitted through a sensor and light emitted from a side portion of the sensor, respectively, using the apparatus of
a) and (b) are graphs showing spectral characteristics of light transmitted through a sensor and light emitted from a side portion of the sensor, respectively, using the apparatus of
a) and (b) are graphs showing spectral characteristics of light transmitted through a sensor and light emitted from a side portion of the sensor, respectively, using the apparatus of
a) and (b) are graphs showing spectral characteristics of light transmitted through a sensor and light emitted from a side portion of the sensor, respectively, using the apparatus of
a) and (b) are graphs showing spectral characteristics of light transmitted through a sensor and light emitted from a side portion of the sensor, respectively, using the apparatus of
a) and (b) are graphs of spectral characteristics of liquids having different refractive indices, the data having been obtained using the apparatus of
a) and (b) are graphs of spectral characteristics of liquids having different refractive indices, the data having been obtained using the apparatus of
Referring initially to
In the embodiment described in
The sensor 22 comprises a waveguide that is in this example provided in the form of an optical fibre comprising a core region 32 and a thin film 26 on the core region 32. The thin film 26 is formed from a material suitable for the generation of surface plasmons and has a surface 28. It is to be appreciated that in alternative embodiments the film 26 may not be deposited directly on the core region, but may be deposited on the thin cladding region.
Further, the film 26 is arranged such that the evanescent field of suitable light such as light produced by the light source 34 guided through the core region 32 penetrates through the surface 28 of the film 26. In this example the film 26 comprises Ag. However it will be appreciated that the film 26 may alternatively comprise Au, Al, Cu or any other material that is suitable for generation of surface plasmons.
To allow the evanescent field of light guided through the core region 32 to penetrate through the surface 28 of the film 26, the film 26 has a thickness within the range of 20-150 nm, such as approximately 50 nm.
In a second step 14 of the method 10, a surface 28 of the sensor 22 is exposed to the sample dielectric material so as to form an interface between the exterior surface 28 of the film 26 and the sample dielectric material.
In a third step 16 of the method 10, light is directed through the sensor 22 so as to generate surface plasmons at the exterior surface 28 of the film 26. An intensity of light the surface 28 is then collected as a function of a wavelength of the light in a fourth step 18.
The method 10 and the apparatus 20 provide the advantage of reducing the dependency of a limit of detection on the thickness of the film compared with conventional methods that detect a sample dielectric material by analysing transmitted light.
Although the above example describes the sensor 22 as comprising a waveguide, it will be appreciated that the sensor 22 may be of any appropriate form.
As mentioned earlier, the film 26 of sensor 22 comprises Ag. Ag is an appropriate material to be used to assist in generating surface plasmons. For example, Ag can be deposited using a chemical reaction based on the reduction of silver nitrate with glucose. A person skilled in the art will appreciate that alternatively suitable physical or chemical vapour depositions techniques may be used. Further, suitable chemical or physical adsorption of metallic nanoparticles may be used to form the Ag film. Now described is a specific example of a sensor 22 and a method of fabricating the sensor 22. The sensor 22 comprises an optical fibre comprising F2 glass (Schott) with a refractive index of 1.62 and having a core diameter of 140 μm. The optical fibre has a polymer cladding having a refractive index of 1.52 (NA=0.56). A small section of the optical fibre, about 4 mm long, is stripped of the cladding and subsequently chemically coated with silver using the so-called “Tollens” reaction.
The Tollens reaction, also known as the silver mirror reaction, comprises adding a solution of silver ammonia to a reducing agent, usually a sugar such as glucose, in order to produce silver nanoparticles that may subsequently be attached to a substrate. The preparation of the Tollens reagents starts with the oxidation of a silver nitrate solution (20 mL of 0.24 mol/L AgNO3) into silver oxide using potassium hydroxide (40 uL of 0.25 mol/L KOH) according to Equation 1 below. This produces a brown precipitate in the initially transparent silver nitrate solution. Ammonia (3 mol/L) is then added drop by drop to dissolve the silver oxide and produce a transparent silver ammonia complex according to Equation 2.
2AgNO3+2KOH→Ag2O(s)+2KNO3+H2O (1)
Ag2O(s)+4NH3+2KNO3+H2O(l)→2Ag(NH3)2NO3+2KOH (2)
A reducing agent comprising a mixture of methanol and glucose (1.9 mol/L) solution is made in the ratio of 1:2 and added in equal parts to the silver ammonia solution, then mixed using a magnetic stirrer. Once the reducing agent is added to the silver ammonia solution, the reaction produces a metallic silver coating according to Equation 3.
CH2OH(CHOH)4CHO+2Ag(NH3)2+3OH−→2Ag(s)+CH2OH(CHOH)4CO2−+4NH3+2H2O (3)
A stripped section of the optical fibre is then placed, at room temperature, into a beaker containing the silver coating solution and left inside the beaker for an appropriate period of time as to form a film of Ag of appropriate thickness. After coating, the optical fibre is rinsed in de-ionized water and then air dried. The thickness of the deposited silver coating may be measured by scanning electron microscopy, transmission electron microscopy or any other suitable method.
The method further comprises the step of detecting a reference dielectric material so that data of the sample dielectric material can be compared with data of the reference dielectric material, such as by subtracting the reference data from the sample data. This may include measuring the sample and reference dielectric media separately with the sensor 22.
Alternatively, the method may comprise exposing a first sensor to the reference dielectric and a second sensor to the sample dielectric. This may be achieved using an apparatus 50 as shown in
The sample dielectric material may contain a biological suspension and the reference dielectric material may for example contain only the solution that suspends a biological sample. In this case the method comprises functionalising the surface such that predominantly a predetermined biological species, such as a virus, specifically interacts with the surface at the surface when the surface is exposed to a suitable dielectric material.
Alternatively, the dielectric material may for example be chemical such as an acid, having molecules with a relatively small molecular weight. In this case the method comprises coating the surface with a coating material that is selected so that the chemical will remove molecules or particles such as microspheres of the coating material from the surface when the surface is exposed to the chemical.
The method comprises exposing the surface of the first sensor 44 to a sample dielectric material and collecting an intensity of light from the surface of the first sensor 44 and exposing the surface of the second sensor 46 to a reference dielectric material and collecting an intensity of light from the surface of the second sensor 46.
Light from the surfaces of the first and second sensors 44, 46 is collected using respective collector elements of a collector (not shown) or by directing the light 44, 46 to a single collector 52 via respective reflective devices 48, 50.
The step of collecting an intensity of light comprises generating electronic data and the method also comprises comparing collected light intensities for the sample dielectric material with those for the reference dielectric material. Processing the electronic data comprises identifying a spectral maximum of the light intensity data for the sample dielectric material compared with light intensity data for the dielectric reference material.
In this example, the light source 34, a “supercontinuum” white light source, is used as a broad band light source and is coupled to the fibre samples using an achromatic lens. An output signal of light transmitted through the sensor 22, referred to as ‘transmission measurements’ in the following, is recorded using a transmitted light collector 38 such as an optical spectrum analyzer, and light from a surface of the film, referred to as ‘evanescent field measurements’ in the following, is collected using a collector 36. In this example the collector 36 comprises an optical fibre bundle and is analysed using a spectrometer.
Apparatus 20 and 40 shown in
In one alternative example the apparatus does not comprise a broadband light source, but comprises instead a single monochromatic light source or a combination of multiple suitable monochromatic light sources. The fluorescent material comprises in this example different types of fluorescent dye molecules that are selected so that together they provide fluorescence light that has a sufficiently broad wavelength range.
In an alternative embodiment a further waveguide may be positioned in the proximity of the evanescent field of the waveguides 32 and 42 so that emitted light may be coupled into the further waveguide. A variation of such an embodiment is illustrated in Figure (a) and (b)
A person skilled in the art will appreciate that
What follows is an example of a method used to detect a dielectric material using sensors of different film thicknesses, with a specific comparison between an embodiment of the present invention that detects a dielectric material by collecting light from the surface of the sensor and a method that characterises the dielectric material by analysing light transmitted through the sensor.
Stripped sections of different optical fibre samples were immersed in a beaker containing an Ag coating solution (prepared as described previously) for different periods of time in order to form sensors 22, each having a different film thickness so as to evaluate the effect of the deposited Ag film thickness on the performance of each sensor 22. After coating, the fibres were rinsed in de-ionized water and then air dried and the thickness of the deposited silver coating was measured by scanning electron microscopy for each sample.
Graph 65 of
Referring to the transmission measurements of graph 65 first, line 66 represents reference measurements taken before immersion into the glycerol solution and these measurements are used as a reference spectrum, line 67 represents sample measurements taken after immersion into the glycerol solution, and line 68 represents results wherein the reference measurements are subtracted from the sample measurements. A dip in the sample transmission measurements, indicated by line 67, can be observed around λ=636 nm of
The transmission results described above fit the numerical simulations performed using the same physical parameters which are shown in
The slight wavelength shift between the transmission measurements of
Referring now to the evanescent field measurements of graph 69, line 70 represents reference measurements taken before immersion into the glycerol solution and these measurements are used as a reference spectrum, line 72 represents sample measurements taken after immersion into the glycerol solution, and line 74 represents results wherein the reference measurements are subtracted from the sample measurements. An additional emission peak is featured in the spectrum associated with light from a side portion of the sensor (“evanescent field”) of
Transmission and evanescent field characterisation of fibres prepared with the same coating techniques but with different silver coating thickness are shown in
These figures show that the Ag coating thickness required to observe a surface plasmon resonance signal in transmission measurements is restricted to a relatively short range of thickness (from 40 to 65 nm) and the evanescent field measurements provides a surface plasmon resonance signal for a much range of Ag thicknesses.
The sensitivity, ˜5.7×10−4 RIU in transmission and ˜6×10−4 RIU for the evanescent field capture mode, are similar for both detection techniques apart for the thinner Ag coating, and in agreement with surface plasmon resonance sensitivity reported in the literature (10−5 to 10−8 RIU). However, the detection limit is a more reliable parameter for the description of a sensor's performance. In that context, the detection limit (DL) is defined as the ration between the resolution (R) of the sensor and its sensitivity (S). The resolution is itself defined as 3σ where σ as the average noise contribution from the signal amplitude (σAmpl), the spectral position (σSpectral) and the thermal noise (σTherm).
3σ=3√{square root over (σAmpl2+σSpectral2+σTherm2)} (5)
The noise on the signal amplitude σAmpl is defined by Equation 6, where SNR is the signal to noise ratio and Δλ the wavelength shift of the surface Plasmon resonance.
In our case, the SNR was defined as
where the noise amplitude is defined as the standard deviation of the experimental data from a log normal fitting model defined by the equation 7 and the amplitude is simply the maximum or minimum on each spectrum depending on the acquisition method.
This fitting model was chosen instead of a Lorentzian as the experimental data present a characteristic asymmetry which was well fitted by Equation 7. The different parameters (y0, A, xc and w) where obtained using a standard fitting routine. The SNR such as that defined above is represented as a function of the deposited Ag thickness for different refractive index liquid measured both in transmission and using the evanescent field capture approach, in
From a rapid observation of
In contrast to conventional transmission methods, the above described evanescent field method works for Ag coating thicknesses below approximately 40 nm or film thicknesses of the order of 40 nm both detection methods yield the same detection limit, whereas for higher Ag thickness, the detection limit of the evanescent field method is two times lower than that for the transmission measurements.
Further tests show that embodiments of the present invention may not be dependent on the manner in which the film 26 is formed. In particular, an alternative coating method was used, in this case thermal evaporation, for Ag deposition. Thermal evaporation has been reported in the literature for surface plasmon sensor fabrication, especially on optical fibre substrates. This method requires a rotational stage to be installed inside an evaporator in order to homogenously coat the entire fibre circumference.
In this example, only half of the surface of the exposed core region of the optical fibre was coated with different thicknesses of Ag. Similar measurements were performed and the same phenomena was observed, hence the evanescent field was enhanced at a spectral position depending on the refractive index liquid into which the fibre sensing region was immersed, corresponding to a surface plasmon resonance signal.
The above results show that using a spectral characterisation of the evanescent field around a section of an optical fibre coated with Ag can provide the same information than the traditional transmission measurements. However, this evanescent field collection mode yields higher signals to noise ratios which implies higher detection limit than its transmission counterpart. Moreover, this technique is less restrictive as a strict control on the thickness of the deposited Ag is no longer required.
The following example outlines test measurements done in respect of the apparatus of
Similar experiments were conducted where the second sensor 46 was kept in air while the first sensor 44 was immersed in different refractive index liquids. Light from the surface of each sensor 44, 46 were measured. Graph 134 of
Referring now to
Surface functionalisition of the sensor will now be described with reference to
After each of the above-described steps, plasmons were generated at the interface and light from the interface was collected in the previously described manner. The first flow cell provided reference data.
The apparatus 200 also allows the use of a different sensing strategy that is especially useful for multiplexing. For example, different fluorescent labels, emitting at different wavelengths, can be used in order not only to detect influenza, as demonstrated in this example, but also to identify a viral strain assuming that specific secondary antibodies are available.
The spacer molecules 260 are arranged for cleaving by a predetermined type of molecule, such as a biological species. Cleaving the spacer molecules results in release of the microspheres, which induces a wavelength shift toward shorter wavelengths of the intensity indicative of surface plasmon generation and a corresponding reduction of the fluorescence intensity. Consequently, the predetermined type of molecule is detectable by measuring correlated spectral changes in intensities indicative of generation of surface plasmons and changes in fluorescence intensity.
The above-described concept can be generalised for multiplexed sensing assuming that different spacer molecules, which react specifically with different chemicals or biological species, are attached at one end to the surface of the interface and at the other end to microspheres containing different fluorescent dyes.
The method may also comprise re-attaching cleaved portions to respective cleaved spacer molecules at the interface such that the spacer molecules are again arranged for coupling to the spacer molecules. In one specific embodiment an antibody or the like may be attached to the loose end of a spacer molecule instead of a label molecule such as a Qdot. After an interaction between the antibody and its antigen counterpart, the spacer molecule may be cleaved by an enzyme and then regenerated by re-attaching the missing part of the spacer molecule such that the sensor can be re-used.
In addition, embodiments of the present invention provide information concerning a preferred orientation of the biological species at the interface. Second Harmonic Generation (SHG) associated with surface plasmon excitations at the interface is used for this purpose suitable laser light is directed to the interface. The laser light has a frequency that corresponds to a fundamental resonance frequency of the plasmonic excitation. In this process, the efficiency of generation of the second harmonic depends on the orientation of the biological species that are localised at the interface. For example, if the biological species are randomly oriented, an intensity of a signal associated with the SHG will be relatively low. Alternatively, if the biological species have a preferred orientation, the signal associated with the SHG is relatively large. Consequently, SHG can be used to probe the orientation of the biological species at the interface.
In the example described in the following the surface of a SPR fibre sensor, embedded into a microfluidic flow cell, was prepared for the specific detection of an enzyme (trypsine) using the cleaving of a specifically engineered spacer as transducing mechanism. The sensor was first coated with polyelectrolyte such as described previously (5 layers: PAH/PSS/PAH/PSS/PAH). The spacer itself is a long peptide chain with a carboxylic function on one end and an amine function on the other end, while the mid section of the spacer presents a chemical function that is design to be specifically cleaved by the enzyme.
Therefore, the spacer was attached to the last PAH layer using amine coupling reagents (EDC/NHS), promoting the reaction between the amine function of the PAH layer and the carboxylic function of the spacer. Following the immobilisation of the spacer, large particles, in this case quantum dots surface functionalised with carboxylic function were attached to the free end of the spacer which presents an amine function, again using amine coupling reagent. At this stage, the sensor is ready to detect specifically the enzyme design to cleave the spacer and release the quantum dots and no blocking reagent are required since the detection is performed through the release of the quantum dots rather than the standard immobilisation onto the sensor surface. The spectral position of the surface plasmon resonance was monitored throughout the different steps of the surface functionalisation process. Following the injection of the enzyme into the flow cell, the wavelength shift of the surface Plasmon resonance was observed toward shorter wavelengths indicating the release of the quantum dots from the surface as shown in the
Although the invention has been described with reference to particular examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms.
Number | Date | Country | Kind |
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2010901110 | Mar 2010 | AU | national |
2011900130 | Jan 2011 | AU | national |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/AU2011/000275 | 3/11/2011 | WO | 00 | 11/21/2012 |