WO2001069174A1 - Plate-forme a grande vitesse, a sensibilite elevee, pour des applications de detection de surface d'onde evanescente - Google Patents

Plate-forme a grande vitesse, a sensibilite elevee, pour des applications de detection de surface d'onde evanescente Download PDF

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Publication number
WO2001069174A1
WO2001069174A1 PCT/US2001/007672 US0107672W WO0169174A1 WO 2001069174 A1 WO2001069174 A1 WO 2001069174A1 US 0107672 W US0107672 W US 0107672W WO 0169174 A1 WO0169174 A1 WO 0169174A1
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optical
measurement
recited
sensing platform
sample
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PCT/US2001/007672
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English (en)
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Ronald J. Rieder
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Satcon Technology Corporation
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Priority to EP01922327A priority Critical patent/EP1266189A1/fr
Priority to AU2001249143A priority patent/AU2001249143A1/en
Publication of WO2001069174A1 publication Critical patent/WO2001069174A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/7703—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
    • G01N21/45—Refractivity; Phase-affecting properties, e.g. optical path length using interferometric methods; using Schlieren methods
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence

Definitions

  • This invention relates generally to optical sensors, and more particularly to high speed, highly sensitive, optical sensing platforms for evanescent wave surface detection applications, i.e., an evanescent interferometer biosensor.
  • Evanescent wave surface detection is an optical technique that has been used in various applications such as the detection of substances in liquid and gaseous samples and the measurement of certain properties of liquid and gaseous samples, including, e.g., changes in refractive indices and ionic concentrations of the samples.
  • the evanescent wave surface detection technique typically includes sensing a change in the local environment at the surface of a waveguide.
  • the waveguide surface is often coated with a chemically or biologically sensitive layer, to which targets within a liquid or gaseous sample are then bound.
  • Light is coupled into the waveguide, which, as it propagates through the waveguide, produces evanescent wave fields that reach out and penetrate the chemically or biologically sensitive layer and the sample bound thereto. Because evanescent wave fields that correspond to different spatial modes of the propagated light typically penetrate at different depths, information relating to the depths of penetration for the different spatial modes can be used to characterize the liquid or gaseous sample provided at the surface of the waveguide.
  • the detection and/or measurement of very small numbers of microorganisms in a sample typically requires amplification, or enrichment, of the target microorganisms population before detecting and/ or measuring the sample is possible. This is often accomplished using culture enrichment techniques that may take up to several days to complete.
  • some evanescent wave surface detection techniques permit direct and rapid detection and /or measurement of very small numbers of target microorganisms by transferring the amplification process from the biological domain to the photonic domain.
  • One such evanescent wave surface detection technique uses fluorescent markers for detecting and/or measuring substances in a liquid or gaseous sample.
  • targets i.e., analytes
  • evanescent wave fields reach out into the tagged sample and excite the fluorescent markers.
  • the target microorganisms in the tagged sample are then detected and/ or measured by monitoring the intensity of the sample's fluorescence.
  • An evanescent wave surface detection technique that uses fluorescent markers has some shortcomings. For example, the expense and complexity of reagents used for tagging the targets affect its utility. Still further, the process of tagging the sample with fluorescent markers has some drawbacks. Specifically, it is often difficult to ensure that only the target analytes are tagged. Frequently, however, random substances bound to the waveguide surface are tagged also, thereby affecting the detection and/ or measurement of the desired targets. Such non-specific binding of random substances can adversely affect, e.g., the signal-to-noise ratio (SNR) of that evanescent wave surface detection technique.
  • SNR signal-to-noise ratio
  • Another optical evanescent wave surface detection technique that can be used to detect and/or measure small numbers of target analytes within a sample involves monitoring changes in the intensity of light related to the evanescent wave fields due to the bound sample.
  • This evanescent wave surface detection technique is used in some commercially available instruments, such as the BIAcoreTM surface plasmon resonance (SPR) instrument manufactured by Amersham Pharmacia Biotech AB, Uppsala, Sweden.
  • SPR surface plasmon resonance
  • the evanescent wave surface detection method used with an SPR instrument typically comprises coating the waveguide surface with a thin layer of metal; immobilizing "selective" receptors to the metal layer; and then capturing the sample onto the receptors.
  • Light is coupled into the waveguide, which, as it propagates through the waveguide, causes evanescent wave fields to reach out into the sample layers on the waveguide surface.
  • the bound targets alter the effective index of refraction (n) of the metal layer.
  • the evanescent wave fields resonantly transfer energy to a surface plasmon, and the intensity of the evanescent wave fields is monitored at an energy matching condition.
  • an evanescent wave surface detection technique used with the SPR instrument has some shortcomings.
  • biochemical and environmental factors such as non-specific binding and temperature variation typically limit the sensitivity and stability of that evanescent wave surface detection technique.
  • the BIAcoreTM brand SPR instrument is commercially expensive; hence, it is often inappropriate for use in low-cost applications.
  • Still another evanescent wave surface detection technique for detecting and/or measuring untagged substances in a bound sample is disclosed in US Pat. 5,120, 131 (the "'131 patent") to Lukosz.
  • a measurement sample is bound to the waveguide surface, and light is coupled into the waveguide, thereby causing evanescent wave fields to reach out into the bound sample.
  • light is coupled into the waveguide so that two mutually coherent, orthogonally polarized modes propagate through the waveguide simultaneously and coaxially.
  • the respective refractive indices of the two guided modes i.e., the transverse electric (TE) and transverse magnetic (TM) change.
  • Relative changes in the refractive index (n) of a measurement sample with respect to the refraction index of a reference sample can be measured with an interferometer, and those measurements can be used for characterizing the bound sample. These changes are manifest as an optical phase change of light traveling through a medium.
  • the evanescent wave surface detection technique disclosed in the '131 patent has some shortcomings.
  • this evanescent wave surface detection technique typically lacks the stability required for accurately detecting and/or measuring very small numbers of targets. This is because the stability of that evanescent wave surface detection technique typically is limited by biochemical and environmental factors, i.e., noise, such as non-specific binding and temperature variation of the bulk liquid, which often result in less than optimal SNR ("signal-to-noise ratio").
  • SNR signal-to-noise ratio
  • thermal and mechanical perturbations which are major sources of noise and which adversely affect the SNR.
  • an improved evanescent wave surface detection technique and device for detecting and/ or measuring substances in liquid and gaseous samples.
  • Such an evanescent wave surface detection technique and device would have the stability and sensitivity required for accurately and directly detecting and/or measuring low levels, e.g., as low as a single microorganism, of small molecules, bio- molecules, and/or microorganisms in a sample.
  • a detection technique would have the stability and sensitivity required for accurately and directly detecting and/ or measuring low levels of small microorganisms without requiring prior amplification, i.e., enrichment, of the target analyte population.
  • the present invention provides a high precision, optical sensing platform that uses a waveguide, which includes at least one first optical path in relatively close spatial proximity to at least one second optical path.
  • the first optical path are exposed to measurement samples containing targets, which are bound to a surface contiguous to the first optical path of the waveguide.
  • the second optical path can be exposed to reference samples, which have known targets bound to a surface contiguous to the second optical path of the waveguide.
  • the measurement and reference samples containing the targets can be either liquid or gaseous samples.
  • light enters a polarization modulator, which facilitates removing low frequency noise signals.
  • the polarization modulator enables exciting two orthogonally polarized spatial, i.e., guided, modes, causing each guided mode to propagate independently and sequentially through the spatially separated measurement and reference optical paths. Subsequently, the light from each path can be coupled out of the waveguide and coherently combined for each polarization mode.
  • An optical phase detector can be used to record any changes in phase for each guided mode and to compare any changes with subsequent measurements.
  • optical phase changes typically, are caused by characteristics of the measurement and reference samples and the targets bound thereto, which samples are contiguous to the surface of the waveguide.
  • optical phase changes caused by the characteristics of the target analytes bound to measurement and reference samples can be detected and/ or measured using a doubly differential surface detection technique, which comprises a set of first differential measurements and a second differential measurement.
  • a set of first differential measurements is obtained from any optical phase change between the reference and measurement samples for each of the individual orthogonal guided modes, each of which propagates sequentially through the first and the second optical paths of the waveguide.
  • a second differential measurement is obtained from a combination of the first differential measurements of optical phase changes for each of the individual guided modes.
  • the set of first differential measurements is obtained by first determining changes in the respective effective refractive indices ( ⁇ n) between the first and second optical paths, which are exposed to a measurement sample and a reference sample, respectively.
  • This "set" of measurements comprises an effective refraction index change for each of the two polarized guided modes, i.e., 5n ⁇ E and onTM, which, because of modulation, propagates through each of the optical paths sequentially rather than simultaneously.
  • this doubly differential surface detection technique provides immunity to environmental effects, such as temperature changes, mechanical vibrations, and biochemical effects, such as non-specific binding, thereby providing the sensitivity and speed required for directly detecting and/or measuring, in real time, very small numbers of bound targets, e.g., small molecules, bio-molecules, and/or microorganisms, including a single target, bound to or contained within the sample.
  • bound targets e.g., small molecules, bio-molecules, and/or microorganisms, including a single target, bound to or contained within the sample.
  • FIG. 1 illustrates a block diagram of an embodiment of an optical sensing platform according to the present invention
  • FIG. 2A illustrates a cross sectional view of an embodiment of an integrated optical sensor used with the optical sensing platform of FIG. 1, taken along a line 2A -2A;
  • FIG. 2B illustrates a cross sectional view of an embodiment of the integrated optical sensor used with the optical sensing platform of FIG. 1, taken along a line 2B -2B;
  • FIG. 2C illustrates a detailed cross sectional view of an embodiment of the integrated optical sensor used with the optical sensing platform of FIG. 1, taken along a line 2C - 2C;
  • FIG. 3 illustrates a block diagram of an embodiment of the phase detector used with the optical sensing platform of FIG. 1;
  • FIG. 4 shows the dramatic effect of polarization modulation on power spectral density
  • FIG. 5 shows an embodiment of modulated time-phase data. DETAILED DESCRIPTION OF THE INVENTION INCLUDING THE PREFERRED EMBODIMENTS THEREOF
  • an optical sensing platform 100 in accordance with one embodiment in the present invention includes a light source, e.g., laser 102, a polarization modulator 103, an integrated optical sensor 105, a beam combiner 113, a phase detector 139, and a computer 141.
  • a light source e.g., laser 102
  • a polarization modulator 103 e.g., an integrated optical sensor 105
  • a beam combiner 113 e.g., a phase detector 139
  • a computer 141 e.g., a computer 141.
  • the light source e.g., laser 102, injects beams 132, 133 into a polarization modulator 103.
  • the polarization modulator 103 rotates, or modulates, the polarization of the incident light to enable the excitement of two orthogonally polarized guided modes, e.g., the TE m (transverse electric) and TM m (transverse magnetic) modes, in each of the measurement and reference paths 130, 131.
  • the polarization modulator 103 causes the two polarized guided modes to propagate sequentially through both the measurement and reference paths 130, 131.
  • the polarization modulator 103 can be of any type that is well known to those of ordinary skill in the art, e.g., a ferro-electric liquid crystal, pockel cell or photoelastic modulator, that switches between S and P polarization to excite the transverse electric and the transverse magnetic guided modes in time, removing low frequency noise and eliminating long-term drift.
  • a ferro-electric liquid crystal, pockel cell or photoelastic modulator that switches between S and P polarization to excite the transverse electric and the transverse magnetic guided modes in time, removing low frequency noise and eliminating long-term drift.
  • Major sources of noise limiting the performance of interferometric systems typically are due to thermal and mechanical perturbations. These noise sources are generally confined to temporal frequencies well under a thousand Hertz.
  • SNR system signal-to-noise ratio
  • the advantage of this method is the ability to suppress unwanted signals within the bandwidth of the desired information.
  • Conventional low-pass filtering is used to suppress unwanted signals outside of the bandwidth of the desired signal. However, with low- pass filtering, any noise occurring within the signal bandwidth cannot be suppressed without also suppressing the desired signal.
  • FIG. 4 shows the noise power spectrum for a typical interferometric system.
  • the curve 42 (shown in Figure 4 as a dashed line) exhibits significantly higher noise power at low frequencies, typically below about 1000 Hertz.
  • the curve 41 (shown in Figure 4 as a straight line) is virtually constant at all frequencies, including in-band frequencies, i.e., frequencies within a desirable bandwidth.
  • polarization modulation filters out low-frequency noise (shown in Figure 4 as the hatched region), typically due to thermal and mechanical perturbations, as would a low-pass filter if applied to the entire spectrum.
  • This common-mode rejection technique is readily applied to an interferometric evanescent wave biosensor.
  • the response to the surface binding of select, e.g., bio-chemical molecules is manifest as a change in the effective refractive index within the guiding region of a single-mode waveguide 111. This index change is dependent on the polarization of the light.
  • the optical response for S- and P- polarized light (respectively propagated as TE and TM spatial modes within a waveguide 11 1) is different, however, many externally applied perturbations are independent of polarization.
  • the modulated light then passes into input couplers 107, 108 of a waveguide 11 1, which causes respective, in-coupled, guided beams (not shown) to propagate, respectively, through the optical paths 131, 130 toward the output couplers 109, 110.
  • the output couplers 109, 120 provide corresponding out-coupled beams 135, 134 to a beam combiner
  • the beam combiner 113 provides a combined beam 137 for each guided mode to a phase detector 139.
  • the phase detector 139 generates measurement data by interpreting optical differences in phase between the first and second optical paths, which, respectively, are exposed to the measurement and reference samples 130, 131 for each guided mode, and sends the measurement data to a computer 141 for subsequent analysis.
  • the integrated optical sensor 105 includes a waveguide 111 that accommodates at least two (2) optical channels or paths 130, 131 on a, e.g., silicon, substrate 202 (see FIG. 2A).
  • the substrate 202 is planar; however, the disclosed invention can be practiced with a non- planar substrate without deviating from the scope and spirit of the disclosure.
  • a measurement path 130 extends between an input coupler 108 and an output coupler 110 and a reference path 131 extends between another input coupler 107 and another output coupler 109.
  • a measurement sample 140 is contiguous to the upper surface of the measurement path 130, e.g., approximately midway between the input coupler 108 and output coupler 110.
  • a reference sample 142 is contiguous to the upper surface of the reference path 131 , e.g., approximately midway between the input coupler 107 and output coupler 109.
  • the measurement and reference samples 140, 142 can be liquid or gaseous samples containing amounts of biological or chemical substances of unknown and known concentration, respectively.
  • Target analytes within these liquid or gaseous samples are located on, i.e., bound to, respectively, the upper surfaces of the measurement and reference paths 130, 131 in manners that are well know to those of ordinary skill in the art.
  • Various surface attachment techniques are presented in "Patterning Multiple Antibodies on Polystyrene” by R.A. Brizzolara that was published in Biosensors and Bioelectronics, 15, pp. 63-68 (2000), which is incorporated herein by reference.
  • respective upper surfaces of the measurement and/or reference paths 130, 131 can be coated appropriately with known chemically or biologically sensitive layers, and the target analytes within the measurement and/ or reference samples 140, 142 then bind to these layers.
  • the measurement and reference paths 130, 131 in association with the input couplers 107, 108 and output couplers 109, 110, provide optical paths for light propagating as gaussian-shaped beams through a thin-film guiding layer, i.e., waveguide 111, of the integrated optical sensor 105.
  • the measurement path 130 and the reference path 131 are rectilinear sections, each having a length on the order of the length of the integrated optical sensor 105.
  • non-rectilinear sections also can be used without deviating from the scope and spirit of this disclosure.
  • the guided beams propagating through the measurement and reference paths 130, 131 in the guiding layer are unconfined in the lateral planar dimension, unwanted scattering of the beams within the guiding layer can be minimized, thereby significantly enhancing the sensitivity of the optical sensing platform 100 in comparison to conventional channel- type paths.
  • the input couplers 107, 108, the output couplers 109, 110, and the optical elements (not shown), e.g., optical fibers, which are used for injecting the beams 132, 133 into the input couplers 107, 108 via the polarization modulator 103, are conventional. Accordingly, specific structures used for implementing these optical elements in the integrated optical sensor 105 are not critical to the present invention, and can take different forms.
  • the laser 102 used as a light source also is conventional and can be implemented as, e.g., a helium-neon (HeNe) laser, a near infrared semiconductor laser, a diode laser or other laser known to those skilled in the art.
  • HeNe helium-neon
  • the detection and/ or measurement of very small numbers of bound targets, including even a single target, from liquid or gaseous samples is accomplished using an interferometric, doubly differential surface detection technique.
  • the light source 102 e.g., laser
  • Two spatially separated, polarized guided beams propagate unconfined in the lateral planar direction through a first and a second optical path 130, 131 within the guiding layer of the waveguide 1 11.
  • Each beam 132, 133 preferably propagates through each optical path 130, 131 in each of two orthogonally polarized waveguide modes, and more preferably in the TEo and TMo modes, which modes are modulated to propagate independently and sequentially.
  • the doubly differential surface detection technique of the present invention then is used to derive a set of first differential measurements, e.g., 5n ⁇ E and 5n ⁇ M, for each of the two polarized guided modes propagating sequentially through each of the measurement and reference paths 130, 131,i.e.,
  • the differential measurements preferably are determined using a programmed microprocessor receiving the data from the fringe detector 304.
  • a microprocessor is programmed readily by a normally skilled programmer.
  • This doubly differential surface detection technique provides substantial immunity, i.e., lack of sensitivity, to both inherent thermal and/ or mechanical perturbations and external thermal variations in the local index of refraction and also compensates for non-specific binding, thereby advantageously providing the sensitivity and stability required for detecting and/or measuring very small concentrations of substances, e.g., small molecules, bio-molecules, and/or microorganisms, in a measurement sample 140.
  • the sensitivity of the disclosed invention can measure even a single bound molecule or pathogen in a measurement sample 140.
  • the measurement path 130 and the reference path 131 can be rectilinear sections.
  • the rectangular cross sectional dimensions of the optical paths 131 , 130, and the wavelength, ⁇ , of the injected beams 132, 133 are specified so that two orthogonally polarized waveguide modes, i.e., TE m and TMm, are allowed to propagate sequentially through each path 131, 130.
  • each of the in-coupled, guided beams preferably propagates through the waveguide 111 of the integrated optical sensor 105 as TE 0 and TMo modes.
  • FIG. 2A shows an embodiment of a cross sectional view of the integrated optical sensor 105 of the present invention, taken along the line 2A - 2A. That view shows the measurement and reference paths 130, 131 through the thin film guided wave layer formed on the substrate 202. Further, the measurement sample 140, containing target analytes of unknown concentration, and the reference sample 142, which can contain target analytes of known concentration, are shown contiguous to the upper surfaces of the measurement and reference paths 130, 131, respectively.
  • FIG. 2B shows an embodiment of a cross sectional view of the integrated optical sensor 105 of the present invention, taken along the line 2B - 2B, i.e., for the measurement path 130.
  • the structure described here for the measurement path 130 is identical to the structure for the reference path. That view shows the input coupler 108, the measurement path 130 and sample 140, and the output coupler 110 of the waveguide 1 1 1 formed on the substrate 202.
  • the injected beam 133 which comes directly from the polarization modulator 103, can propagate through the input coupler 108 and the measurement path 130.
  • the out- coupled beam 134 propagates through the output coupler 110.
  • FIG. 2C shows a second embodiment of a detailed cross sectional view of the integrated optical sensor 105 taken along the line 2C - 2C, i.e., for the measurement path 130. It should be noted that the structure described here for the measurement path 130 is identical to the structure for the reference path.
  • FIG. 2C also shows sidewalls 206 and a well base 208, which are optionally provided on the substrate 202 to produce a well for holding, e.g., a liquid measurement sample 140.
  • the substrate 202 and the wave guiding layer, including the optical paths 130, 131 of the waveguide 1 1 1 can be made of, e.g., glass with a high refractive index, which can be a doped silicon glass, and, more particularly, silicon nitride (Si 3 N 4 ).
  • the input couplers 107, 108, the output couplers 109, 110, the sidewalls 206 and the well base 208 can be made of, e.g., silicon dioxide (Si0 2 ).
  • a waveguide 111 used in optical sensing applications generally has a refractive index (n) that is greater than the refractive index of the substrate 202 on which it is formed, thereby providing a refractive index difference that is large enough to substantially ensure total internal reflection of light propagating through the waveguide 111.
  • the waveguide 111 of the integrated optical sensor 105 preferably satisfies some additional requirements.
  • the measurement and reference paths 130 and 131 preferably support only the TEo and TMo modes, which modes can best detect and/or measure very small concentrations of substances in the measurement sample 140 using the evanescent wave surface detection technique.
  • the substances in the measurement sample 140 are detected and/or measured by measuring differences in the effective refractive indices ( ⁇ n) between the first and second optical paths 130, 131 for both polarized guided modes, TEo and TM 0 , i.e.,
  • ⁇ nTE nTE measurement - TE reference
  • ⁇ nTM nTM measurement - TM reference
  • the polarization modulator 103 causes to propagate sequentially through the measurement and reference paths 130, 131.
  • These differences are proportional to the penetration depths of the evanescent wave fields, corresponding with the TEo and TMo modes, into the measurement and reference samples 140, 142, which are contiguous to the measurement and reference paths 130, 131, respectively. Accordingly, the differences in the effective refractive indices of the modes, ⁇ nTE and ⁇ nTM, are measured with the highest resolution when both of the injected beams 132, 133 propagate through the measurement and reference paths 130, 131 in the TEo and TMo modes only.
  • the wave guiding layer used for propagating light through the integrated optical sensor 105 is preferably as optically uniform as possible. This reduces optical scattering and enhances the sensitivity of the platform 100.
  • At least one measurement sample 140 is located contiguous to the upper surface of the measurement path 130, e.g., approximately midway between the optical couplers 108, 110 and, a reference sample 142 is located contiguous to the upper surface of the reference path 131, e.g., approximately midway between the optical couplers 107, 109.
  • the measurement and reference samples 140, 142 can be liquid or gaseous samples that contain, respectively, known and unknown concentrations of biological or chemical substances. Furthermore, the respective upper surfaces of the measurement and reference paths 130, 131 can be coated with a known chemically or biologically sensitive layer so that the target analytes contained in the measurement and reference samples 140, 142 bind appropriately thereto.
  • a light source e.g., laser 102, injects light beams 132, 133 into the polarization modulator 103, which alternately and sequentially sends one of the two orthogonal, polarized modes, i.e., guided or excited modes, to each input coupler 107, 108 of the waveguide 111.
  • the respective modulated, in-coupled, guided beams are excited and propagate through the measurement and reference paths 130, 131.
  • the light beams 132, 133 are guided by total internal reflection alternately in the excited TEo and TMo modes through the paths 130, 131.
  • the evanescent wave fields corresponding with the TEo and TMo modes reach out into the substrate 202 and the target analytes within the measurement and reference samples 140, 142 that are bound to the upper surface of the measurement path 130 and reference path 131, respectively.
  • the guided beams then propagate to the output couplers 109, 110, which provide the corresponding out-coupled beams 135, 134 to the beam combiner 113.
  • the beam combiner 113 combines the beams from each optical path 130, 131, which is to say, that the reference pattern for a TEo mode is combined with a corresponding measurement pattern for the TEo mode and a reference pattern for the TMo mode is combined with a corresponding measurement pattern for the TMo mode.
  • the combined reference patterns are sent to the phase detector 139 for generating measurement data.
  • the measurement data generated by the phase detector 139 includes optical information relating to relative phase shifts of the TEo and TMo excited modes in the out-coupled beams 134, 135.
  • the phase detector 139 provides the measurement data to a computer 141 for deriving the above-mentioned set of first differential measurements and the second differential measurement.
  • the set of first differential measurements i.e., ⁇ n E and ⁇ nTM, are obtained, respectively, for each of the two polarized modes, TEo and TMo, which propagate sequentially through both the measurement and reference paths 130, 131.
  • ⁇ n E and ⁇ nTM the two polarized modes
  • TEo and TMo the two polarized modes
  • evanescent wave fields reach out into the substrate 202 and penetrate the bound samples 140, 142.
  • the difference in the refractive indices of the excited TEo mode i.e., ⁇ n E and ⁇ nTM
  • the set of first differential measurements i.e., ⁇ nTE and ⁇ nTM, derived from the two polarized guided modes, TEo and TMo, propagating through the measurement and reference paths 130, 131, has the effect of subtracting out biochemical instabilities, i.e., noise, resulting from non-specific binding, and also any external thermal dependence of
  • each polarized light beam propagating sequentially through the measurement and the reference paths 130, 131 outputs one of two orthogonal polarization components, S and P, wherein the S component is generated independently by the TEo mode and the P component is generated independently by the TMo mode.
  • the S component is linearly polarized parallel to the plane of the waveguide 1 11 and the P component is linearly polarized in the direction normal to the plane of the waveguide 111.
  • the independent measurements of the S and P components have a phase difference, ⁇ (t), between them that is dependent upon the relative concentrations of substances, e.g., microorganisms, in the measurement and reference samples 140, 142 located on, i.e., bound to, the upper surfaces of the measurement and the reference paths 130, 131, respectively.
  • ⁇ (t) is dependent upon the relative penetration depths of the evanescent wave fields corresponding with the TEo and TMo modes within the measurement and the reference paths 130, 131 into the measurement and the reference samples 140, 142.
  • the optical path lengths for the two polarization components, S and P are substantially equal and, further, that ⁇ (t) is not changed during the propagation.
  • a measurement sample 140 is provided with a variable concentration of target analytes, which analytes are bound to the surface of the measurement path 130 while a reference sample 142 is provided with a specific concentration of target analytes, which analytes are bound to the surface of the reference path 131.
  • Each of the measurement and reference samples 140, 142 also includes a quantity of non-specific binding elements.
  • the light beams propagating through the measurement and the reference paths 130, 131 are outputted as beams 134, 135 by the output couplers 110, 109, combined by the beam combiner 113, and the relative phase shifts of the TEo and TMo modes in out-coupled beams 134, 135 (due to the penetration of the evanescent wave fields into the target analytes within the measurement and reference samples 140, 142 that are bound to the upper surface of the measurement and reference paths 130, 131) are detected and measured by the phase detector 139, which provides the measurement data to a computer 141 for deriving the above-mentioned set of first differential measurements and the second differential measurement.
  • the set of first differential measurements i.e., ⁇ nTE and ⁇ nTM
  • the first differential measurement optically subtracts out the biochemical instabilities resulting from the nonspecific binding, thereby measuring only the concentration of target analytes in the measurement sample 140 relative to the concentration of target analytes, if any, in reference sample 142.
  • immunity i.e., lack of sensitivity, to environmental effects, such as temperature changes and mechanical vibrations, and biochemical effects, such as non-specific binding, advantageously makes the optical sensing platform 100 of the present invention highly stable with a significantly higher system SNR than conventional systems.
  • preferred embodiments of the present invention can produce a degree of accuracy that is at least about an order of magnitude greater than devices of the prior art.
  • the propagation modulator 103 effectively filters out low frequency, e.g., less than about 1000 Hertz, perturbations from noise sources, thereby significantly increasing the system signal-to-noise ratio (SNR).
  • SNR system signal-to-noise ratio
  • FIG. 3 shows a detailed view of an embodiment of the phase detector 139, which preferably includes a fringe imaging lens 302, a fringe detector 304, and a processor 306 that produces a phase value in digital format and provides the value to the computer 141 for data logging and/or subsequent processing.
  • the beam combiner 113 combines the out-coupled beams
  • the fringe-imaging lens 302 focuses the optical interference pattern onto the fringe detector 304, which is, e.g., a matched photodiode array detector or an un-matched CCD detector. Because the integrated optical sensor 105 preferrably supports the TEo and TMo modes at ⁇ equal to about 0.4 to 1.0 ⁇ m, the matched photodiode array can be suitably implemented as a silicon array.
  • the fringe detector 304 selectively samples the optical interference pattern to detect any translational shifts in fringe positions relative to the overall interference pattern. These translational shifts are proportional to changes in the relative phase, ⁇ (t). As described above, these phase shifts, are caused by and are proportional to the penetration of the evanescent wave fields into the concentration of bound analytes within the measurement and reference samples 140, 142.
  • the optical phase detector 304 can be constructed using a linear array of photodetectors.
  • a spatial heterodyne fringe pattern is focused onto the detector array without regard to the number of pixels sampling a complete fringe.
  • Each detector pixel is sampled and the respective signals are processed.
  • the processing typically consists of calculating the amplitude and conjugate phase for each spatial frequency of the sampled imaged interference pattern. In a preferred embodiment, this can be accomplished by calculating the Fourier transform of the array of sampled light intensities, identifying the frequency of the fringe pattern, and recording its corresponding phase value, ⁇ . This recorded phase value is equal to the optical phase, ⁇ , of the interferometer.
  • FIG. 5a shows an exemplary graph, which is shown as phase (in cycles) versus time, of typical results from the first set of differential measurements. Indeed, FIG 5a shows the amplitude and conjugate phase for each spatial frequency, i.e., TE and TM, of the sampled imaged interference pattern.
  • FIG. 5b shows, further, an exemplary graph of a typical phase-time relationship using doubly differentiated results.
  • the frequency of the interference pattern is carefully matched with the spacing of the detector pixels.
  • the intensity (I) sampled by a pixel is given by
  • I(x) I DC + I Ac cos ( ⁇ + ⁇ N )
  • ⁇ is the optical phase and ⁇ N is the position of the sampled pixel with respect to spatial frequency of the interference pattern.
  • one complete fringe can be sampled by four detector pixels as describe by Helmers et al., Applied Optics, 35(4), p 676, 1996, which is incorporated herein by reference.
  • ⁇ N is equal to 0, ⁇ /2, ⁇ , and 3 ⁇ /2.
  • one complete fringe can be sampled by three detector pixels as described by US Pat. 5,530,543 to Hercher, here ⁇ N is equal to 2 ⁇ /3, 0, and -2 ⁇ /3.
  • the preferred phase detector 139 has the capability of recovering relative phase information, ⁇ (t), at a level of precision equal to about 10 7 cycles/VHz. This is an important advantage for the detection and/or measurement of small molecules, bio-molecules, and/or microorganisms because it provides the high sensitivity necessary for rapidly and directly detecting, e.g., a single bound target analyte, which is typically a very time consuming task using conventional detection techniques, and for the discrimination or binding of small molecules to significantly larger complexes.
  • the preferred optical sensing platform 100 of the present invention therefore has both the sensitivity and speed required for directly detecting and/or measuring substances such as small molecules, bio- molecules and/ or microorganisms in real time applications.
  • the optical sensing platform 100 of the present invention includes an integrated optical sensor 105 with a measurement path 130 and a reference path 131.
  • the optical sensing platform 100 alternatively can be configured as a Mach-Zehnder interferometer, an embodiment of which is disclosed in, e.g., U.S. Patent 4,515,430 to Johnson.
  • a Mach-Zehnder interferometer includes structure for bifurcating a light beam into two separate and distinct optical paths, and then combining the two light beams. Hence, a Mach-Zehnder- type interferometer configuration can be used for performing the double differential measurements required by the present invention.
  • Channel-type waveguides which are well known to those of ordinary skill in the art, also can be used. However, it should be understood that because the guided beams propagating through the optical paths of some prior art channel-type waveguides are not unconfined in the lateral dimension as in a preferred embodiment of the integrated optical sensor 105 shown in FIG. 1 , the scattering of the light beams within the guiding layer is not optimally minimized. Therefore, channel-type waveguides may not provide the optimal level of sensitivity for measuring very small numbers of targets in the measurement sample 140.
  • suitable optical fibers can be used for injecting the light beams 132, 133 into the propagation modulator 103 and/or as a waveguide.
  • the respective light beams alternatively can be injected into the propagation modulator 103 via propagation in free-space.
  • the out-coupled light beams 134, 135 can be provided to the beam combiner 113 via propagation in free-space.
  • the analytes in the measurement and reference samples 140, 142 can be bound to the upper surfaces of the measurement and reference paths 130, 131 using a chemically or biologically sensitive layer.
  • certain compatible probes e.g., antibody or nucleic acid probes
  • the probes can be attached to the upper surface of the measurement path 130, e.g., either covalently or by using an intermediate linker.
  • suitable probes can be used for binding specific concentrations of, e.g., small molecules, bio-molecules, and/or microorganisms to the upper surface of the reference path 131.
  • the reference path 131 advantageously serves as both a biochemical and an environmental reference, thereby subtracting out interfering biological and/ or chemical binding and removing extraneous temperature effects.
  • the optical sensing platform 100 of the present invention includes an integrated optical sensor 105. However, this was also merely an illustrative example.
  • the optical sensing platform 100 alternatively can be implemented using a plurality of separate waveguides 111. Further, it was described that the waveguide 111 used with the optical sensing platform 100 includes one measurement path 130 and one reference path 131. However, this was merely an illustrative example.
  • the optical sensing platform 100 of the present invention alternatively can be implemented with more than one measurement path 130 and/ or more than one reference path 131, depending upon the requirements of the optical sensing application.

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Abstract

L'invention concerne une plate-forme de détection (100) à grande vitesse, à sensibilité élevée, ainsi qu'un procédé destiné à détecter et/ou à mesurer des caractéristiques d'une substance dans un échantillon (140) à mesurer. La plate-forme comprend au moins une paire de chemins optiques (130, 131) formés dans un guide d'ondes (111), une source de rayonnement lumineux (102) destinée à injecter des faisceaux optiques le long des chemins optiques, un modulateur de rayonnement lumineux (103) permettant d'exciter des ondes de guide électrique et magnétique transversales, et un détecteur de phase (139) destiné à détecter des différences de phase entre les faisceaux qui se propagent le long des chemins optiques.
PCT/US2001/007672 2000-03-13 2001-03-12 Plate-forme a grande vitesse, a sensibilite elevee, pour des applications de detection de surface d'onde evanescente WO2001069174A1 (fr)

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AU2001249143A AU2001249143A1 (en) 2000-03-13 2001-03-12 High speed, highly sensitive platform for evanescent wave surface detection applications

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
GB2409034A (en) * 2003-12-12 2005-06-15 Partha Ghose Interaction-free object detection
NL2003743A (en) * 2009-11-02 2011-04-04 Ostendum Holding B V Method for detection of an analyte in a fluid sample.

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US5120131A (en) * 1988-02-14 1992-06-09 Walter Lukosz Method and apparatus for selecting detection of changes in samples by integrated optical interference
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2409034A (en) * 2003-12-12 2005-06-15 Partha Ghose Interaction-free object detection
NL2003743A (en) * 2009-11-02 2011-04-04 Ostendum Holding B V Method for detection of an analyte in a fluid sample.
WO2011053147A1 (fr) * 2009-11-02 2011-05-05 Ostendum Holding B.V. Procédé de détection d'un analyte dans un échantillon de liquide
CN102713578A (zh) * 2009-11-02 2012-10-03 奥斯坦德姆控股有限公司 液体样品中的分析物的检测方法

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