Patent Publication Number: US-2002003627-A1

Title: Doubly-differential interferometer and method for evanescent wave surface detection

Description:
CROSS REFERENCE OR RELATED APPLICATIONS  
     [0001] This application claims the benefit of U.S. Provisional Application No. 60/188,808 filed Mar. 13, 2000. 
    
    
     
       BACKGROUND OF THE INVENTION  
       [0002] 1. Field of the Invention  
       [0003] 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.  
       [0004] 2. Discussion of Related Art  
       [0005] 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.  
       [0006] 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.  
       [0007] For example, the detection and/or measurement of very small numbers of microorganisms in a sample, using evanescent wave surface detection techniques, 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. However, 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.  
       [0008] One such evanescent wave surface detection technique uses fluorescent markers for detecting and/or measuring substances in a liquid or gaseous sample. Specifically, targets, i.e., analytes, within a sample, which are bound to the surface of a waveguide, are tagged, or labeled, with fluorescent markers.  
       [0009] Light is coupled into the waveguide. As the light propagates through the waveguide, 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&#39;s fluorescence.  
       [0010] An evanescent wave surface detection technique that uses fluorescent markers, however, 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.  
       [0011] 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 BIAcore™ surface plasmon resonance (SPR) instrument manufactured by Amersham Pharmacia Biotech AB, Uppsala, Sweden.  
       [0012] Specifically, 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.  
       [0013] However, an evanescent wave surface detection technique used with the SPR instrument has some shortcomings. For example, 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. Furthermore, the BIAcore™ brand SPR instrument is commercially expensive; hence, it is often inappropriate for use in low-cost applications.  
       [0014] Still another evanescent wave surface detection technique for detecting and/or measuring untagged substances in a bound sample is disclosed in U.S. Pat. No. 5,120,131 (the “&#39;131 patent”) to Lukosz. According to that disclosed invention, 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. Specifically, light is coupled into the waveguide so that two mutually coherent, orthogonally polarized modes propagate through the waveguide simultaneously and coaxially. As a result of the interaction between the propagated light and the bound sample, 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.  
       [0015] However, the evanescent wave surface detection technique disclosed in the &#39;131 patent has some shortcomings. For example, 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”). Moreover, thermal and mechanical perturbations, which are major sources of noise and which adversely affect the SNR.  
       [0016] It would be desirable, therefore, to provide 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. Moreover, such 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. It would also be desirable to have an evanescent wave surface detection technique and device for detecting and/or measuring small numbers of small molecules, biomolecules, and/or microorganisms in a sample that provides results quickly and can be implemented at relatively low cost. Furthermore, it would be desirable to have an evanescent wave surface detection technique and device that removes noise associated with thermal and mechanical perturbations to maximize the SNR.  
       SUMMARY OF THE INVENTION  
       [0017] 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.  
       [0018] In a preferred embodiment, 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. These 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.  
       [0019] According to a preferred embodiment of the present invention, 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. Specifically, 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.  
       [0020] More specifically, the set of first differential measurements is obtained by first determining changes in the respective effective refractive indices (δn) 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., δn TE  and δn TM , which, because of modulation, propagates through each of the optical paths sequentially rather than simultaneously. Further, the second differential measurement is obtained by determining an overall change in the effective refractive indices (ΔN) between the respective polarized guided modes, i.e., ΔN=δn TE -δn TM . Changes in the effective refractive indices of the first and second optical paths are caused by (a) the characteristics of the target analytes bound to the measurement and reference surfaces of the waveguide and also (b) by the polarized guided mode propagating through the waveguide.  
       [0021] Advantageously, this doubly differential surface detection technique, as implemented on the optical sensing platform of the present invention, 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.  
       [0022] Because change in refractive index (δn) is dependent on the polarization of the light, the optical response of the polarized light is different for each of the orthogonally guided modes that propagate within the waveguide. Modulating the polarization of the incident light enables exciting two guided modes at modulation frequencies above that of a predetermined unwanted noise distribution. Moreover, subtracting temporally adjacent measurements in the reference and measurement samples results in the common-mode rejection of in-band noise. Thus, noise due to thermal and mechanical perturbations, which is generally restricted to temporal frequencies similar to those of the target signal (less than about a few thousand Hertz) is substantially eliminated, significantly improving the SNR.  
       [0023] Still further aspects and advantages of the present invention will become apparent from a consideration of the ensuing description and drawings. 
     
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
     [0024] The invention will be better understood by reference to the following more detailed description and accompanying drawings, wherein like elements correspond to like elements, in which:  
     [0025]FIG. 1 illustrates a block diagram of an embodiment of an optical sensing platform according to the present invention;  
     [0026]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  2 A- 2 A;  
     [0027]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  2 B- 2 B;  
     [0028]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  2 C- 2 C;  
     [0029]FIG. 3 illustrates a block diagram of an embodiment of the phase detector used with the optical sensing platform of FIG. 1;  
     [0030]FIG. 4 shows the dramatic effect of polarization modulation on power spectral density; and  
     [0031]FIG. 5 shows an embodiment of modulated time-phase data.  
    
    
     DETAILED DESCRIPTION OF THE INVENTION INCLUDING THE PREFERRED EMBODIMENTS THEREOF  
     [0032] As shown in FIG. 1, 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 .  
     [0033] 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 . Moreover, 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.  
     [0034] Modulating the polarization of the incident beams  132 ,  133 , alternately exciting the TE m  and TM m  guided modes at modulation frequencies above that of a predetermined unwanted noise distribution and subtracting adjacent measurements, e.g., from the reference sample  142 , results in the common-mode rejection of the low frequency noise sources. 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. Thus, obtaining the desired information from a differential measurement and employing active commonmode rejection techniques significantly suppresses these external disturbances to increase the system signal-to-noise ratio (SNR). 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.  
     [0035] The dramatic effect of polarization modulation is shown in FIG. 4, which shows the noise power spectrum for a typical interferometric system. Without modulation, the curve  42  (shown in FIG. 4 as a dashed line) exhibits significantly higher noise power at low frequencies, typically below about 1000 Hertz. With polarization modulation, however, the curve  41  (shown in FIG. 4 as a straight line) is virtually constant at all frequencies, including in-band frequencies, i.e., frequencies within a desirable bandwidth. Indeed, polarization modulation filters out low-frequency noise (shown in FIG. 4 as the hatched region), typically due to thermal and mechanical perturbations, as would a lowpass filter if applied to the entire spectrum.  
     [0036] This common-mode rejection technique is readily applied to an interferometric evanescent wave biosensor. In such a device 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. Moreover, the optical response for S- and P- polarized light (respectively propagated as TE and TM spatial modes within a waveguide  111 ) is different, however, many externally applied perturbations are independent of polarization. By modulating the polarization of the incident light at a rate at least two times the signal bandwidth to excite alternatively the TE and TM guided modes and subtracting corresponding values of  φTE  and  φTM  in time, where φ is the optical phase, in-band perturbations common to both  φTE  and  φTM  is removed leaving only the desired signal and thus a robust system.  
     [0037] The modulated light then passes into input couplers  107 ,  108  of a waveguide  111 , 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  113 . 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.  
     [0038] 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). Preferably, 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 . Further, 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 .  
     [0039] For example, 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. For example, 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.  
     [0040] Preferably, 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 . In one embodiment, 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 . However, it should be understood that non-rectilinear sections also can be used without deviating from the scope and spirit of this disclosure. Because 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.  
     [0041] It should be noted that 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.  
     [0042] In preferred embodiments, 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. Preferably, using the above-described optical sensing platform  100 , the light source  102 , e.g., laser, injects beams  132 ,  133  into input couplers  107 ,  108 , respectively, via the polarization modulator  103 , which enables exciting a plurality of orthogonally polarized spatial modes. 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  111 . 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 TE 0  and TM 0  modes, which modes are modulated to propagate independently and sequentially.  
     [0043] As described in greater detail later in this specification, the doubly differential surface detection technique of the present invention then is used to derive a set of first differential measurements, e.g.,  n TE  and  n TM , for each of the two polarized guided modes propagating sequentially through each of the measurement and reference paths  130 ,  131 , i.e.,  
         n   TE   =n   TE measurement   −n   TE reference    and  n   TM   =n   TM measurement   −n   TM reference ,  
     [0044] and a second differential measurement (ΔN) from a combination of the first differential measurements for each guided mode, i.e.,  
       ΔN= n   TE   − n   TM .  
     [0045] The differential measurements preferably are determined using a programmed microprocessor receiving the data from the fringe detector  304 .  
     [0046] Such a microprocessor is programmed readily by a normally skilled programmer.  
     [0047] This doubly differential surface detection technique, as implemented on the optical sensing platform  100 , 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 . Indeed, the sensitivity of the disclosed invention can measure even a single bound molecule or pathogen in a measurement sample  140 .  
     [0048] As mentioned above, in one embodiment, the measurement path  130  and the reference path  131  can be rectilinear sections. In this embodiment, the rectangular cross sectional dimensions of the optical paths  131 ,  130 , and the wavelength, λ, of the injected beams  132 ,  133 , are specified so that two orthogonally polarized waveguide modes, i.e., TE m  and TM m , are allowed to propagate sequentially through each path  131 ,  130 . Further, each of the in-coupled, guided beams preferably propagates through the waveguide  111  of the integrated optical sensor  105  as TE 0  and TM 0  modes.  
     [0049]FIG. 2A shows an embodiment of a cross sectional view of the integrated optical sensor  105  of the present invention, taken along the line  2 A- 2 A. 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.  
     [0050]FIG. 2B shows an embodiment of a cross sectional view of the integrated optical sensor  105  of the present invention, taken along the line  2 B- 2 B, 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. That view shows the input coupler  108 , the measurement path  130  and sample  140 , and the output coupler  110  of the waveguide  111  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 .  
     [0051]FIG. 2C shows a second embodiment of a detailed cross sectional view of the integrated optical sensor  105  taken along the line  2 C- 2 C, 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 .  
     [0052] The substrate  202  and the wave guiding layer, including the optical paths  130 ,  131  of the waveguide  111  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 (SiO 2 ).  
     [0053] 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 .  
     [0054] Furthermore, in order to provide 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 the measurement sample  140 , the waveguide  111  of the integrated optical sensor  105  preferably satisfies some additional requirements. For example, the measurement and reference paths  130  and  131  preferably support only the TE 0  and TM 0  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.  
     [0055] The substances in the measurement sample  140  are detected and/or measured by measuring differences in the effective refractive indices (δn) between the first and second optical paths  130 ,  131  for both polarized guided modes, TE 0  and TM 0 , i.e.,  
     
       
       n 
       TE 
       =n 
       TE 
       measurement 
       −n 
       TE reference  
       and  
     
         n   TM   =n   TM measurement   −n   TM reference ,  
     [0056] which 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 TE 0  and TM 0  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, δn TE  and δn TM , 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 TE 0  and TM 0  modes only.  
     [0057] 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 .  
     [0058] The doubly differential surface detection technique of the present invention, implemented using the optical sensing platform  100 , will now be described in detail as follows. Initially, 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 .  
     [0059] As mentioned above, 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.  
     [0060] 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 . Specifically, the light beams  132 ,  133  are guided by total internal reflection alternately in the excited TE 0  and TM 0  modes through the paths  130 ,  131 . As a result, the evanescent wave fields corresponding with the TE 0  and TM 0  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.  
     [0061] 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 TE 0  mode is combined with a corresponding measurement pattern for the TE 0  mode and a reference pattern for the TM 0  mode is combined with a corresponding measurement pattern for the TM 0  mode. The combined reference patterns are sent to the phase detector  139  for generating measurement data.  
     [0062] Preferably, the measurement data generated by the phase detector  139  includes optical information relating to relative phase shifts of the TE 0  and TM 0  excited modes in the out-coupled beams  134 ,  135 . Finally, 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.  
     [0063] The set of first differential measurements, i.e., δn TE  and δn TM , are obtained, respectively, for each of the two polarized modes, TE 0  and TM 0 , which propagate sequentially through both the measurement and reference paths  130 ,  131 . For example, as the TE 0  polarized mode propagates through each optical path  130 ,  131  of the waveguide  111 , 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 TE 0  mode,  
         n   TE   =n   MEASUREMENT   −n   REFERENCE ,  
     [0064] changes as a result of the respective penetration of the evanescent wave fields into the bound target analytes within the measurement and reference samples  140 ,  142 . Likewise, as the TM 0  polarized mode propagates through each optical path  130 ,  131  of the waveguide  111 , 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 TM0 mode,  
       n TM   =n   MEASUREMENT   −n   REFERENCE ,  
     [0065] also changes as a result of the respective penetration of the evanescent wave fields into the bound target analytes within the measurement and reference samples  140 ,  142 . The set of first differential measurements, i.e., δn TE  and δn TM , derived from the two polarized guided modes, TE 0  and TM 0 , 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 δn TE  and δn TM .  
     [0066] These changes in the refractive indices, i.e., δn TM  and δn TE , correspond with the above-mentioned set of first differential measurements and are provided by a suitably programmed computer  141 , e.g., a microprocessor, using the measurement data provided by the phase detector  139 . There are many ways to detect the optical phase change, e.g., by using an array detector spatially matched to an optical interference pattern or by using an unmatched, charge-couple device (“CCD”) detector, both of which are well-known to those of ordinary skill in the art. The advantages and disadvantages of these two detectors are reciprocal. Indeed, a CCD detector benefits from having fewer interference pattern alignment problems but requires more difficult data processing. A matched array detector, on the other hand, is more difficult to align, but data processing is simplified.  
     [0067] Very small concentrations of substances, e.g., small molecules, bio-molecules, and/or microorganisms, in the measurement sample  140  can be detected and/or measured by measuring an overall change in the effective refractive indices corresponding to each polarized guided mode, i.e., ΔN=δn TE −δn TM . This overall change in the effective refractive indices, δN, corresponds to the above-mentioned second differential measurement and is proportional to relative difference in the individual phase shifts of the TE 0  and TM 0  polarized guided modes in the out-coupled beams  134 ,  135 .  
     [0068] Specifically, 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 TE 0  mode and the P component is generated independently by the TM 0  mode. The S component is linearly polarized parallel to the plane of the waveguide  111  and the P component is linearly polarized in the direction normal to the plane of the waveguide  111 .  
     [0069] The independent measurements of the S and P components have a phase difference, δΦ(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. Specifically, δΦ(t) is dependent upon the relative penetration depths of the evanescent wave fields corresponding with the TE 0  and TM 0  modes within the measurement and the reference paths  130 ,  131  into the measurement and the reference samples  140 ,  142 .  
     [0070] It is known to those of ordinary skill in the art that, for the propagation of light through non-birefringent media, e.g., many of the optical elements described above that make up the optical sensing platform  100 , the optical path lengths for the two polarization components, S and P, are substantially equal and, further, that  Φ(t) is not changed during the propagation. This means that, in the absence of target analytes within samples  140 ,  142  bound to the upper surfaces of the measurement and reference paths  130 ,  131 , the  Φ(t) between the S and P components is the same everywhere in the waveguide  111 , even in the presence of inherent thermal and mechanical perturbations. The second differential measurement, i.e., ΔN=n TEl − n   TM , also relates to the phase difference,  Φ(t), between the S and P components.  
     [0071] As an illustrative example, 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.  
     [0072] Accordingly, 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 TE 0  and TM 0  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.  
     [0073] Specifically, the set of first differential measurements, i.e., δn TE  and δn TM , optically subtracts out the biochemical instabilities and any external thermal dependence; and, the second differential measurement, i.e., ΔN=δn TE −δn TM , optically subtracts out the inherent instabilities due to temperature changes and mechanical vibrations, which are common to both guided modes. More specifically, the first differential measurement optically subtracts out the biochemical instabilities resulting from the non-specific 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 . Thus, 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. Indeed, 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.  
     [0074] Moreover, 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).  
     [0075]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.  
     [0076] Preferably, the beam combiner  113  combines the out-coupled beams  134 ,  135 , producing a combined beam  137 , from which produces an optical interference pattern, commonly known as a fringe or a spatial heterodyne pattern, which generally includes a series of uniform, sinusoidally distributed, light and dark areas. 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 unmatched CCD detector. Because the integrated optical sensor  105  preferrably supports the TE 0  and TM 0  modes at λ equal to about 0.4 to 1.0 μm, the matched photodiode array can be suitably implemented as a silicon array.  
     [0077] Next, 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, δΦ(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 .  
     [0078] For example, the optical phase detector  304  can be constructed using a linear array of photodetectors. In one method, e.g., 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, Φ. This recorded phase value is equal to the optical phase, Φ, of the interferometer.  
     [0079]FIG. 5 a  shows an exemplary graph, which is shown as phase (in cycles) versus time, of typical results from the first set of differential measurements.  
     [0080] Indeed, FIG. 5 a  shows the amplitude and conjugate phase for each spatial frequency, i.e., TE and TM, of the sampled imaged interference pattern. FIG. 5 b  shows, further, an exemplary graph of a typical phase-time relationship using doubly differentiated results.  
     [0081] In a second method, 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(φ+Δ N )  
     [0082] where φ is the optical phase and AN is the position of the sampled pixel with respect to spatial frequency of the interference pattern.  
     [0083] In one embodiment, 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. Here Δ N  is equal to 0, π/2, π, and 3π/2. In another embodiment, one complete fringe can be sampled by three detector pixels as described by U.S. Pat. No. 5,530,543 to Hercher, here Δ N  is equal to 2π/3, 0, and −2π/3. A distinct advantage of the first method is nominal constraints on alignment compared to the second method. In contrast, the corresponding processing requirements are significantly greater compared with the second method.  
     [0084] The preferred phase detector  139  has the capability of recovering relative phase information, δΦ(t), at a level of precision equal to about 10 −7  cycles/{square root}Hz. 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, biomolecules and/or microorganisms in real time applications.  
     [0085] It can be appreciated that numerous alternative embodiments or variations can be made. For example, it was described that 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 . However, this was merely an illustrative example, and other configurations are available. For example, 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. Pat. No. 4,515,430 to Johnson.  
     [0086] 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.  
     [0087] 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 .  
     [0088] It was described that suitable optical fibers can be used for injecting the light beams  132 ,  133  into the propagation modulator  103  and/or as a waveguide. However, this was also merely an illustrative example. The respective light beams alternatively can be injected into the propagation modulator  103  via propagation in free-space. Similarly, the out-coupled light beams  134 ,  135  can be provided to the beam combiner  113  via propagation in free-space.  
     [0089] It was described that 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. It was also described that wells can be formed in the surface of the integrated optical sensor  105  for holding the measurement and reference samples  140 ,  142 . However, these were merely illustrative examples. Specifically, because the optical sensing platform  100  of the present invention is particularly useful for detecting and/or measuring target analytes in a measurement sample  140 , a plurality of highly specific probes alternatively can be immobilized on the surface of the measurement path  130 .  
     [0090] For example, certain compatible probes, e.g., antibody or nucleic acid probes, can be used for binding variable concentrations of small molecules, bio-molecules or microorganisms in the measurement sample  140  to the upper surface of the measurement path  130 . Further, the probes can be attached to the upper surface of the measurement path  130 , e.g., either covalently or by using an intermediate linker.  
     [0091] Similarly, 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 . In this way, 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.  
     [0092] It was described that 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.  
     [0093] The present invention has been described in detail including the preferred embodiments thereof. However, it should be appreciated that those skilled in the art, upon consideration of the present disclosure, may make modifications and/or improvements of this invention that are within the scope and spirit of this invention as set forth in the following claims.