Abstract:
A device, including sample and reference channels through which first and second solutions flow, respectively, the first solution including an analyte, the channels having a metal film in contact with the first and second solutions, the metal film configured with a linker to selectively bind the analyte; a light source whose output is modulated by an optical system, so that light is directed from the optical system alternately towards the sample and reference channels, surface plasmons within the metal film being created; a first photodetector that monitors the strength of the output from the light source; a second photodetector that collects optical signals reflected from the metal film; electronics that monitors output from the first and the second photodetectors, thereby detecting a noise-compensated difference in signals from the two channels; and a computer processor that determines, from analysis of the noise-compensated difference, presence of the analyte in the first solution.

Description:
[0001]    This invention was made with government support under ECCS-0823827 awarded by the National Science Foundation. The government has certain rights in the invention. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates to a method of optical sensing, and an associated apparatus, using surface plasmon resonance (SPR) to achieve ultrasensitive interfacial detection of gas and solution-phase biological and chemical analytes. 
       BACKGROUND OF THE INVENTION 
       [0003]    Biosensors directed towards disease and cancer detection often target biomarkers such as proteins that are not present in healthy individuals. The buildup of these biomarkers correlates with the progression of the particular disease. Unfortunately, the number of available treatment options and the overall prognosis decrease considerably as the disease progresses from early to advanced stages. Further, many diseases are asymptomatic until the advanced stages, at which point options are limited. For these reasons, early detection is critical for successful treatment. 
         [0004]    The gold standard for biosensing has been ELISA (enzyme-linked immunosorbent assay), which is utilized in commercial products such as pregnancy tests, as well as for detection of antibiotics in milk and the presence of salmonella. Similarly, sandwich assays can be performed with fluorescently-labeled secondary antibodies. In both cases, however, antibody labeling is a requirement. While these labeled methods typically provide adequate signal-to-noise ratio, the production of labeled antibodies is expensive, time consuming, and can influence molecular binding. Further, since the sandwich assay requires multiple incubation steps, these assays do not permit real-time detection. 
         [0005]    Many techniques have been developed to overcome the shortcomings of bio sensors that require molecular labeling. These techniques typically probe the interfacial binding of a particular protein by means of changes in interfacial refractive index or mass. Surface plasmon resonance (Knoll, W., Interfaces and thin films as seen by bound electromagnetic waves,  Annual review of physical chemistry  1998, 49 (1), 569-638) and ellipsometry (Ostroff, R. M.; Maul, D.; Bogart, G. R.; Yang, S.; Christian, J.; Hopkins, D.; Clark, D.; Trotter, B.; Moddel, G., Fixed polarizer ellipsometry for simple and sensitive detection of thin films generated by specific molecular interactions: applications in immunoassays and DNA sequence detection,  Clin Chem  1998, 44 (9), 2031-2035) are common techniques for monitoring interfacial refractive index, while quartz crystal microbalance (Liss, M.; Petersen, B.; Wolf, H.; Prohaska, E., An Aptamer-Based Quartz Crystal Protein Biosensor,  Analytical chemistry  2002, 74 (17), 4488-4495) measures interfacial mass change. A number of different modifications have been proposed to reduce the noise in surface plasmon resonance (SPR) sensors such as polarization interferometry (Sun, Z. L.; He, Y. H.; Guo, J. H., Surface plasmon resonance sensor based on polarization interferometry and angle modulation,  Applied Optics  2006, 45 (13), 3071-3076) and differential phase change (Wu, S.; Ho, H.; Law, W.; Lin, C.; Kong, S., Highly sensitive differential phase-sensitive surface plasmon resonance biosensor based on the Mach-Zehnder configuration,  Optics letters  2004, 29 (20), 2378-2380). A very low (or possibly the lowest known) detection limit for SPR based biosensing has been demonstrated by Li et al. (Li, Y. C.; Chang, Y. F.; Su, L. C.; Chou, C., Differential-phase surface plasmon resonance biosensor,  Analytical chemistry  2008, 80 (14), 5590-5595). Li et al. use differential-phase-sensitive surface plasmon resonance to monitor the interaction between mouse IgG and antimouse IgG at 67 attomolar concentration. While this is an impressive achievement, there are a number of disadvantages with the experimental setup of Li et al. One important disadvantage relates to the sensitivity of the apparatus. It is well known that for phase-sensitive SPR measurements, the largest sensitivity to phase change occurs at the minimum angle of reflectivity. However, operating at minimum reflection where only a small percentage of photons reach the detector severely reduces the signal-to-noise ratio. Operating outside of the minimum reflection or changing the gold thickness can increase reflectivity but only at the expense of the phase sensitivity. Increasing laser power is also not a viable option, since the adsorbed photons dissipate as heat in the dielectric material of interest and at high powers can create temperature gradients in the sample. Further, phase measurements are sensitive to the roughness of the gold film. Achieving ultrasmooth gold films and substrates for gold deposition can be a daunting task that is required for this configuration to maintain the optimum sensitivity. In addition, there is an extremely narrow dynamic range for phase detection. Thus, only extremely low interfacial concentrations or smaller molecules can be measured. For real-world applications, such as detection in serum, non-specific adsorption alone would likely cause departure from the usable detection range. 
       SUMMARY OF THE INVENTION 
       [0006]    The present invention provides a device, comprising: 
         [0007]    sample and reference channels through which first and second solutions flow, respectively, wherein the first solution includes an analyte of interest, the channels having a metal film in contact with the first and second solutions, a surface of the metal film configured with a linker to selectively bind the analyte to the surface of the metal film; 
         [0008]    a light source whose output is modulated by an optical system, so that light is directed from the optical system alternately towards the sample and reference channels, wherein surface plasmons within the metal film are created; 
         [0009]    a first photodetector that monitors the strength of the output from the light source; 
         [0010]    a second photodetector that collects optical signals reflected from the metal film; 
         [0011]    electronics that monitors output from both the first and the second photodetectors, thereby detecting a noise-compensated difference in signals from the two channels; and 
         [0012]    a computer processor that determines, from analysis of the noise-compensated difference, that the analyte is present in the first solution. 
         [0013]    The present invention provides an apparatus for detecting an analyte, said apparatus comprising: 
         [0014]    a reference channel through which a reference fluid is flowing; 
         [0015]    a sample channel through which a sample fluid is flowing, said sample fluid comprising the reference fluid and the analyte, said reference fluid comprising a molar concentration of the analyte that is no more than 50% of the analyte that is in the sample fluid, said reference channel and said sample channel being different channels; 
         [0016]    a metal layer in contact with the sample fluid and the reference fluid, a surface of the metal layer configured with a linker to selectively bind the analyte to the surface of the metal layer; 
         [0017]    an optical system; 
         [0018]    a reference photodetector coupled to the optical system; 
         [0019]    a sample photodetector; 
         [0020]    noise reduction electronics coupled to the reference photodetector and the sample photodetector; 
         [0021]    a lock-in amplifier coupled to the noise reduction electronics; and 
         [0022]    a computer processor coupled to the lock-in amplifier; 
         [0023]    said optical system configured to receive a scanning beam from a laser, said scanning beam comprising laser noise generated in the laser; 
         [0024]    said optical system configured to split the scanning beam into a reference beam and a sample beam, said sample beam and said reference beam each comprising the laser noise; 
         [0025]    said optical system configured to direct the reference beam to the reference photodetector causing the reference photodetector to send a resultant reference signal containing the laser noise to the noise reduction electronics; 
         [0026]    said optical system configured to direct the sample beam alternately toward the sample channel and the reference channel under conditions where surface plasmon resonance (SPR) occurs in the metal layer, said directed sample beam being alternately reflected from the surface of the metal layer at the sample and reference channels; 
         [0027]    said optical system configured to direct the reflected sample beam to the sample photodetector causing the sample photodetector to send a resultant sample signal containing the laser noise to the noise reduction electronics; 
         [0028]    said noise reduction electronics configured to (i) implement a reduction of the laser noise from the sample signal via utilization of the reference signal and (ii) generate an output signal comprising the sample signal after the laser noise has been removed from the sample signal; 
         [0029]    said lock-in amplifier configured to (i) lock in to the output signal from the noise reduction electronics and (ii) determine, from processing different portions of cycles of the output signal from the noise reduction electronics, a difference in amplitude (ΔA) between the alternately directed beams reflected at the metal layer, said ΔA being determined after the laser noise has been cancelled from the sample signal; 
         [0030]    said computer processor configured to determine, from analysis of the difference in amplitude (ΔA) determined by the lock-in amplifier, that the analyte is present in the sample fluid flowing in the sample channel. 
         [0031]    The method of optical sensing, and an associated apparatus, of the present invention offers the advantage of being both label-free (e.g., free of fluorescent tags) and having considerably lower baseline noise than alternative label-free techniques. Since detection is directly related to the signal to noise ratio, reduction of the noise floor increases precision and can be used to achieve detection at the lower concentrations present during early stages of disease. Further, the method and apparatus of the present invention can be used in a unique self-referencing configuration to eliminate the undesired contribution of non-specific interfacial adsorption (e.g., serum proteins), making the apparatus particularly useful for ultrasensitive measurements in the presence of complex media (e.g., blood). 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0032]      FIG. 1A  depicts all specific and non-specific interactions from an interfacial measurement using a surface plasmon resonance (SPR) sensor with modulation, in accordance with embodiments of the present invention. 
           [0033]      FIG. 1B  depicts a protein of interest from a self-referenced interfacial measurement using a SPR sensor with modulation, in accordance with embodiments of the present invention. 
           [0034]      FIG. 2A  depicts adjacent streams within a single channel of a fluid cell at an initial time and a later time, in accordance with embodiments of the present invention. 
           [0035]      FIG. 2B  depicts adjacent streams within a flow cell, in accordance with embodiments of the present invention. 
           [0036]      FIG. 3A  depicts a top view of an apparatus or device comprising an optical system that includes a scanning minor for alternately directing a scanning beam from a laser onto a sample channel and a reference channel of a flow cell, in accordance with embodiments of the present invention. 
           [0037]      FIG. 3B  depicts a vertical cross section of the optical system of  FIG. 3A , in accordance with embodiments of the present invention. 
           [0038]      FIG. 4A  depicts a top view of an apparatus or device comprising an optical system that includes a calcite beam splitter for splitting a scanning beam from a laser into beams alternately directed to a sample channel and a reference channel of a flow cell, in accordance with embodiments of the present invention. 
           [0039]      FIG. 4B  depicts a vertical cross section of the optical system of  FIG. 4A , in accordance with embodiments of the present invention. 
           [0040]      FIG. 5  is a flow chart depicting a method for detecting an analyte, in accordance with embodiments of the present invention. 
           [0041]      FIG. 6  depicts a graph of SPR response voltage versus time for a fluid whose refractive index is dynamically varied, in accordance with embodiments of the present invention. 
           [0042]      FIG. 7  is an exemplary SPR curve of reflected intensity versus angle of incidence of a scanning beam. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0043]    The present invention uses surface plasmon resonance (SPR) to monitor the reflected intensity with modulation to achieve the shot-noise detection limit. However, unlike phase detection which operates at the minimum reflection angle, the present invention uses intensity measurements that are most sensitive away from the minimum reflection angle, which increases the achievable signal-to-noise ratio while not heating the sample considerably. To understand why modulation is used, the reduction of noise through modulation and how to implement modulation in the present invention are discussed next. 
         [0044]    In general, the detection limit of a specific analyte is determined by the biosensor&#39;s sensitivity and noise. The biosensor&#39;s sensitivity is related to the change in interfacial refractive index which depends upon analyte binding and which results in a change in reflected beam intensity (see  FIG. 7 , discussed infra). For intensity measurements using surface plasmon resonance signal, the sensitivity depends on the excitation wavelength and interfacial architecture. The presence of the analyte is monitored by the change in output signal relative to the system noise. Since the sensitivity to analyte is predetermined, improvement in detection is accomplished by the reduction of noise. 
         [0045]    For surface plasmon resonance, laser noise (defined as noise in excess of the shot noise, spurious modulation, and power drift) is the major source of noise. The amplitude of the laser noise is less at higher frequencies. Therefore, the present invention implements splitting a laser beam into a sample beam and a reference beam to significantly reduce laser noise. Shot noise is the fundamental noise limit as determined by photon statistics and is the theoretical quantum noise for the emitted light power from the laser. Achieving the shot-noise limited detection may be realized by compensating for the sources of laser noise or operating at a frequency where laser noise levels are minimal compared to shot noise. Unfortunately, the latter requires operating frequencies from one to several hundred megahertz depending on the laser source, which is not readily achievable. 
         [0046]    The method described herein of shot-noise limited surface plasmon resonance-based detection is accomplished by spatially manipulating the laser beam to rapidly switch between a reference channel (which remains fixed with time) and a sample channel (where the interfacial biochemical reaction or adsorption of interest occurs). In addition, a photodetector is used to achieve the shot-noise limited detection by compensating for the sources of laser noise or operating at a frequency where laser noise levels are minimal compared to shot noise. The photodetector uses an all-electronic noise reduction scheme which subtracts a photocurrent associated with the reference beam from a photocurrent associated with the sample beam. Furthermore, for the photodetector to suppress laser noise, the intensity on the photodetector must not deviate during the transition between the channels as would occur with beam chopping. The present invention provides a first embodiment and a second embodiment for spatial beam manipulation in a manner that permits the use of balanced photodetection to achieve the fundamental shot-noise detection limit. 
         [0047]    Further, this modulation configuration provides a unique characteristic relative to standard labeled or label-free sensors. This modulation scheme has the inherent advantage of being entirely self-referencing in one embodiment by having the modulation itself reference out (i.e., remove from consideration) non-specific adsorption (e.g., adsorption of a background protein) and generate a signal for only an analyte of interest (e.g., a protein of interest). Therefore, while non-specific adsorption is often a problem for standard interfacial detection, which limits commercial viability, modulation according to the present invention examines differences between signals alternately reflected from sample and reference channels and could isolate specific recognition in the presence of non-specific adsorption. As described above, one channel (i.e., the reference channel) remains unchanged throughout implementation of the methods of the present invention. In the modulated system, the reference channel requires a buffer flow (see  FIG. 1A ) since the modulated signal continuously examines the difference between the sample and reference channels. 
         [0048]      FIG. 1A  depicts all specific and non-specific interactions from an interfacial measurement using a SPR sensor with modulation, in accordance with embodiments of the present invention. Thus in  FIG. 1A , the modulated signal, which is alternately directed toward the sample channel and the reference channel at a frequency of alternation (i.e., modulation frequency), represents all interfacial interactions, both specific and non-specific. 
         [0049]      FIG. 1B  depicts a protein of interest from a self-referenced interfacial measurement using a SPR sensor with modulation, in accordance with embodiments of the present invention. Thus in  FIG. 1B , the background protein is present in both the reference channel and the sample channel, and the protein of interest is present in only the sample channel. Thus, the non-specific adsorption is referenced out by the modulation, and a signal is generated by the protein of interest in one embodiment. A sensor that utilizes a specific interaction (aptamer or antibody) upstream of the reference measurement may be employed to filter the protein of interest from the reference channel to test for the presence of a specific biomarker. 
         [0050]    A first embodiment and a second embodiment of the present invention for increasing the sensitivity of surface plasmon resonance are described infra. Surface plasmon polaritons are electromagnetic waves confined to the interface between a metal and a dielectric. The intensity of these electromagnetic waves decays exponentially into the bulk medium of the metal making the electromagnetic waves exquisitely sensitive to change in refractive index of the interface between the metal and the dielectric. Surface plasmon resonance is an optical technique that utilizes surface plasmon polaritons to monitor small changes occurring at the interface such as the adsorption or desorption of proteins. 
       First Embodiment 
       [0051]      FIG. 2A  depicts adjacent streams  31  and  33  within a single channel of a fluid cell at a first position  36  corresponding to an initial time and a second position  37  corresponding to a later time, in accordance with embodiments of the present invention. At the first position  36 , the adjacent streams  31  and  33  are separate streams because insufficient time has elapsed for mixing of the streams to occur, as depicted in the projection  38  of the adjacent streams  31  and  33 . At the second position  37 , a mixed fluid region  32  has formed between the adjacent streams  31  and  33  due to turbulent mixing of the adjacent streams  31  and  33 , as depicted in the projection  39  of the streams  31 ,  32 , and  33 . 
         [0052]      FIG. 2B  depicts adjacent streams  41  and  43  within a flow cell  45 , in accordance with embodiments of the present invention. The flow cell  45  has channel dimensions for achieving a low Reynolds number that produces laminar flow (i.e., flow having a Reynold&#39;s number less than 2000) under which fluid mixing occurs by diffusion rather than by the convection that characterizes the standard turbulent flow illustrated in  FIG. 2A . Therefore since diffusion is relatively slow, the adjacent streams  41  and  43  must travel a considerable distance before a mixed fluid region can form. 
         [0053]      FIG. 2B  also depicts a metal film (e.g., a gold film)  44  on which reflects a scanning beam  46 . The metal film  44  is on adjacent streams  41  and  43 . Most of the light from the scanning beam  46  is reflected, except under a condition of surface plasmon resonance in which the light from the scanning beam  46  is essentially totally absorbed into the metal film  44  and may be subsequently detected as a signal of decreased energy as compared with the energy of the reflected beam under conditions in which surface plasmon resonance is absent. The scanning beam  46  is depicted as beams  47  and  48  which alternate between streams  41  and  43  at a frequency of alternation. This alternation is a modulation which may be used to eliminate or substantially reduce noise from a laser that generates the scanning beam  46 . 
         [0054]    The first embodiment of the present invention, as illustrated in  FIGS. 3A and 3B  described infra, utilizes a scanning minor in combination with a single flow cell to rapidly sweep a scanning beam between the reference and sample channels within the flow cell. The flow cell provides a seamless transition in refractive index while still maintaining distinctly isolated channels. If these channels were physically separated by a barrier, the refractive index difference between the barrier and the buffer solution would produce a considerable change in signal intensity. Similar to beam chopping, the intensity change resulting from this refractive index difference causes a considerable deterioration in the noise suppression. For typical flow cells, removing the barrier leads to turbulent mixing of the solutions. However, using a barrierless flow cell with the appropriate channel dimensions to achieve a low Reynolds number of less than 2000, produces laminar flow. Under laminar flow constraints of the Reynold&#39;s number being less than 2000, fluid mixing occurs via diffusion rather than via convection as with the standard turbulent flow illustrated in  FIG. 2A . Therefore, adjacent streams within a single channel can travel a considerable distance before interfacial mixing occurs, thus eliminating the need for a physical barrier that would change the refractive index between the channels. A reflected beam can pass across the solution interface without signal disruption created by a barrier, which will allow the system noise to achieve shot-noise limited detection. One of the primary benefits is that this noise suppression scheme can be realized without complex modifications to the original surface plasmon resonance setup. A flow cell for use with the present invention may be fabricated out of polydimethylsiloxane using standard soft lithography methodology. A scanning minor is introduced into the beam path to oscillate the beam between the reference channel and the sample channel. 
         [0055]      FIG. 3A  depicts a top view of an apparatus or device comprising an optical system  51  that includes a scanning mirror  6  for alternately directing a scanning beam  20  from a laser  1  onto a sample channel and a reference channel of a barrierless flow cell  9 , in accordance with embodiments of the present invention. In one embodiment, the fluid in the sample channel and the fluid in the reference channel are essentially separated from each other.  FIG. 3B  depicts a vertical cross section of the optical system  51  of  FIG. 3A , in accordance with embodiments of the present invention. 
         [0056]    The scanning beam  20  includes a laser noise component whose amplitude reflects the laser noise (i.e., noise in excess of the shot noise, spurious modulation, and power drift) generated in the laser  1 . The scanning beam  20 , after passing through a polarizer  2  and a lens  3 , is split by a beam splitter  5  into a reference beam  21  and a sample beam  22 . Both the reference beam  21  and the sample beam  22  include the laser noise component of the scanning beam  20 . 
         [0057]    The reference beam  21  is collected by a reference photodetector  10  which outputs a resultant reference signal  27  that is directed to noise reduction electronics  13 . The reference signal  27  includes the laser noise component of the reference beam  21 . 
         [0058]    In one embodiment, the reference photodetector  10  is a photodiode, wherein the reference signal  27  is a photocurrent. 
         [0059]    In one embodiment, the reference photodetector  10  is a wireless device, wherein the reference signal is a wireless signal. 
         [0060]    The sample beam  22  is directed to a spherical mirror  4  which reflects and redirects the sample beam  22  toward a scanner mirror  6 . After being redirected toward the scanner mirror  6 , the sample beam is denoted by reference numeral  23 . 
         [0061]    The scanner mirror  6  engages in a rotation about its axis to direct the sample beam  23  alternately toward the sample channel and the reference channel of the flow cell  9  at a modulation frequency determined by the motion of the scanner minor  6 . 
         [0062]    The alternately directed beams are reflected at the metal layer  8  in contact with the flow cell  9 . The reflections occur alternately at the sample channel and the reference channel. The reflected beams are directed toward the spherical mirror  4 B.  FIG. 3B  shows the reflected beams striking the spherical mirror  4 B at two different elevations to make the reflected beams parallel to each other as parallel beams  25 A and  25 B, after which the beams  25 A and  25 B are collected by a sample photodetector  11  which outputs a resultant sample signal  28  that is directed to the noise reduction electronics  13 . The sample signal  28  includes the same laser noise component that is included in the reference signal  27 . 
         [0063]    In one embodiment, the sample photodetector  11  is a photodiode, wherein the sample signal  28  is a photocurrent. 
         [0064]    In one embodiment, the sample photodetector  11  is a wireless device, wherein the sample signal  28  is a wireless signal. 
         [0065]    The noise reduction electronics  13  receives the sample signal  28  and the reference signal  27  either as: (i) photocurrents (e.g., if the sample photodetector  11  and the reference photodetector  10  are photodiodes); or (ii) electric currents into which the sample signal  28  and the reference signal  27  have been converted (e.g., if the sample photodetector  11  and the reference photodetector  10  are wireless devices). The symbols I SAMP  and I REF  denote the time varying electric currents associated with the sample signal  28  and the reference signal  27 , respectively. The time varying electric currents I SAMP  and I REF  are received and processed by the noise reduction electronics  13 . 
         [0066]    The noise reduction electronics  13  generates an output signal  29 , denoted as I OUT , which comprises the sample signal  28  after the laser noise component has been removed from the sample signal  28  by being cancelled by the laser noise component of the reference signal  27 . 
         [0067]    In one embodiment, the noise reduction electronics  13  determines the output signal  29  as I OUT =I SAMP −(I REF −I REFDC ) wherein I REFDC  is the DC value of I REF . The preceding determination of I OUT  may be implemented using the electronic noise reduction scheme described in Hobbs, P. C. D., Shot Noise Limited Optical Measurements at Baseband with Noisy Lasers,  SPIE Laser Noise  1990, 1376, 216-221. 
         [0068]    In one embodiment, the noise reduction electronics  13  contains a feedback loop that splits the electric current I REF  in each feedback cycle to generate a reduction current I SUB  such that the laser noise component is identified and subsequently cancelled from I SAMP  upon determining that the DC value of I SUB  equals the DC value of I SAMP-RC  (which occurs during one of the feedback cycles), wherein I SAMP-RC  is the value of a portion of I SAMP  corresponding to the reflected sample beam from the reference channel. 
         [0069]    In one embodiment, the noise reduction electronics  13  may implement a determination of a ratio of I SAMP  to I REF  which approximately eliminates the presence of the laser noise component in the reduced-noise value of I SAMP  that is used in the determination of I OUT . 
         [0070]    The electronic noise reduction scheme in the noise reduction electronics  13  enables achievement of the shot-noise limited detection of analyte in the sample channel of the flow cell  9 . 
         [0071]    The output signal  29  from the noise reduction electronics  13  is processed by a lock-in amplifier  14  that locks in (i.e., selects) the output signal  29  having the modulation frequency determined by the scanner mirror  6 . Noting that the output signal  29  is periodic in accordance with the frequency of alternation (i.e., modulation frequency), the lock-in amplifier  14  determines, from processing different portions of cycles of the output signal  29 , the difference in amplitude (ΔA) between the alternately directed beams reflected at metal layer  8 . The difference in amplitude (ΔA) is included in the output signal  30  from the lock-in amplifier  14 . 
         [0072]    The output signal  30  from the lock-in amplifier  14  is sent to a computer system  15  for further processing by a computer processor of the computer system, which includes determining the presence and amount of analyte in the fluid of the sample channel. In one embodiment, the amount of analyte present in the fluid of the sample channel may be determined from a calibration curve that plots the difference in amplitude (ΔA) versus amount or concentration of analyte present. The calibration curve is specific to the analyte of interest being considered. In one embodiment, the maximum molar concentration of analyte that may be in the reference fluid is about 50% of that in the sample fluid. 
       Second Embodiment 
       [0073]      FIG. 4A  depicts a top view of an apparatus or device comprising an optical system  52  that includes a polarizing beam splitter  63  for alternately directing a scanning beam  20  from a laser  1  onto a sample channel and a reference channel of a flow cell  9 , in accordance with embodiments of the present invention. In one embodiment, the fluid in the sample channel and the fluid in the reference channel are essentially separated from each other.  FIG. 4B  depicts a vertical cross section of the optical system  52  of  FIG. 4A , in accordance with embodiments of the present invention. 
         [0074]    The scanning beam  20  includes a laser noise component whose amplitude reflects the spurious noise (i.e., in excess of the shot noise) generated in the laser  1 . The scanning beam  20 , after passing through a polarizer  2 , is split by a beam splitter  5  into a reference beam  21  and a sample beam  26 . Both the reference beam  21  and the sample beam  22  include the laser noise component of the scanning beam  20 . 
         [0075]    The reference beam  21  is collected by a reference photodetector  10  which outputs a resultant reference signal  27  that is directed to a noise reduction electronics  13 . The reference signal  27  includes the laser noise component of the reference beam  21 . 
         [0076]    In one embodiment, the reference photodetector  10  is a photodiode, wherein the reference signal  27  is a photocurrent. 
         [0077]    In one embodiment, the reference photodetector  10  is a wireless device, wherein the reference signal is a wireless signal. 
         [0078]    The sample beam  26  passes through a photoelastic modulator  61  which changes the polarization of the sample beam  26  at a specific frequency defined by the photoelastic modulator  61  to modulate (i.e., alternate) the polarization rapidly (e.g., at 50,000 Hz) between circularly polarized light and P polarized light. 
         [0079]    After passing through the photoelastic modulator  61 , the sample beam  26  passes through a quarter-wave plate  62  that changes the circularly polarized light to linearly polarized light, which results in the sample beam  26  being modulated between two polarizations, namely S polarized light and P polarized light, with respect to the reflecting SPR surface of the metal layer  8 . 
         [0080]    After passing through the quarter-wave plate  62 , the sample beam  26  passes through the polarizing beam displacer (e.g., a calcite beam displacer)  63  which displaces the S and P polarizations by a distance determined by the geometry of the polarizing beam displacer  63 , which generates displaced beams  68  and  69 . The polarizing beam displacer  63  allows one of the two polarizations to pass directly through without being displaced and causes the other of the two polarizations to be displaced by a certain distance. Thus, the displaced beams  68  and  69  alternate back and forth between the two spatial positions as the photoelastic modulator  61  changes the polarization. Although any polarizing beam displacer would work, most polarizing beam displacers require more alignment than does the calcite beam displacer  63  which may be used for simplicity. 
         [0081]    The two displaced beams  68  and  69  emerging from the polarizing beam splitter  63  are then rotated by 45 degrees by a half-wave plate  64  such that the overall P polarization between the two displaced beams  68  and  69  remains constant with time. 
         [0082]    Then, a linear polarizer  65  removes the S polarization which is not needed for surface plasmon resonance, which creates a beam with constant overall intensity that alternates between two positions in space allowing for the use of two separate flow cells, said two positions corresponding to where the sample channel and the reference channel are located. 
         [0083]    The two displaced beams  68  and  69  are reflected at metal layer  8  in contact with the flow cell  9 . The reflections occur alternately at the sample channel and the reference channel. The reflected beams are directed toward the spherical mirror  4 B and then striking spherical mirror  4 B at two different elevations to make the two displaced beams parallel to each other as described supra in conjunction with  FIG. 3B , after which the two displaced beams are collected by a sample photodetector  11  which outputs a resultant sample signal  28  that is directed to the noise reduction electronics  13 . The sample signal  28  includes the same laser noise component that is included in the reference signal  27 . 
         [0084]    In one embodiment, the sample photodetector  11  is a photodiode, wherein the sample signal  28  is a photocurrent. 
         [0085]    In one embodiment, the sample photodetector  11  is a wireless device, wherein the sample signal  28  is a wireless signal. 
         [0086]    The noise reduction electronics  13  receives the sample signal  28  and the reference signal  27  either as: (i) photocurrents (e.g., if the sample photodetector  11  and the reference photodetector  10  are photodiodes); or (ii) electric currents into which the sample signal  28  and the reference signal  27  have been converted (e.g., if the sample photodetector  11  and the reference photodetector  10  are wireless devices). The symbols I SAMP  and I REF  denote the time varying electric currents associated with the sample signal  28  and the reference signal  27 , respectively. The time varying electric currents I SAMP  and I REF  are received and processed by the noise reduction electronics  13 . 
         [0087]    The noise reduction electronics  13  generates an output signal  29 , denoted as I OUT , which comprises the sample signal  28  after the laser noise component has been removed from the sample signal  28  by being cancelled by the laser noise component of the reference signal  27 . 
         [0088]    In one embodiment, the noise reduction electronics  13  determines the output signal  29  as I OUT =I SAMP −(I REF −I REFDC ) wherein I REFDC  is the DC value of I REF . The preceding determination of I OUT  may be implemented using the electronic noise reduction scheme described in Hobbs, P. C. D., Shot Noise Limited Optical Measurements at Baseband with Noisy Lasers,  SPIE Laser Noise  1990, 1376, 216-221. 
         [0089]    In one embodiment, the noise reduction electronics  13  contains a feedback loop that splits the electric current I REF  in each feedback cycle to generate a reduction current I SUB  such that the laser noise component is identified and subsequently cancelled from I SAMP  upon determining that the DC value of I SUB  equals the DC value of I SAMP-RC  (which occurs during one of the feedback cycles), wherein I SAMP-RC  is the value of a portion of I SAMP  corresponding to the reflected sample beam from the reference channel. 
         [0090]    In one embodiment, the noise reduction electronics  13  may implement a determination of a ratio of I SAMP  to I REF  which approximately eliminates the presence of the laser noise component in the reduced-noise value of I SAMP  that is used in the determination of I OUT . 
         [0091]    The electronic noise reduction scheme in the noise reduction electronics  13  enables achievement of the shot-noise limited detection of analyte in the sample channel of the flow cell  9 . 
         [0092]    The output signal  29  from the noise reduction electronics  13  is processed by a lock-in amplifier  14  that locks in (i.e., selects) the output signal  30  having the modulation frequency determined by the scanner mirror  6 . Noting that the output signal  29  is periodic in accordance with the frequency of alternation (i.e., modulation frequency), the lock-in amplifier  14  determines, from processing different portions of cycles of the output signal  29 , the difference in amplitude (ΔA) between the alternately directed beams reflected at metal layer  8 . The difference in amplitude (ΔA) is included in the output signal  30  from the lock-in amplifier  14 . 
         [0093]    The output signal  30  from the lock-in amplifier  14  is sent to a computer system  15  for further processing by a computer processor of the computer system, which includes determining the presence and amount of analyte in the fluid of the sample channel. In one embodiment, the amount of analyte present in the fluid of the sample channel may be determined from a calibration curve that plots the difference in amplitude (ΔA) versus amount or concentration of analyte present. The calibration curve is specific to the analyte of interest being considered. In one embodiment, the maximum molar concentration of analyte that may be in the reference fluid is about 50% of that in the sample fluid. 
       Inventive Method 
       [0094]      FIG. 5  is a flow chart depicting a method for detecting an analyte, in accordance with embodiments of the present invention. The method of  FIG. 5 , which includes steps  71 - 78 , encompasses both the first embodiment ( FIGS. 3A ,  3 B) and the second embodiment ( FIGS. 4A ,  4 B) of the present invention, described supra. 
         [0095]    Step  71  provides a sample channel through which a sample fluid is flowing, a reference channel through which a reference fluid is flowing, and a metal layer in contact with the sample fluid and the reference fluid. The sample fluid comprises the reference fluid and the analyte. In one embodiment, the maximum molar concentration of analyte that may be in the reference fluid is about 50% of that in the sample fluid. In one embodiment, the reference fluid does not comprise the analyte. The reference channel and the sample channel are different channels. 
         [0096]    In step  72 , an optical system receives a scanning beam from a laser. The scanning beam comprises laser noise generated in the laser. 
         [0097]    In step  73 , the optical system splits the scanning beam into a reference beam and a sample beam. The reference beam comprises information pertaining to the laser noise. 
         [0098]    In step  74 , the optical system directs the reference beam to a reference photodetector causing the reference photodetector to send a resultant reference signal (e.g., a photocurrent or a wireless signal) to noise reduction electronics. 
         [0099]    In step  75 , the optical system directs the sample beam alternately toward the sample channel and the reference channel at a frequency of alternation (i.e., a modulation frequency). The directed sample beam is reflected from the metal layer such that surface plasmon resonance (SPR) is triggered in the metal layer. The reflected sample beam is directed to a sample photodetector causing the sample photodetector to send a resultant sample signal (e.g., a photocurrent or a wireless signal) to the noise reduction electronics. 
         [0100]    In step  76 , noise reduction electronics (i) implements a reduction of the laser noise by subtracting the laser noise component of the reference photocurrent from the sample photocurrent and (ii) generates an output signal comprising the sample signal after the laser noise component has been removed from the sample signal by being cancelled by the laser noise component of the reference signal. 
         [0101]    Step  77  determines the difference in amplitude (ΔA) between the alternately directed beams reflected at the metal layer alternately at the sample channel and the reference channel. In step  77 , the output signal from the noise reduction electronics is processed by a lock-in amplifier that locks in (i.e., selects) the output signal from the noise reduction electronics having the modulation frequency determined by the scanner mirror. The lock-in amplifier determines, from processing different portions of cycles of the output signal from the noise reduction electronics, the difference in amplitude (ΔA) between the alternately directed beams reflected at the metal layer. The difference in amplitude (ΔA) is included in the output signal from the lock-in amplifier. 
         [0102]    Step  78  determines the presence of analyte in the sample channel. In step  78 , the output signal from the lock-in amplifier is sent to a computer system for further processing by a computer processor of the computer system, which includes determining the presence and amount of analyte in the fluid of the sample channel. In one embodiment, the amount of analyte present in the fluid of the sample channel may be determined from a calibration curve that plots the difference in amplitude (ΔA) versus amount or concentration of analyte present. The calibration curve is specific to the analyte of interest being considered. In one embodiment, the maximum molar concentration of analyte that may be in the reference fluid is about 50% of that in the sample fluid. 
       Demonstration of Technique 
       [0103]      FIG. 6  depicts a graph of SPR response voltage versus time for a fluid whose refractive index is dynamically varied, in accordance with embodiments of the present invention.  FIG. 6  demonstrates the technique of the present invention by examining the difference in refractive index between two bulk solutions: deionized water and a 25 mM sodium chloride (NaCl) solution (the reference and sample channel, respectively). The sample beam is modulated at 500 Hz in accordance with the first embodiment based on  FIG. 3A  as described supra. The fluid is deionized water initially with an accompanying baseline response voltage of zero volts. At about 70 seconds, 25 mM NaCl is added to the fluid in the sample channel and the response voltage increases to about 4 volts at about 130 seconds when a steady state is reached, which reflects a change in refractive index (Δn) of the fluid due to the addition of NaCl. The sharp decrease in SPR response voltage at approximately 160 seconds is an experimental artifact due to a pressure difference introduced in the sample channel fluid by the addition of the 25 mM NaCl to the sample channel fluid. This pressure difference caused some of the fluid in the reference channel to move to the sample channel. This experimental artifact is not relevant to the present invention and therefore may be ignored. The NaCl is removed beginning at about 170 seconds which causes the response voltage to decrease continuously until the response voltage is the baseline value of zero volts at about 275 seconds, which reflects a return of the refractive index to its value for deionized water. 
         [0104]    While surface plasmon resonance is typically used for monitoring interfacial changes, it is sensitive to any change within the evanescent wave of the surface plasmon which includes changes in the bulk refractive index. In this example, the addition of sodium chloride changes the bulk refractive index in the sample channel. The modulated signal between the reference and sample channels is fed into a lock-in amplifier which isolates the signal at the scanning frequency. A step change of known refractive index as shown in  FIG. 6  is a simple method to determine the resolution of the system. The addition of sodium chloride to water, resulting in a 25 mM solution, changes the refractive index of the solution by ˜2.4E-4 refractive index units (RIUs) for the wavelength of this experiment. The measured change in lock-in voltage output (ΔV) is proportional to the known change in refractive index (Δn) due to the NaCl (see Equation (1) infra). Thus, the smallest refractive index change detectable by the apparatus (n noise ), measured in refractive index units, can be determined in accordance Equation (1) from the known refractive index change (Δn), the noise in the lock-in output (V noise ), and the change in lock-in voltage output (ΔV). 
         [0000]        n   noise   =Δn*V   noise   /ΔV   (1)
 
         [0000]    Equation (1) is valid for the linear regime of the SPR curve such as the SPR curve of  FIG. 7  which is described infra. The refractive index noise (n noise ) for the system used in the experiment of  FIG. 6  in its current state is ˜2E-7, which is approximately one order of magnitude less than with commercially available systems. 
         [0105]    The minimum achievable concentration of a specific biomarker is determined largely based on the interactions between the biomarker and the surface (e.g., a gold surface may be used). Typically, the surface is configured with a linker to selectively bind the biomarker of interest. For example, for a biomarker that is a specific protein, an antibody may be placed on the surface to serve as a linker for the specific protein because the antibody has a selective affinity for the particular protein. The lowest concentration that could be resolved is the minimum resolvable concentration for the specific biomarker under the conditions of the experiment, which corresponds to the signal change (due to Δn) being equal to the noise level. 
       Determining Sensitivity 
       [0106]      FIG. 7  is an exemplary SPR curve  80  of reflected intensity versus angle Θ of incidence of a scanning beam. The scanning beam is an unmodulated laser beam (i.e., unmodulated in contrast with the modulation of sample beams  23  and  26  in  FIGS. 3A and 3B , respectively) having a 830 nm wavelength. The laser beam is reflected off the sample surface (analogous to surface  49  in  FIG. 2B ), and the angle Θ is varied with respect to the surface normal to the sample surface. The angle Θ is the angle of incidence of the scanning beam  47  incident upon the surface  49  of the metal film  44  as depicted in  FIG. 2B . The metal film is a gold film having a thickness of 48 nm. An adhesion layer of chromium of thickness 1.5 nm adheres the gold film to a SF11 (high refractive index glass) prism. The sample channel comprises MilliQ water, which is ultra pure water. The collected scanning beam exhibits a dip in reflected intensity which is associated with exciting surface plasmon polaritons in the metal film  44 . The lock-in voltage output (V) is associated with the reflected intensity.  FIG. 7  also depicts a derivative curve  90  which is the derivative of the reflected intensity  80 . The SPR curve  80  in  FIG. 7  has a linear regime, between points  81  and  83 , which is approximately linear with an approximately constant slope and contains an inflection point  82  where the derivative curve  90  is minimized in  FIG. 7 . The derivative of the reflected intensity allows one to easily identify the inflection point where sensitivity of change in reflected intensity to angular change is maximized. 
         [0107]    The minimum detection limit is typically defined as a signal-to-noise (SNR) ratio of 1. Therefore, knowledge of the noise level enables a determination of the sensitivity in refractive index units (RIUs). The minimum detectable RIU is given by Equation (2): 
         [0000]        RIU=V   noise /(slope of  SPR  linear regime*angular sensitivity to refractive index)  (2)
 
         [0000]    wherein slope of SPR linear regime=δV/δΘ wherein δV and δΘ denote a change in V and a change in Θ, respectively at a specific angle Θ in the linear regime  81  of the SPR curve and, in one embodiment, may be taken at the inflection point  82  to maximize sensitivity. The ratio of angular sensitivity to refractive index is equal to ΔΘ/ΔRIU. These values can be determined theoretically or experimentally using a calibration with known refractive index samples. 
         [0108]    The minimum detectable concentration of a protein can be determined from the differential refractive index, dn/dc via Equation (3). 
         [0000]      Minimum detectable concentration= RIU   min   *dn/dc   (3)
 
       CONCLUSION 
       [0109]    The present invention provides a method, and an associated apparatus, based on surface plasmon resonance to achieve ultrasensitive interfacial detection of gas and solution-phase biological and chemical analytes. Increased sensitivity is achieved by signal modulation with active electronic noise suppression. The first embodiment utilizes the barrierless construct accomplished with the incorporation of a flow cell. This allows a single laser source to transverse between the two channels without signal contamination in the frequency domain that would increase noise. The second embodiment utilizes polarization modulation to rapidly alternate a laser beam between two flow channels. Further, each embodiment operates in a self-referencing mode to eliminate the undesired contribution of non-specific interfacial adsorption (e.g., serum proteins) making the apparatus particularly useful for ultrasensitive measurements in the presence of complex media (e.g., blood). 
         [0110]    While particular embodiments of the present invention have been described herein for purposes of illustration, many modifications and changes will become apparent to those skilled in the art. Accordingly, the appended claims are intended to encompass all such modifications and changes as fall within the true spirit and scope of this invention.