Polarization modulation interrogation of grating-coupled waveguide sensors

An optical interrogation system and a GCW sensor are described herein that are used to determine whether a biological substance (e.g., cell, molecule, protein, drug) is located in a sensing region of the GCW sensor. The optical interrogation system includes a light source, a polarization modulator and a detection system. The light source outputs a polarized light beam and the polarization modulator modulates the polarized light beam and outputs a polarization-modulated light beam. The GCW sensor receives and converts the polarization-modulated light beam into an amplitude modulated light beam that is directed towards the detection system. The detection system receives the amplitude modulated light beam and demodulates the received amplitude modulated light beam by responding to signals at a modulation frequency of the polarization-modulated light beam and ignoring noise affecting the signals outside the modulation frequency to detect a resonant condition (e.g., resonant angle, resonant wavelength). The detected resonant condition that has a one-to-one relationship with the refractive index of the superstrate containing the biological substance is analyzed to determine whether or not the biological substance is located in the sensing region of the GCW sensor.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates in general to a grating-coupled waveguide (GCW) sensor and, in particular, to an optical interrogation system and method for using polarization-modulated light beams to interrogate a GCW sensor in order to determine whether or not a biological substance is located within a sensing region of the GCW sensor.

2. Description of Related Art

Grating-coupled waveguide (GCW) sensors are fast becoming the technology of choice to enable accurate label-free detection of a biological substance (e.g., cell, drug, chemical compound). This technology typically involves the use of a waveguide evanescent field to sense changes in the refractive index of a GCW sensor caused by the presence of a biological substance in a sensing region of the GCW sensor. To generate the evanescent field, an optical interrogation system is used which has a light source that couples a light beam into a waveguide of the GCW sensor. The optical interrogation system also includes a detector that receives a light beam coupled out from the waveguide that is analyzed to determine the effective refractive index of the waveguide. In determining the effective refractive index of the GCW sensor it should be understood that the light beam received by the detector had interacted with the waveguide under a resonant condition, where the wavevectors of a diffraction grating, incoming light beam, and guided mode all sum to zero. And, that this resonant condition occurs only for a specific wavelength and angle of the incoming light where changes in this angle or wavelength corresponds to changes in the effective refractive index of the waveguide caused by the presence of the biological substance in the sensing region of the GCW sensor. Thus, the optical interrogation system is used to sense a change in the effective index of the GCW sensor which enables one to determine whether or not a biological substance is located within the sensing region of the GCW sensor.

For this technology to be viable, one must have an optical interrogation system and in particular a detector capable of accurately monitoring the resonant angle, wavelength, or both. In particular, the optical interrogation system must emit a light beam that interacts with the GCW sensor, and must in turn receive the light beam coupled-out off the GCW sensor and process that light beam to detect in real time any changes in it's resonant angle and/or wavelength. While there are many approaches for accomplishing these tasks, each has unique implementation challenges, since the light beam output from the GCW sensor is relatively weak and there are multiple sources of noise that degrade this light beam especially in high-throughput screening applications.

GCW sensors are particularly attractive for use in high-throughput screening applications, where the absence of fluorescent tags and the possibility of reduced false-negatives would provide a large cost advantage. For this reason, the microplate has been targeted as the platform for such sensors, where 96 or 384 individual wells provide the high-throughput access demanded by the industry. In this application, the waveguide and diffraction grating of the GCW sensor are located in the bottom of each well; e.g., the diffraction grating may be stamped into the well bottom, and the waveguide subsequently grown on top of the diffraction grating. The wells themselves are typically composed of an optically transparent, low-birefringence, low-cost plastic that is typically several millimeters thick. To probe the GCW sensor in the well bottom while leaving the tops of the wells open for fluid handling, etc., the optical light beam is emitted into the bottom of the microplate and passes through the well plastic before striking the GCW sensor. One source of noise for this type of optical interrogation system is produced by the Fresnel reflection emanating from the bottom surface of each well. Due to the large number of wells, one ideally tries to design the GCW sensor to operate with incoming light beams near normal incidence. As a result, this spurious Fresnel reflection which acts as noise is often inextricably mixed with the light beam output from the GCW sensor that contains the desired information about the resonant angle and/or wavelength. In addition to the Fresnel reflection caused by the bottom surface of the microplate, the top surface of the waveguide inserts yet another Fresnel reflection into the output light beam that mingled with the light beam that propagated as a waveguide mode and exited the GCW sensor through the diffraction grating.

In addition to these direct optical noise sources, the traditional optical interrogation system is susceptible to other electrical and optical noises. For example, the wavelength or angle of the output light beam is often monitored with detectors such as charge-coupled device (CCD) cameras or spectrographs that observe the signal in a DC fashion. All of the DC electrical and optical (stray light) noise can impede the detection of the resonant angle and/or wavelength in the output light beam. Accordingly, there is a need for an optical interrogation system and method that can avoid the aforementioned problematical noise sources when interrogating one or more GCW sensors. This need and other needs are satisfied by the optical interrogation system, GCW sensor and method of the present invention.

BRIEF DESCRIPTION OF THE INVENTION

The present invention includes an optical interrogation system capable of interrogating a GCW sensor to determine whether a biological substance (e.g., cell, molecule, protein, drug) is located in a sensing region of the GCW sensor. The optical interrogation system includes a light source, a polarization modulator and a detection system. The light source outputs a polarized light beam and the polarization modulator modulates the polarized light beam and outputs a polarization-modulated light beam. The GCW sensor receives and converts the polarization-modulated light beam into an amplitude modulated light beam that is directed towards the detection system. The detection system receives the amplitude modulated light beam and demodulates the received amplitude modulated light beam by responding to signals at a modulation frequency of the polarization-modulated light beam and ignoring noise affecting the signals outside the modulation frequency to detect a resonant condition (e.g., resonant angle, resonant wavelength). The detected resonant condition that has a one-to-one relationship with the refractive index of the superstrate containing the biological substance is analyzed to determine whether or not the biological substance is located in the sensing region of the GCW sensor.

DETAILED DESCRIPTION OF THE DRAWINGS

Referring toFIG. 1, there is shown a diagram of the basic components of a GCW sensor100and an optical interrogation system120in accordance with the present invention. Basically, the optical interrogation system120interrogates the GCW sensor100to determine whether a biological substance102(e.g., cell, molecule, protein, drug, chemical compound, nucleic acid, peptide, carbohydrate) is located in a sensing region103(superstrate103) of the GCW sensor100. The optical interrogation system120includes a light source122, a polarization modulator127(e.g., photoelastic modulator127, photorefractive modulator127, liquid crystal modulator127) and a detection system124. The light source122outputs a polarized light beam125and the polarization modulator127modulates the polarized light beam125and outputs a polarization-modulated light beam126. The GCW sensor100receives and converts the polarization-modulated light beam126into an amplitude modulated light beam128that is directed towards the detection system124. The detection system124receives the amplitude modulated light beam128and demodulates the received amplitude modulated light beam128by responding to signals at a modulation frequency of the polarization-modulated light beam126and ignoring noise affecting the signals outside the modulation frequency to detect a resonant condition (e.g., resonant angle, resonant wavelength). The detected resonant condition has a one-to-one relationship with the refractive index of the superstrate containing the biological substance102that indicates whether the biological substance102is located in the sensing region103of the GCW sensor100. How the GCW sensor100converts the polarization-modulated light beam126into the amplitude modulated light beam128which enables the optical interrogation system120to demodulate the amplitude modulated light beam128in a manner that avoids the problems caused by noise sources (e.g., Fresnel reflections, DC electrical and stray light noise) is described in greater detail below after a brief discussion about the basic structure and functionality of the GCW sensor100.

As shown inFIG. 1, the GCW sensor100includes a thin (˜100 nm) layer of material106(e.g., waveguide film106) deposited on the surface of a diffraction grating108which together form a waveguide110. The waveguide film106is preferably made of a dielectric material such as Ta2O5, TiO2, TiO2—SiO2, HfO2, ZrO2, Al2O3, Si3N4, HfON, SiON, scandium oxides or mixtures thereof. The diffraction grating108is formed within a substrate112or waveguide film106by embossing, holography, or other methods. The diffraction grating108can thereby be located above, below, or even within the waveguide film106. Moreover, the diffraction grating108need not be in direct physical contact with a waveguide film106, simply near enough to cause optical influence on the waveguide mode. Furthermore, due to effective-index waveguiding, the diffraction grating108itself can be fabricated with appropriately high enough index to serve as the waveguide itself without the need for an additional waveguide film deposition. The substrate112is preferably made of glass or plastic such as cyclic-olefin copolymer (COC). For example, the GCW sensor100can have a cyclic-olefin substrate112which has an index ns=1.53, a grating pitch Λ=538 mm, a grating thickness is tg=10 mm, a waveguide index nf=2.01, a waveguide thickness tf=110 mm, and a superstrate index that is nominally the index of water (the solvent in which most experiments are performed, nc≧1.33). This GCW sensor100is referred to herein as the exemplary GCW sensor100.

The biological substance102which may be located within a bulk fluid is introduced to the superstrate103(sensing region) of the GCW sensor100and it is the presence of this biological substance102that alters the index of refraction at the surface104of the GCW sensor100. Thus, to detect the biological substance102, the GCW sensor100is probed with a light beam126emitted from the light source122and then a reflected light beam128received at the detection system124is analyzed to determine if there are any changes (˜1 part per million) in the refractive index caused by the presence of the biological substance102in the sensing region103of the GCW sensor100. In one embodiment, the top surface104may be coated with biochemical compounds (not shown) that only allow surface attachment of specific complementary biological substances102which enables an GCW sensor100to be created that is both highly sensitive and highly specific. In this way, the optical interrogation system120and GCW sensors100may be used to detect a wide variety of biological substances102and if the GCW sensors100are arranged in arrays they may be used to enable high throughput drug or chemical screening studies (seeFIG. 15).

The sensitivity of the GCW sensor100may be best understood by analyzing the structure of the diffraction grating108and the waveguide110. The light beam126shone on the diffraction grating108can only be coupled into the waveguide110if its wave vector satisfies the following resonant condition as shown in equation no. 1:
k′x=kx−κ  [1]
where kx′ is the x-component of the incident wave vector, kxis the guided mode wave vector, and κ is the grating vector. The grating vector κ is defined as a vector having a direction perpendicular to the lines of the diffraction grating108and a magnitude given by 2π/Λ where Λ is the grating period (pitch). This expression may also be written in terms of wavelength λ and incident angle θ as shown in equation no. 2:

2⁢π⁢⁢nincλ⁢sin⁢⁢θ=2⁢π⁢⁢neffλ-2⁢πΛ[2]
Where θ is the angle of incidence of the light beam126, nincis the index of refraction of the incident medium, λis the wavelength of the light126, and neffis the effective index of refraction of the waveguide110. The effective index of the waveguide110is a weighted average of the indices of refraction that the optical waveguide mode field or fundamental mode “sees” as it propagates through the waveguide110. The fundamental mode preferably has a spatial extent that is much wider than the waveguide110itself, the extent depending on the refractive index difference between the waveguide110and the substrate112, as well as between the waveguide110and the superstrate103. In particular, the fundamental mode has an evanescent wave/tail that extends into the superstrate103(sensing region) which “sees” any surface changes created when the biological substance102approaches or comes in contact with the top surface104of the GCW sensor100.

The previous expression shown in equation no. 2 may be rewritten in the more convenient form shown in equation no. 3:

sin⁢⁢θ=neff-λΛ[3]
which is the equation of a line where sin θ being the y axis, λ being the x-axis, Λneffthe x-intercept, and −1/Λ the slope. To obtain equation no. 3, ninchas been set to 1 so that it could be removed from equation no. 2. This approximation is used since air (n˜1.0003) is the most common incident medium. This relation is pictured in the graph shown inFIG. 2. When a biological substance102binds to the surface104, the effective index of the waveguide110is altered which leads to the shifting the resonant wavelength or resonant angle of the GCW sensor100. This shifting can be seen as a shift of the x-intercept in the line shown inFIG. 2.

The resonant condition (e.g., resonant wavelength or resonant angle) of such a GCW sensor100may be interrogated to determine refractive index changes by observing the reflected light128from the GCW sensor100(seeFIG. 1). There are two different modes of operation for monitoring refractive index changes—spectral interrogation or angular interrogation. In spectral interrogation, a nominally collimated, scanned-wavelength beam of modulated polarized light126is sent into the GCW sensor100and the reflected amplitude modulated light128is collected and monitored by a photodiode (for example) within a detection system124. By observing the spectral location of the resonant wavelength (peak), one can monitor binding or refractive index changes on or near the surface104of the GCW sensor100. The spectral interrogation concept is graphically represented in the graph shown inFIG. 3. Conversely, in angular interrogation, a nominally single wavelength of modulated polarized light126is angle-scanned to create a range of illumination angles and directed into the GCW sensor100. The reflected amplitude modulated light128is monitored by a photodiode (for example) within the detection system124. By monitoring the position of the resonant angle reflected by the GCW sensor100, one can monitor binding or refractive index changes on or near the surface104of the GCW sensor100. The angular interrogation concept is graphically represented in the graph shown inFIG. 4.

To maintain simplicity and efficiency of operation, the GCW sensors100employed in biosensing applications can be designed such that only the zeroth diffracted orders of the incident light126propagate in free space, while what would be the ±1 orders couple to the fundamental mode of the waveguide110. The higher diffraction orders are avoided by designing a sub-wavelength diffraction grating108which has a grating pitch Λ smaller than the desired operating wavelength λ of the incident light126. In this case, the coupling efficiency of the waveguide110is large since multiple orders do not remove power from the GCW sensor100. Moreover, since only the zeroth reflected and transmitted beams exist in free space, the GCW sensor100can thereby produce nearly total reflection or transmission of the desired (anomalous) wavelength λ of the incident light126.FIG. 5shows a GSOLVER (rigorous coupled-wave analysis, or RCWA code) analysis of an exemplary GCW100where the TE input light126angle is 3° and the reflected light beam128which is at 3° from the normal has a resonance502in the vicinity of 824 nm when the substance (water) in the superstrate103has an index of 1.33.

As mentioned above, GCW sensors100are used in biosensing applications because they enable one to determine the location of the resonance angle/wavelength502which directly corresponds to the refractive index of the superstrate103and thereby allows the monitoring of biological substance102binding on the GCW sensor100. This is all possible because the evanescent tail of the propagating fundamental mode in the waveguide110senses index changes in the superstrate103caused by the presence of the biological substance102. The index change in the supersrate103changes the resonance condition of the GCW100according equation no. 1 and then the resonance502shifts to a new wavelength or angle location. The location of the shifted resonance indicates the current index of the superstrate103which indicates whether or not the biological substance102is in the superstrate103of the GCW100. It has been shown that the resonance502can shift hundreds of nanometers for a unit change in the refractive index of the superstrate103(seeFIG. 2). The relationship between angle and wavelength is displayed in the graph shown inFIG. 6for a BIOS-1 GCW sensor100. The different curves in the graph show behavior for both TE and TM polarizations with two different cover indices.

Referring toFIGS. 7-15, there are illustrated five embodiments of the optical interrogation system120shown inFIG. 1that utilize different approaches to interact with one or more GCW sensors100in order to detect the presence of a biological substance102. Although only five embodiments of the optical interrogation system120are described herein, it should be understood that other configurations of the optical interrogation system120could be used to interact with the GCW sensor100in order to detect the presence of the biological substance102. Accordingly, the optical interrogation system120should not be construed in a limited manner.

Referring toFIG. 7, there is a diagram illustrating the basic components of a first embodiment of the optical interrogation system120ain accordance with the present invention. The optical interrogation system120autilizes an angular scanning approach to scan the polarization-modulated light beam126that is directed into the GCW sensor100in order to enable the detection of the biological substance102.

As shown, the optical interrogation system120aincludes a light source122athat outputs a polarized light beam125athat is received by a polarization modulator127a(e.g., photoelastic modulator127a). The polarization modulator127amodulates the polarized light beam125aby causing a time-varying polarization alternation between TE and TM modes (see the wobbling vectors inFIG. 7). The polarization modulator127aoutputs the polarization-modulated light beam126ato an acousto-optic modulator702awhich changes the angle of the polarization-modulated light beam126aby mixing it with an acoustic wave. The magnitude of the angular deflection depends upon the drive frequency of the acousto-optic modulator702aand therefore the acoustic wave. In effect, the acousto-optic modulator702aenables one to control the angular scanning of the polarization-modulated light beam126a. Since the polarization-modulated light beam126ashould intersect the microwell plate704ain a constant location, a lens706aand beamsplitter708aare placed between the acousto-optic modulator702aand the microwell plate704a, where the distance from the lens706ato each GCW sensor100is a focal length. In this manner, the angle of the polarization-modulated light beam126aincident upon the microwell plate704achanges with the frequency of the acousto-optic modulator702a, while the location of the incidence remains constant. After interacting with the GCW sensor100in the microwell plate704a, the polarization-modulated light beam126awhich has only one of the two polarization states resonant within the GCW sensor100is converted into the amplitude modulated light beam128athat is modulated at the same frequency as the polarization-modulated light beam126a. The amplitude modulated light beam128ais received by the detection system124aand in particular by a detector710a(e.g., photodiode710a) which converts the amplitude modulated light beam128ainto an electrical signal that is then demodulated by a lock-in amplifier712aor a similar instrument capable of detecting electrical signals at specific frequencies (e.g., an RF mixer). The lock-in amplifier712ademodulates the electrical signal by responding to signals at the modulation frequency of the polarization-modulated light beam126aand ignoring noise affecting the signals outside the modulation frequency. A computer716athen analyzes the demodulated electrical signal (e.g., a DC voltage proportional to the original modulated reflected optical signal strength) to determine a resonant angle that correlates to the superstrate103refractive index and therefore the biological substance content102at the sensing region103of the GCW sensor100. The frequency being detected by the lock-in amplifier712acan be derived from a function generator714athat also drives the polarization modulator127a.

The use of the acousto-optic modulator702ais attractive since it utilizes no moving parts, however other angular scanning techniques can be used in the present invention. For example, a simple rotating plate of glass could be used that deflects the polarization-modulated light beam126athrough refraction at the glass/air interface. The deflection angle depends upon the rotation angle of the glass plate such that the rotating plate causes a smoothly varying angle. The results of several experiments using the optical interrogation system120aand the exemplary GCW sensor100are provided below with respect toFIGS. 8-11.

Referring toFIG. 8, there is a graph that shows the resonance observed when testing an experimental optical interrogation system120awhere the amplitude recorded by the lock-in amplifier712aversus the frequency of the acousto-optic modulator702arepresents the signal. This scan took 0.5 s-2 min. to acquire, and resulted in the very high signal-to-noise curve shown inFIG. 8. It should be noted that the light source122a(e.g., He/Ne laser122a) had to be attenuated greatly to avoid saturating the detector710a(e.g., Si photodiode710a) and the lock-in amplifier712astill had many (>5) orders of magnitude of input gain settings to accommodate still lower laser levels.

It should be appreciated that the actual sensitivity of the optical interrogation system120ato biological events depends upon the GCW sensor100, instrument, angular stability, etc. Thus, to obtain some estimate of the performance of the experimental optical interrogation system120a, index fluids were placed on the top surface104of the GCW sensor100to illicit a change in the resonant angle and thereby determine the refractive index unit sensitivity.FIG. 9shows the result of this study. Noise levels were determined to be as low as ˜10−5(single standard deviation), and further work indicates that the desired 10−6sensitivity is easily achieved with more accurate scanning electronics.

Another important aspect of the present invention, is that the lock-in amplifier712aprovides an extra observable beyond the simple amplitude resonance pictured above inFIG. 8. As a phase-locked technique, the lock-in amplifier712aprovides not only the amplitude of the resonant signal, but also its phase, as referenced to a stable frequency signal such as the electrical function driving the polarization modulator127a. It should be noted that this information could be obtained with most phase-sensitive detectors (other than a lock-in amplifier112), or even with a device that can separately monitor the TE and TM polarizations of the amplitude modulated light beam128aoutput from the GCW sensor100.

To understand what information the phase signal might present, one can examine the amplitude signal more closely.FIG. 10shows a diagram of the amplitude resonance1002with the characteristic trough/peak shape seen in experiments and theory, where the TE mode has been phase-matched to the GCW sensor100. The negatively valued dip (source of the arrow on the bottom left of the figure) represents a region where the TE power is below the TM ambient signal, caused by resonantly transmitting the TE light through the GCW sensor100(seeFIG. 1).) The peak represents the region where the TE light resonantly reflects from the GCW sensor100, enhancing the power on the detector710a. Since the TM mode does not interact with the waveguide110, its level is constant for both the trough and peak. Remembering that the light is modulated between TE and TM modes in time, the curves drawn inFIG. 10therefore represent the time-varying signal present at the lock-in amplifier712aat the modulation frequency. By comparing the signals from the peak and trough regions, one can see that the waves are 180° phase-shifted from one another. Moreover, this phase shift should occur at the instant that the amplitude crosses from negative to positive in the center of the resonance.

This simple understanding serves to accurately predict what was observed during the experiments with the experimental optical interrogation system120a.FIG. 11shows the phase of the resonance1102superimposed on the amplitude plot shown inFIG. 8. Since the lock-in amplifier712aoutputs a −10-10 volt signal to the computer716ato register 360° in phase, the change from ˜8 V to ˜−2 V represents the 180° phase change indicated inFIG. 11. The transition occurs very close to zero amplitude, where the small difference is actually due to filtering of the amplitude signal that shifts it relative to the phase signal that is acquired at a different time. It should be noticed that this phase transition is extremely steep compared to the resonance amplitude. Moreover, it is akin to a digital indication (on/off) of the resonance location, and therefore represents the simplest signal to detect from a signal processing standpoint. The steepness results from the fact that the transition occurs in a trigger fashion, depending on the relative intensities of the TE/TM modes. Because this signal is so narrow, it could possibly relax the constraints on the resonance width as they relate to detection and location of the resonance. Because the phase signal is inextricably related to the amplitude resonance however, it is not apparent that the localization of the resonance peak will improve using the phase signature, but one may be able to take advantage of signal processing techniques (e.g. a Schmidt trigger) to derive some sensitivity advantage from this phase signal1102. Assuming this is the case, the width of the amplitude resonance may become less relevant, and allow a whole new class of sensor designs where the width constraints are greatly relaxed.

Referring toFIG. 12, there is a diagram illustrating the basic components of a second embodiment of the optical interrogation system120b. The optical interrogation system120butilizes an angular scanning approach to scan the amplitude modulated light beam128bemitted from the GCW sensor100in order to enable the detection of the biological substance102. As shown, the optical interrogation system120bincludes a light source122bthat outputs a polarized light beam125bthat is received by a polarization modulator127b(e.g., photoelastic modulator127b). The polarization modulator127bmodulates the polarized light beam125bby causing a time-varying polarization alternation between TE and TM modes (see the wobbling vectors inFIG. 12). The polarization modulator127boutputs the polarization-modulated light beam126bto a lens1206band beamsplitter1208b. The lens1206band beamsplitter1208bdirect the polarization-modulated light beam126bto the GCW sensor100within the microwell plate1204b. After interacting with the GCW sensor100in the microwell plate1204b, the polarization-modulated light beam126bwhich has only one of the two polarization states resonant within the GCW sensor100is converted into the amplitude modulated light beam128bthat is modulated at the same frequency as the polarization-modulated light beam126b. The amplitude modulated light beam128bis received by the detection system124band in particular by a scanning pinhole plate1202bthat scans the angle of the amplitude modulated light beam128b. The detector1210b(e.g., photodiode1210b) converts the scanned amplitude modulated light beam128binto an electrical signal that is then demodulated by a lock-in amplifier1212b. The lock-in amplifier1212bdemodulates the electrical signal by responding to signals at the modulation frequency of the polarization-modulated light beam126band ignoring noise affecting the signals outside the modulation frequency. A computer1216bthen analyzes the demodulated electrical signal (e.g., a DC voltage proportional to the original modulated reflected optical signal strength) to determine a resonant angle that correlates to the superstrate103refractive index and therefore the biological substance content102at the sensing region103of the GCW sensor100. The frequency being detected by the lock-in amplifier1212bcan be derived from a function generator1214bthat also drives the polarization modulator127b.

Referring toFIG. 13, there is a diagram illustrating the basic components of a third embodiment of the optical interrogation system120c. The optical interrogation system120cutilizes a wavelength scanning approach to scan the polarization-modulated light beam126cdirected into the GCW sensor100in order to enable the detection of the biological substance102. As shown, the optical interrogation system120cincludes a broadband light source122cthat outputs a polarized light beam125cthat is received by a polarization modulator127c(e.g., photoelastic modulator127c). The polarization modulator127cmodulates the polarized light beam125cby causing a time-varying polarization alternation between TE and TM modes (see the wobbling vectors inFIG. 13). The polarization modulator127coutputs the polarization-modulated light beam126cto a tunable filter1306c. The tunable filter1306cscans the wavelength of the polarization-modulated light beam126cand directs the scanned polarization-modulated light beam126cto a beamsplitter1308c. The beamsplitter1308cdirects the polarization-modulated light beam126cto the GCW sensor100within the microwell plate1304c. After interacting with the GCW sensor100in the microwell plate1304c, the polarization-modulated light beam126cwhich has only one of the two polarization states resonant within the GCW sensor100is converted into the amplitude modulated light beam128cthat is modulated at the same frequency as the polarization-modulated light beam126c. The amplitude modulated light beam128cis received by the detection system124band in particular by a detector1310c(e.g., photodiode1310c) which converts the amplitude modulated light beam128cinto an electrical signal that is then demodulated by a lock-in amplifier1312c. The lock-in amplifier1212cdemodulates the electrical signal by responding to signals at the modulation frequency of the polarization-modulated light beam126cand ignoring noise affecting the signals outside the modulation frequency. A computer1316cthen analyzes the demodulated electrical signal (e.g., a DC voltage proportional to the original modulated reflected optical signal strength) to determine a resonant angle that correlates to the superstrate103refractive index and therefore the biological substance content102at the sensing region103of the GCW sensor100. The frequency being detected by the lock-in amplifier1312ccan be derived from a function generator1314cthat also drives the polarization modulator127c.

Referring toFIG. 14, there is a diagram illustrating the basic components of a fourth embodiment of the optical interrogation system120d. The optical interrogation system120dutilizes an wavelength scanning approach to scan the amplitude modulated light beam128demitted from the GCW sensor100in order to enable the detection of the biological substance102. As shown, the optical interrogation system120dincludes a broadband light source122dthat outputs a polarized light beam125dthat is received by a polarization modulator127d(e.g., photoelastic modulator127d). The polarization modulator127dmodulates the polarized light beam125dby causing a time-varying polarization alternation between TE and TM modes (see the wobbling vectors inFIG. 14). The polarization modulator127doutputs the polarization-modulated light beam126dto a beamsplitter1408d. The beamsplitter1408ddirects the polarization-modulated light beam126dto the GCW sensor100within the microwell plate1404d. After interacting with the GCW sensor100in the microwell plate1404d, the polarization-modulated light beam126dwhich has only one of the two polarization states resonant within the GCW sensor100is converted into the amplitude modulated light beam128dthat is modulated at the same frequency as the polarization-modulated light beam126d. The amplitude modulated light beam128dis received by the detection system124dand in particular by a scanning filter1406dthat scans the wavelength of the amplitude modulated light beam128d. The detector1410d(e.g., photodiode1210d) converts the scanned amplitude modulated light beam128dinto an electrical signal that is then demodulated by a lock-in amplifier1412d. The lock-in amplifier1412ddemodulates the electrical signal by responding to signals at the modulation frequency of the polarization-modulated light beam126dand ignoring noise affecting the signals outside the modulation frequency. A computer1416dthen analyzes the demodulated electrical signal (e.g., a DC voltage proportional to the original modulated reflected optical signal strength) to determine a resonant angle that correlates to the superstrate103refractive index and therefore the biological substance content102at the sensing region103of the GCW sensor100. The frequency being detected by the lock-in amplifier1412dcan be derived from a function generator1414dthat also drives the polarization modulator127d.

Referring toFIG. 15, there is a diagram illustrating the basic components of a fifth embodiment of the optical interrogation system120e. The optical interrogation system120eutilizes a parallel angle/wavelength scanning approach to scan multiple polarization-modulated light beams126edirected to multiple GCW sensors100to enable the high-throughput detection of biological substances102. As shown, the optical interrogation system120eincludes a light source122ethat outputs a polarized light beam125ethat is received by a polarization modulator127e(e.g., photoelastic modulator127e). The polarization modulator127emodulates the polarized light beam125eby causing a time-varying polarization alternation between TE and TM modes and outputs the polarization-modulated light beam126eto a diffractive optic1502e. The diffractive optic1502eemits an array of polarization-modulated light beams126eto a beamsplitter1508e. The beamsplitter1508edirects the polarization-modulated light beams126eto the GCW sensors110within the microwell plate1504d. After interacting with the GCW sensors100in the microwell plate1504e, the polarization-modulated light beams126dare converted into the amplitude modulated light beams128ethat are modulated at the same frequency as the polarization-modulated light beams126e. The amplitude modulated light beams128eare received by the detection system124eand in particular by an array of detectors1510e(e.g., photodiodes1510e) which converts the scanned amplitude modulated light beams128einto electrical signals that are then demodulated by an array of lock-in amplifiers1512e. The lock-in amplifiers1512edemodulate the electrical signals by responding to signals at the modulation frequency of the polarization-modulated light beam126eand ignoring noise affecting the signals outside the modulation frequency. A computer1516ethen analyzes the demodulated electrical signal (e.g., a DC voltage proportional to the original modulated reflected optical signal strength) to determine a resonant angle that correlates to the superstrate103refractive index and therefore the biological substance content102at the sensing region103of the GCW sensor100. The frequency being detected by the lock-in amplifiers1512ecan be derived from a function generator1514ethat also drives the polarization modulator127e. It should be appreciated that the configuration of this optical interrogation system120eis only one of many different possible configurations that can be used in high-throughput screening applications. Basically, this invention can be applied to all multiplexed, scanning, etc. systems that are compatible with polarization modulation.

AlthoughFIG. 15does not explicitly show the components required to scan the angle or wavelength as per one of the many embodiments shown inFIGS. 7,12,13, or14. It is assumed that one of these scanning techniques (or a suitable substitute) would be employed together with the components ofFIG. 15to achieve a system that scans the GCW100resonance over every well. For the embodiments ofFIGS. 7 and 13, only one scanning element (acousto-optic modulator or filter) need be placed before the diffractive optic, thereby copying the scanning behavior to each separate beam. In the embodiments ofFIGS. 12 and 14, the scanning apparatus (pinhole or filter) would likely be required in duplicate for each beam reflected from each well of the plate.

Referring toFIG. 16, there is a flowchart illustrating the basic steps of a preferred method for using the optical interrogation system120and GCW sensor100to detect a biological substance102in accordance with the present invention. Although the GCW sensor100and optical interrogation system120are described herein as being used to detect the presence of biological substances102like cells, molecules, proteins, drugs, chemical compounds, nucleic acids, peptides or carbohydrates on the surfaces104of the GWC sensor100, it should be understood that the GCW sensor100and optical interrogation system120can be used to perform a wide variety of studies. For example, the GCW sensor100and optical interrogation system120can be used to perform cell migration assays, drug permeability assays, drug solubility studies, virus detection studies and protein secretion studies.

Beginning at step1602, the light source122and polarization modulator127are used to direct a polarization-modulated light beam126to the GCW sensor100. At step1604, the detection system124receives an amplitude modulated light beam128from the GCW sensor100. Then at step1606, the detection system124demodulates and analyzes the received amplitude modulated light beam128to detect a resonant wavelength or resonant angle which corresponds to a superstrate103refractive index that indicates whether a biological substance102is located on the surface104of the GCW sensor100.

Following are some advantages and uses of the optical interrogation system120and GCW sensors100of the present invention:The use of polarization modulation in accordance with the present invention enables one to use large-area photodiodes that provides a cost advantage and robustness to the instrument design not currently available with traditional optical interrogation systems. Moreover, the use of large area detectors greatly relaxes the performance (flatness, etc.) required from the sensor wellplate. Basically, the reflected beam simply has to hit the photodiode somewhere in its area to be correctly detected and decoded. The only constraint in this situation is that the beams from separate wells do not “cross” each other in the farfield. In other words, each beam should retain its relationship relative to its neighbors, although neighbor proximity may vary from well to well. This is a much less stringent condition than would be required for coupling back into optical fiber, for example, or imaging the reflections onto CCDs used by traditional optical interrogation systems.The use of polarization modulation in accordance with the present invention can be applied to nearly every instrument designed to interrogate grating-coupled biosensors. The invention involves the overlay of polarization modulation and coherent (phase-sensitive) detection onto the structures of the traditional optical interrogation systems. This is true regardless of whether angle, wavelength or some other parameter is being scanned, either on the input or output of the GCW sensor100.The present invention removes a large number of noise sources by taking advantage of optical beam polarization.The present invention drastically reduces the required power for detection of biological substances. As a result, the milliwatts of power typically required of the output beam for sensitive detection by a CCD element is reduced several orders of magnitude under this invention. This is very important for the high-throughput screening market, since 96, 384, or even 1536 wells may be interrogated in parallel with the same optical (laser) source.In addition to the signal-to-noise improvements associated with the present invention, there are several other equally important benefits of the technology. First, because the polarization modulation technique obtains high signal-to-noise data from ordinary photodiodes, the cost and complexity of the instrumentation can be greatly reduced compared to the expensive CCD or spectrograph solutions used by traditional optical interrogation systems. Moreover, the scale-up of the optical interrogation system120to accommodate 96 or 384 wells in a plate becomes much easier since one only needs to use more inexpensive detectors and lock-in amplifiers.In addition to the cost and complexity advantage of the present invention, the use of lock-in amplifiers provides phase information about the resonance previously unavailable. The presence of both amplitude and phase information from the phase-sensitive detection provides an extra observable. As discussed above, this phase information actually contains a convenient and unique signal that indicates the resonance location.The polarization modulation concept of the present invention can be implemented within most interrogation schemes (e.g., angular or wavelength-based approaches). It simply requires that the polarization be modulated in order to distinguish the waveguide output from noise, somewhat independent of the other variables of the system. In one embodiment of this invention, one could modulate the input beam polarization much faster than the angular scanning rate, such that the angular position is essentially constant during the demodulation process for each angular step.As described above, the preferred modulation method is photoelastic, where a quartz plate is vibrated to produce time-variable birefringence due to the photoelastic effect. This technology is preferred both for the purity of polarization modulation as well as high available modulation frequency, 100 kHz.Although the preferred embodiments of the present invention described above utilized a reflected light beam to enable the detection of the biological substance, it should be readily appreciated that a transmitted beam and even a beam exiting the side of the sensor could also be used to detect the biological substance. Of course, minor changes to the set-up of the system would be required to detect the transmitted beam or the beam exiting the side of the sensor.