Abstract:
A system and method for sensitive measurements of molecular binding as a function of time and temperature provided by a surface plasmon resonance (SPR) instrument producing a stable temperature gradient over the sensor surface. Continuous monitoring of reflected light intensity from different locations of the sensor allows simultaneous measurement of time and temperature dependence of binding interactions.

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application claims the benefit of U.S. provisional application 62/257,876 filed Nov. 20, 2015 and U.S. provisional application 62/206,083 filed Aug. 17, 2015 both of which are hereby incorporated by reference. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     This invention was made with United States government support awarded by the following agency: National Science Foundation (NSF) 1152042, The United States government has certain rights in this invention. 
    
    
     BACKGROUND OF THE INVENTION 
     The present invention relates to instruments and methods for the temperature dependent analysis of nucleic acids, peptides, carbohydrates, and the like (hereinafter biological oligomers) and in particular to an instrument that can assist in quantitative analysis of binding interactions of such oligomers. 
     Measuring the thermodynamics of interactions involving nucleic acids and peptides is a vital component of biomolecular studies. Conventionally, these properties are analyzed in solution by temperature dependent spectroscopic measurement (e.g., UV-vis, CD, fluorescence) or thermal methods (ITC, DSC). See generally (1) Mergny, J. L.; Lacroix, L. Oligonucleotides 2003, 13, 515-537 and Jelesarov, I.; Bosshard. H. R. J. Mol. Recognit. 1999, 12, 3-18. For example, DNA thermal denaturation profiles obtained by monitoring changes in UV absorption as the temperature of the solution is incremented can be used to generate robust models for predicting the stabilities of DNA structures in solutions. 
     Surface-based diagnostics, such as surface plasmon resonance (SPR), have been used to provide analysis of binding kinetics and affinities of biomolecular interactions at a single temperature. In SPR, a nanoscale thin metallic film is illuminated from the back “reflecting side” of the film. At a certain angle, known as the plasmon angle, the energy from the illumination is coupled into electromagnetic waves creating a resonant condition (surface plasmon resonance) that is highly sensitive to surface conditions on the “sensing side” of the film opposite to the reflecting side. To the extent that SPR can directly detect linking between probe molecules on the film and target molecules in a solution, largely eliminates the need to use fluorescent or enzymatic tags for monitoring such linking. 
     Specifically, in SPR imaging, multiple probe spots of a micro-array may be analyzed in parallel, with different biological oligomer probes attached over the surface. In this technique of SPR imaging, p-polarized light impinges on a prism, a gold thin film, and a flow cell assembly at a fixed angle. The reflected light is passed through a narrow band pass filter and collected by a CCD camera. In this technique, probe molecules are covalently linked to discrete positions on the sensing side of the film to selectively bind with target molecules in the solution to be analyzed. The binding of the targets to the surface bound probes causes localized changes in the index of refraction at those spots, which are detected by the CCD camera. This data can then be used to identify the presence and composition of the target molecules by the location of the detected binding event. 
     SPR experiments can be repeated at different temperatures to provide a better understanding of interactions between biological oligomers, for example, indicating the temperatures at which binding interactions occur or providing plots showing how sensor binding kinetics varies with temperature. Because SPR measurements are highly sensitive to temperature variation, for each experiment the SPR apparatus is normally stabilized to a uniform temperature. Changing this uniform temperature between measurements is normally accompanied by an extended stabilization time during which the apparatus acclimates to a uniform and precise temperature greatly slowing the acquisition of data. 
     SUMMARY OF THE INVENTION 
     The present invention provides an SPR instrument that can create a stable and precise thermal gradient across its sensing surface allowing parallel measurements at different temperatures to be simultaneously received, greatly speeding the measurement process. Although the temperature across the sensing surface varies, the temperature at any specific location can be held constant allowing established SPR techniques to quantify biological oligomer interactions by separating out changes in reflectivity caused by molecular associations and disassociations from changes in reflectivity caused by changes in temperature. The temperature profile of the gradient is determined through a calibration process developed for the specific apparatus. 
     Specifically then, one embodiment of the invention provides an apparatus for evaluating interactions between biological oligomers having a light source, a camera, and an optical coupler positioned between the light source and the camera to receive light from the light source for reflection from an active face of the optical coupler to be received by the camera. A metal film extends along a measurement plane proximate to the active face for holding oligomers subject to interaction on a first face of the metal film, and a flow channel opens against the first face of the metal film for receiving a liquid flow in a direction therealong. First and second thermal stabilizers are positioned in thermal communication with the metal film and separated along the measurement plane near opposite edges of the metal film and adapted to establish a predetermined and substantially constant temperature gradient along the metal film. 
     It is thus a feature of at least one embodiment of the invention to provide a mechanism for rapid acquisition of temperature-related phenomenon using a temperature gradient that is sufficiently stable to prevent the introduction of confounding temperature errors. By providing a sufficiently constant flow rate through the flow channel and low thermal impedance thermal stabilizers, the present inventor has determined that a sufficiently stable temperature gradient can be produced for this purpose. 
     The apparatus may include a controller attached to the first and second thermal stabilizers for controlling the first and second thermal stabilizers in a first state to have different temperatures to establish a substantially constant temperature gradient along the metal film and in a second state to have identical temperatures to establish a gradient-free, substantially constant temperature along the metal film. 
     It is thus a feature of at least one embodiment of the invention to provide a versatile temperature control that allows both the measurement of temperature-sensitive binding experiments and that permits development of the necessary calibration curves enabling this precise measurement. 
     The thermal stabilizers may be separated along the direction of fluid flow to establish a temperature gradient in the direction of fluid flow. The thermal stabilizers may also be separated in a direction orthogonal to the direction of fluid flow to establish a temperature gradient across the sensor. 
     It is thus a feature of at least one embodiment of the invention to provide longitudinally consistent temperature gradients perpendicular to the fluid flow for simplifying data averaging and the like. 
     The apparatus may include an electronic computer operating to: (a) image reflectivity from the active face of the optical coupler at multiple points over at least one dimension on the surface of the second face aligned with the temperature gradient; and (b) compare the imaged reflectivity to gradient calibration data indicating a temperature associated with each of the multiple points to provide an indication of binding as a function of temperature. 
     It is thus a feature of at least one embodiment of the invention to accommodate arbitrary but temporally constant gradient shapes through the use of calibration curves indicating the temperature at different points on the surface of the active face of the optocoupler. 
     The camera may image reflectivity of the active face of the optical coupler in two dimensions, a first dimension parallel to a temperature gradient, and a second dimension perpendicular to the temperature gradient, and the computer may combine data along the second dimension before analysis. 
     It is thus a feature of at least one embodiment of the invention to provide more robust measurements through the ability to combine data sampled across the fluid flow such as experience a similar temperature. 
     The flow cell may include at least one flow path along the metal film and where the flow cell is split into additional channels separated by a wall preventing mixing of liquid between each flow path. Each additional channel may include a binding material to interact with a biological oligonucleotide on the test surface. The electronic computer may further image reflectivity from the active face of the optical coupler at multiple points over at least one dimension on the active face aligned with the temperature gradient images for each of the flow paths and may use multiple points to deduce temperature along each flow path for establishing the gradient calibration data. 
     It is thus a feature of at least one embodiment of the invention to allow different analyte solutions to be flown over the sensor at the same time, facilitating parallel measurement of solutions (efficiency). 
     A first channel may have a binding material and a second channel may not contain binding material, thus, the second channel is used as reference for background subtraction. 
     It is thus a feature of at least one embodiment of the invention to permit measurements for generating temperature gradient calibration data. One of the flow cell channels can be used as a reference channel to act as “background” or reference. The background subtraction accounts for things like change in brightness of the light source or sensitivity of the camera over time. 
     The stored program may provide a display outputting information providing indication of binding as a function of both time and temperature. 
     It is thus a feature of at least one embodiment of the invention to permit simultaneous measurements over a range of temperatures as a function of time. 
     The first and second thermal stabilizers may be associated with temperature sensors and control circuitry to operate the first and second thermal stabilizers in closed loop fashion to provide predetermined temperatures. 
     It is thus a feature of at least one embodiment of the invention to provide extremely precise temperature control possible with high gain feedback. 
     The flow channel may have a thermal conductivity of at least 0.8 W/mK. 
     It is thus a feature of at least one embodiment of the invention to increase the thermal connection between the thermal stabilizers and the flowing liquid by pre-tempering the liquid flowing past the test surface for greater flexibility in temperature range measurement. 
     These particular objects and advantages may apply to only some embodiments falling within the claims and thus do not define the scope of the invention. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a cross-sectional view of an apparatus constructed according to the present invention showing an optical coupler communicating between a light source and camera to measure reflected light in the optical coupler adjacent to metal film where metal film is exposed to a flow channel, and showing thermal stabilizers positioned along the flow channel for establishing a thermal gradient at the metal film, and further showing the temperature profile of the thermal gradient, a simplified image collected by the camera, and measured reflectance from that image, as well as a connected computer and display for displaying output data; 
         FIG. 2  is a simplified depiction of different oligomer binding behavior as a function of temperature along the metal film; 
         FIG. 3  is a flowchart of the steps for calibrating and using the apparatus of  FIG. 1 ; 
         FIG. 4  is an example graph output on the display of  FIG. 1  providing both binding kinetics and strength measurements; 
         FIG. 5  a perspective view of the apparatus of  FIG. 1  showing the use of separate flow channels for dynamic calibration; 
         FIG. 6  is a perspective representation of an alternative thermal stabilizer using heat exchanger blocks; 
         FIG. 7  is a diagram of a pump system for providing constant flow rates to the flow channel of  FIG. 1 ; 
         FIG. 8  is a perspective view and fragmentary cross-section of a bottom surface of the optical coupler providing a striping of probe molecules for refined parallel binding measurements; and 
         FIG. 9  is a figure similar to that of  FIG. 1  showing an alternative flow cell arrangement useful for surface plasmon enhanced fluorescence spectroscopy (SPFS) and otherwise identical to  FIG. 1 . 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     Referring now to  FIG. 1 , a temperature gradient surface plasmon resonance (SPR) device  10  may include a flow conduit  12  providing a channel  14  conducting a fluid along an x-axis direction. An upper wall of the channel  14  opens toward a transparent optical coupler  16 , for example, a right angle prism, whose base  18  defines a measurement plane  20 . 
     Attached to or proximate to the base  18  is a metal film  22  (shown greatly exaggerated for clarity), for example, of metallic gold. The metal film  22  is sized to support plasma waves on its lower active surface  24  as stimulated by evanescent waves from light internally reflected off of the base  18  of the optical coupler  16 . 
     The upper wall of the channel  14  is sized so that a lower active surface  24  of the metal film  22  facing the channel  14  is in contact with a fluid within the channel  14 . This lower active surface  24  may be spotted at probe locations with probe molecules  26  such as biological oligomers in a regular pattern over two dimensions within the measurement plane  20 . For a 15 nucleotide probe sequence as the probe molecule  26 , an average spacing of about three nanometers between probes provides the optimal combination of sensor activity and hybrid stability. This spacing can be identified by using the present invention to monitor binding strength as a function of spacing. A spacing may then be enforced for example through co-adsorption with a second component that acts as a lateral spacer. 
     A collimated light source  30  (for example, white light) is positioned to direct light through a first upward sloping face of the optical coupler  16  to illuminate an active area of the base  18  stimulating plasmon activity in the metal film  22  adjacent to the base  18  over which the probe molecules  26  are dispersed. Before being received by the optical coupler  16 , the light passes through a monochromator/polarizer  32  which filters the light to a desired narrowband frequency and provides polarization as is generally understood in the art. The light from the light source  30  strikes the base  18  at an adjustable angle θ with respect to a normal  34  to the measurement plane  20 . Either or both of the angle θ and the frequency of light from the collimated light source  30  may be adjusted, the latter, for example, by using various bandpass filters (e.g. 770 nanometers, 780 nanometers, 790 nanometers) or other frequency adjustments. These changes may be used to optimize the percent reflectivity value over the temperature gradient as discussed below. 
     A portion of the light striking the base  18  is coupled into plasmons in the metal film  22 . The remaining light reflects internally off the base  18  at an angle θ′ equal and opposite to angle θ, to be received through a narrow band pass filter  36  by a CCD camera  38 . The CCD camera  38  is focused on the measurement plane  20  of the base  18  to provide an image of the reflected light from the base  18 . 
     In one embodiment, the light source  30  may be a tungsten halogen light source and the monochromator/polarizer  32  may provide for a  1200  groove/mm grating with a 750 nm blaze to provide a narrow band light within the range from 770 to 800 nanometers. The light source  30  may include optics to collimate the light source to an approximately 1 in. diameter beam. The CCD camera  38  may provide for a CMOS image sensor running a resolution of approximately 600 by 490 lines with a 35 mm lens. The filter  36  may provide a 785 nm bandpass filter (FWHM=45 nm) to reduce stray light, while still allowing use of monochromated light ranging from 770 to 800 nm. 
     The flow conduit  12  may be constructed of stainless steel having a total channel volume of less than 20 μL to provide uniform flow of liquid at flow rates of less than 50 UL/min using a peristaltic pump (not shown). The peristaltic pump may be associated with flow-smoothing elements such as accumulators and flow restrictors of a type known in the art to provide a nearly constant flow through the flow conduit  12 . 
     The CCD camera  38  may communicate with an electronic computer  40  including a processor  42  reading and writing data with a memory  45  holding a stored program  48  whose operation will be described below. Memory  45  may also include calibration curves  47  and measurement data  49  to be acquired by the SPR device  10  as will be discussed below. This measurement data  49  may be displayed on an attached display  50  providing for graphic display capabilities. 
     Positioned against the lower surface of the flow conduit  12 , in thermal communication therewith and thus in thermal communication with the liquid  46  in the flow conduit  12  and the metal film  22 , are first and second thermal stabilizers  44   a  and  44   b . In one embodiment the first and second thermal stabilizers  44   a  and  44   b  may be Peltier devices receiving DC electrical power controlled in polarity and/or voltage by a temperature controller  54  providing feedback regulation of the voltage and current to the Peltier devices (and hence constant temperature) under the higher level control of the electronic computer  40 . The thermal stabilizers  44   a  and  44  are displaced along the measurement plane  20  to be positioned beneath opposite sides of the metal film  22  so to apply a thermal gradient to the metal film  22  along the measurement plane  20  in the direction of fluid flow through the flow conduit  12 . 
     Generally thermal stabilizer  44   a  will be biased to cool the lower surface of the flow conduit  12  on an entrance side of the flow conduit  12  that receives liquid  46 , whereas thermal stabilizer  44   b  will be biased to provide heat to a lower surface of the flow conduit  12  on a exit side of the flow conduit  12  out of which liquid flows. Temperature sensing elements  51  may be positioned at an interface between the thermal stabilizers  44  and the flow conduit  12  to measure temperature of the flow conduit  12  and to allow closed-loop temperature control of the flow conduit  12 . For this purpose, the temperature sensing elements  51  provide feedback signals to the temperature controller  54  which may then control the thermal stabilizers  44 , for example, by controlling the voltage or duty cycle of the voltage, independently for each thermal stabilizer  44  to provide a desired temperature value. 
     The construction of a flow conduit  12  from highly conductive metal such as stainless steel having a thermal resistance of at least 12 W/mK, or an even a more conductive material, and the positioning of portions of the channel  14  before and after its contact with the metal film  22  near the thermal stabilizers  44 , helps provide for a pre-cooling and post-heating of the liquid  46  flowing through the channel  14  by the thermal stabilizers  44 . Glass having a thermal resistance of 0.8 W/mK may also be used. This pre-cooling and post-heating reduces the effect of liquid flow on the temperature gradient established between the thermal stabilizers  44  and provides greater versatility in the starting and ending temperatures of the temperature gradient. 
     In one embodiment, thermal stabilizers  44  may each be 24-watt rectangular thermoelectric/Peltier modules mounted underneath the flow cell and separated from each other by approximately 6 mm, with the temperature of each monitored using 10 kΩ thermistors embedded in the body of the flow conduit  12  directly above each thermal stabilizer  44 . 
     A water cooling block  60  having an internal channel  62  for receiving a flow of temperature regulated water is mounted in thermal contact with the lower surface of the thermal stabilizers  44  opposite its contact with the flow conduit. This water cooling block  60  provides a source or sink for excess heat. Heating of the opposite ends of the flow conduit  12  over the range 20 to 80° C. (±0.02° C.) may be achieved. 
     In this way, the SPR device  10  may be operated during flow of liquid  46  to provide a temperature gradient  64  as a function of distance along the x-axis along the lower surface of the metal film  22  varying between approximately 20 degrees centigrade at the leftmost edge (as depicted) of the metal film  22  exposed to the channel  14  to about 60 degrees centigrade at the rightmost edge (as depicted) of the metal film  22  exposed the channel  14 . The temperature gradient may be approximately linear, but linearity is not required. 
     The light source  30  (and optics) and camera  38  (and optics) are attached to independent motorized rotation stages (not shown) to allow adjustment of the angles θ of the light and its frequency under control of electronic computer  40 . 
     Referring now to  FIG. 2 , during operation, the liquid  46  flowing past the lower active surface  24  of the metal film  22  may suspend biological oligomers  66  that may bind with the probe molecules  26  attached to the lower surface of the metal film  22 . The amount of binding will depend on the local temperature of the liquid and probe molecules  26  and  66  along the temperature gradient along the metal film  22 . 
     The different amounts of binding will be detectable in changes in the index of refraction in the environment adjacent to the lower surface of the metal film  22  in the vicinity of the probe molecules  26 . These changes in index of refraction will change the amount of reflected light at the base  18  of the optical coupler  16  through the mechanism of surface plasmon resonance. In turn, this change in reflected light will be captured in a reflection image  68  by the camera  38  measuring at different pixels of the reflection image  68  different intensities of reflected light. Generally these intensities will vary along the x-axis because of the effect of the temperature gradient on the binding interaction. 
     The quantitative assessment of this image  68  along any line parallel to the x-axis will yield a reflection curve  70  showing generally (for example) greater reflection with greater binding. Analysis of this reflection curve  70 , for example, may be used to determine the temperatures at which binding occurs by looking for a location along the x-axis showing an abrupt change in light reflection related to binding and relating that x-axis location to a temperature per the temperature gradient. This temperature provides insight into the binding strength of the probe molecules  26  and  66 . 
     The time evolution of the image  68  permits a second measurement of binding kinetics, for example, showing how the binding changes with time as fluid flows through channel  14 . 
     Data related both to binding kinetics and binding strength may be simultaneously displayed on the display  50  (shown in  FIG. 1 ) in the form of a three-dimensional representation  72  providing a measure of binding (for example, per reflection) along a vertical axis and time in temperature along two orthogonal horizontal axes. 
     Referring now to  FIGS. 1 and 3 , because the light reflected off of the base  18  will be dependent both on binding to the sensor and on the temperature of the liquid  46  at each point along the base  18 , it is necessary to know the temperature gradient  64  with high accuracy so as to be able to separate these two effects. For this reason, use of the SPR device  10  will be preceded by a calibration procedure producing one or more calibration curves  47  that can be held in the memory  45  and used to provide necessary calibration to the SPR device  10 . Generally these calibration curves  47  will provide for data necessary to accurately determine the temperature gradient  64  and will also include other measurements used to convert reflectivity into a measure of binding as will be discussed. 
     As indicated by process block  80 , a first set of calibration curves  47  establishes the relationship between the temperature of the liquid  46  and reflectivity necessary to measure temperature gradient  64  across the lower active surface  24  using reflectivity. In this calibration process both thermal stabilizers  44   a  and  44   b  are controlled to provide the same temperature eliminating the gradient across the metal film  22 . A solution approximating that which will be used during binding experiments (that is, without the biological oligomers) is then passed through the channel  14  and reflectance measured using the camera  38 . After each reflectance measurement, the temperature of the thermal stabilizers  44   a  and  44   b  are incremented so that a set of reflectance values may be obtained for a range of temperatures, for example, from 20 to 60 degrees Celsius. 
     The liquid  46 , for this purpose may be an aqueous solution of specific ionic strength and acidity. An example is NaCl-TE containing between 0.1 and 1.0 moles per liter of sodium chloride and trace amounts of Tris-HCL and EDTA adjusted to a pH of 7. 
     As indicated by process block  82  these temperature/reflectance values are then used to measure the actual temperature gradient  64  to be provided by the thermal stabilizers  44   a  and  44   b  by adjusting each of the thermal stabilizers  44  to different temperatures (for example, to 20 degrees for thermal stabilizer  44   a  and to 60 degrees for thermal stabilizer  44   b , respectively) and flowing the same liquid NaCl-TE along the metal film  22  to collect reflectivity measurements. At this time, the reflectivity will generally vary in the image  68  as a function of position along the x-axis. These reflectivity measurements may then be matched to corresponding reflectivities for particular temperatures per the previously acquired calibration data  47  to deduce the temperature gradient  64 . This temperature gradient  64  may provide a temperature gradient calibration data  47  also stored in the memory  45 . 
     At process block  84 , measurement of sensor binding may employ successive measurements made with three different liquids  46   a ,  46   b  and  46   c . The first two liquids  46   a  and  46   b  each provide an oligomer-free solution, for example, of sodium chloride as described above. A first of these two liquids  46   a  will be substantially identical to the third liquid  46   c  used during the binding process being investigated while a second of these two liquids  46   b  will have a slightly different index of refraction, for example, containing an additional 0.050 moles per liter of sodium chloride. The refractive indices of these liquids  46   a  (and  46   c ) and  46   b  may be determined by separate measurements (for example, using an Abbe refractometer) to determine the refractive indices n 1  and n 2 . 
     A third measurement is then made using liquid  46   c  being substantially identical to the first of the two liquids  46   a  but also including the reacting oligomer  66  shown in  FIG. 2 . Each of these measurements produces an image  68  that may be stored in memory  45 . 
     A change of index Δn hyb  each point of the images  68  may then be computed by combining the intensities of reflectivities measured by each of these images  68  as follows: 
     
       
         
           
             
               
                 
                   
                     Δ 
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     
                       n 
                       hyb 
                     
                   
                   = 
                   
                     
                       ( 
                       
                         
                           
                             I 
                             hyb 
                           
                           - 
                           
                             I 
                             1 
                           
                         
                         
                           
                             I 
                             2 
                           
                           - 
                           
                             I 
                             1 
                           
                         
                       
                       ) 
                     
                     × 
                     
                       ( 
                       
                         
                           n 
                           2 
                         
                         - 
                         
                           n 
                           1 
                         
                       
                       ) 
                     
                   
                 
               
               
                 
                   ( 
                   1 
                   ) 
                 
               
             
           
         
       
     
     where I 1 , I 2  and I hyb  are the measured intensities of reflection (at corresponding points in each image  68 ) for each of the oligomer-free liquids  46   a ,  46   b , and  46   c , respectively, and n 1  and n 2  are the indices of refraction of the oligomer-free liquids  46   a  (and  46   c ) and  46   b , respectively. 
     At process block  86  an amount of sensor binding, may be computed using the following formula (for example, for a DNA hybridization measurement where probe molecules  26  and  66  are DNA oligomers and sensor binding is reported as hybrids-cm 2 ): 
     
       
         
           
             
               hybrids 
               · 
               
                 cm 
                 
                   - 
                   2 
                 
               
             
             = 
             
               
                 ( 
                 
                   
                     l 
                     d 
                   
                   2 
                 
                 ) 
               
               × 
               
                 ( 
                 
                   
                     Δ 
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     
                       n 
                       DNA 
                     
                   
                   
                     
                       n 
                       DNA 
                     
                     - 
                     
                       n 
                       b 
                     
                   
                 
                 ) 
               
               × 
               
                 ( 
                 
                   
                     
                       ρ 
                       DNA 
                     
                     × 
                     
                       N 
                       A 
                     
                   
                   
                     M 
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     W 
                   
                 
                 ) 
               
             
           
         
       
     
     where: 
     l d  is the decay length of the evanescent wave after reflection of light in the optical coupler  16  (estimated to be 350 nanometers if the light wavelength is 780 nanometers); 
     n a -n s  is the difference in index of refraction between the liquid  46   c  (n a ) containing the oligomer  66  and liquid (n s ) not containing the oligomer  66  (estimated to be 1.7); 
     ρ DNA  is the bulk density of the oligomer (estimated to be 1.7 grams/cm 3  for DNA); 
     MW is the molecular weight of the oligomer. 
     At process block  88 , these values hybrids·cm −2  characterized for each point of the image  68  (or another reflectance-based value) may be output, for example, in the form of the graph shown in  FIG. 4 . Quantitative representations of these values hybrids·cm −2  in the form of tables or the like or two-dimensional graphs representing cross-sections through the three-dimensional figure shown in  FIG. 4  may also be provided. 
     During each of these measurements of reflectance, the angles θ and θ′ may be adjusted to provide maximum sensitivity to temperature changes in the central portion of the gradient. Desirably the angles θ are set to provide a percentage of reflectivity of between 30 and 60 percent of its maximum value. It will be appreciated that optical systems can be developed to allow separate angles to be provided for different x-axis portions of the gradient. Alternatively, measurements may be made at multiple angles or wavelengths of light for different portions of the gradient. 
     Each of these measurements also may be repeated using different wavelengths of light, for example, 780 nanometers, 770 nanometers, and 790 nanometers affecting each of the process blocks  80 ,  82 , and  84  for providing higher accuracy at the beginning and ending of the gradient. Data from multiple measurements may be combined using statistical techniques such as averaging. Further, data sampled along points of constant x-axis (for example, along the y-axis) may be combined, for example, by averaging to improve these measurements. When computing the temperature gradient  64 , the temperature data may be fit to a sigmoidal equation to provide a temperature for each point along the y-axis. 
     Referring now to  FIG. 5 , in one embodiment, the channel  14  may be split into two parallel channels  14   a  and  14   b  divided by a wall  90  extending longitudinally along the direction of fluid flow and separating the channels  14   a  and  14   b  to prevent intermixing of the fluid flow in the parallel channels  14   a  and  14   b . Reflectance data above each of parallel channels  14   a  and  14   b  may be separately imaged as image portions  68   a  and  68   b  shown in  FIG. 1 . 
     During process block  84 , when the sensor binding experiment is being performed, the oligomer  66  may be introduced only in the channel  14   a , and channel  14   b  used for the purpose of monitoring real time changes attributed to small temperature variations or other instrumental parameters. 
     Referring now to  FIG. 6 , it will be appreciated that the thermal stabilizers  44  need not be Peltier devices but may make use of any known thermal stabilization technique, for example, using heat exchangers  92  receiving circulated liquid through conduits  94  communicating with a reservoir  96  of temperature stabilize liquid, the latter temperature controlled, for example, by conventional heater thermostat systems or chillers known in the art. 
     Referring now to  FIGS. 1 and 7 , the stability of the temperature gradient  64  is strongly influenced by the constancy of the flow rate of solution along the metal film  22 . In one embodiment, the invention contemplates a hybrid pump system  100  providing liquid  46  for introduction into the channel  14 . The hybrid pump system  100  includes a peristaltic pump  102  feeding liquid  46  from a reservoir through a flexible tube  104  progressively compressed by a roller mechanism  106  to pinch off the tube  104  and propel the contained liquid toward a diverter valve  108  which may be set to send the liquid to the channel  14 . The roller mechanism  106  may be powered by a constant speed DC electric motor  109 . This mode may be useful for calibration processes where constant temperature liquid is required and hence slight pulsation or variation in the flow rate may be tolerated. During temperature gradient measurements, diverter valve  108  is changed in position to provide a source of liquid to the channel  14  from a syringe pump  110  having a syringe  112  containing liquid  46 . The plunger  114  of the syringe  112  is advanced by means of a motor  116 , for example, driving a lead screw  118  and follower  120  or the like to expel liquid from the syringe  112 . The general linear and continuous nature of the syringe pump  110  is believed to provide a more consistent flow rate without the need for accumulating reservoirs or the like such as increase the needed sample size. 
     Referring now to  FIG. 8 , the flow channels  14   a  and  14   b  provided in the embodiment of  FIG. 5  can contain multiple linear regions  122  of probe molecules  26  separated by empty exclusion rows  124  that serve to establish multiple zones providing the image portions  68   a - 68   e  (only a limited number of portion shown for clarity) for the purpose of temperature correction described above with respect to  FIG. 5 . That is, imaging of the exclusion rows  124  may be used to monitor changes in background values and imaging of the individual linear regions  122  may be used to simultaneously determine binding of the probe molecules  26  in each region. For this purpose, the electronic computer  40  uses a mask applied to the acquired image and separating these two regions for different analysis purposes. In addition, each of the linear regions  122  may have the same or different probe molecules  26  for parallel analysis of different systems. 
     The linear regions  122  may be created, for example, by selective application of materials and probe molecules  26  to the bottom of the optical coupler  16 . In one embodiment, the process may be an additive process employing a mask. For example, a two nanometer titanium layer  126  and a 50 nanometer gold layer  128  may be applied selectively to linear regions  122  of the lower surface of the optical coupler  16  through the use of a shadow mask. Alternatively, these linear regions  122  may be created by an etching process using a photoresist and projected mask. The probe molecules  26  are then adsorbed onto the surface of the gold layer  128 . Alternatively, linear regions  122  may be created, for example, by applying a passivating layer  130  that is resistant to nonspecific adsorption of DNA such as oligo(ethylene)glycol or mercaptohexanol, and exposing the sample to UV light through a mask to degrade the passivating layer. The probe molecules  26  are then adsorbed onto the surface of the exposed gold layer  128 . These probes may then be used in the process described above with respect to  FIGS. 1-4 . 
     Referring now to  FIG. 9 , the present invention can also be adapted for use with surface plasmon enhanced fluorescence spectroscopy (SPFS) measurements by constructing a lower wall  132  of the conduit  12  beneath the metal film  22  from an optically transparent material. This optically transparent material allows light from fluorescent materials associated with the probe molecules  26  to pass downward through the lower wall  132 , for example, to a reflector  134  to be received by a camera  136 . SPFS senses fluorescing molecules that are in the region beyond the Förster radius but within the evanescent wave penetration depth (i.e., between approximately 10 nm and 200 nm from the surface  24  of the gold metal film  22 ). For example, SPFS measurements of probe molecules  26  in the form of hairpin DNA probes  140 , can have a distal end functionalized with a fluorescent tag  142  such as WellRED D2 or IRDye800. This fluorescent tag  142  would be expected to yield a small fluorescence signal at low temperatures where the hairpin is closed (shown by probe molecule  26 ′) because of quenching from the gold of metal film  22 . The fluorescence signal would increase at high temperature when the hairpin opens because the fluorophore tagged end of the strand extends further from the surface  24 . SPFS can also be used to measure sensor responses from fluorescently labelled analytes, and in such instances would be complementary to reflectivity measurements. 
     Generally, the above described invention is not limited to the analysis of DNA or hybridization but may make measurements of other binding energies and drug interactions. For example, the present invention may be used to characterize the binding between small molecules and hybridized DNA structures. Small molecules that intercalate into the double helix structure of DNA are important for biological processes including gene regulation and have pharmacological applications as antitumor agents. The present invention may also be used to characterize binding of nanoparticles functionalized to bind with probes on the gold surface  24  such as will cause an abrupt change in index of refraction. 
     Certain terminology is used herein for purposes of reference only, and thus is not intended to be limiting. For example, terms such as “upper”, “lower”. “above”, and “below” refer to directions in the drawings to which reference is made. Terms such as “front”, “back”, “rear”, “bottom” and “side”, describe the orientation of portions of the component within a consistent but arbitrary frame of reference which is made clear by reference to the text and the associated drawings describing the component under discussion. Such terminology may include the words specifically mentioned above, derivatives thereof, and words of similar import. Similarly, the terms “first”, “second” and other such numerical terms referring to structures do not imply a sequence or order unless clearly indicated by the context. 
     The term “constant gradient” should be understood to refer to a constant spatial gradient shape with respect to time and is not limited to a gradient with a constant first derivative or linear slope. 
     When introducing elements or features of the present disclosure and the exemplary embodiments, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of such elements or features. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements or features other than those specifically noted. It is further to be understood that the method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed. 
     References to “a microprocessor” and “a processor” or “the microprocessor” and “the processor,” can be understood to include one or more microprocessors that can communicate in a stand-alone and/or a distributed environment(s), and can thus be configured to communicate via wired or wireless communications with other processors, where such one or more processor can be configured to operate on one or more processor-controlled devices that can be similar or different devices. Furthermore, references to memory, unless otherwise specified, can include one or more processor-readable and accessible memory elements and/or components that can be internal to the processor-controlled device, external to the processor-controlled device, and can be accessed via a wired or wireless network. 
     It is specifically intended that the present invention not be limited to the embodiments and illustrations contained herein and the claims should be understood to include modified forms of those embodiments including portions of the embodiments and combinations of elements of different embodiments as come within the scope of the following claims. All of the publications described herein, including patents and non-patent publications are hereby incorporated herein by reference in their entireties.