Abstract:
An apparatus and method for detection of anything to which an antibody can be raised, or to which a chemical receptor can be fashioned, based on surface plasmon resonance. The apparatus and method have the capability to detect proteins, viruses, bacteria, toxins, pathogens, contaminants, chemical compounds, or nucleic acids based on surface plasmon resonance and surface receptor technologies which may include antibodies or chemical receptors. The device is field deployable and utilizes a single use sample holder card which includes the sample to be tested, test channels, waste reservoir and a functionalized test surface.

Description:
CROSS-REFERENCES TO RELATED APPLICATIONS 
     This application claims the benefit of U.S. Provisional Patent Application No. 61/335,297 filed Jan. 4, 2010, the disclosure of which patent application is incorporated by reference as if fully set forth herein. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Grant No. W81XWH-06-1-0275 awarded by the United States Army. 
    
    
     THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT 
     Not Applicable 
     INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC 
     Not Applicable 
     BACKGROUND OF THE INVENTION 
     This invention relates to testing in the fields of chemistry, biology, and biotechnology. In particular, the invention relates to testing for pathogens and other analytes using a surface plasmon resonance (SPR) process. 
     Surface plasmon resonance (SPR) is a powerful analytical principle which is very sensitive to changes on an interrogated surface. Surface plasmons are generated in certain metals when light is incident upon the prism metal interface at a specific angle. As the properties of the surface change, e.g., the binding of a molecule to the surface, the angle at which SPR occurs is measurably shifted. The presence of a surface plasmon reduces the amount of light reflected at the specific angle, so a binding event on the surface results in a direct translation of the reflection minima: a parameter that is readily measurable. 
     In the background art, instruments utilizing SPR as a method of detection have been limited to laboratory use due to bulky size, limited field portability, and complicated operating procedures. Despite their shortcomings, SPR instruments are highly sensitive to extremely small changes at the interrogated surface. This makes the principle ideal for ultra-trace level detection. What is needed, however, is a compact detection system, with a simple user interface and operation, as well as pre-functionalized surfaces which together limit the required user skills to pressing buttons, performing simple sample injections upon prompting, and reading the result from a screen. 
     The Spreeta™ SPR transducer is an inexpensive SPR transducer that was originally developed by Texas Instruments, Inc. and is currently manufactured by Sensata Technologies, Inc. of Attleboro, Mass. The Spreeta™ SPR transducer has become competitive with other SPR methods and instruments, rivaling other SPR-based designs. The Spreeta™ SPR transducer contains a light source, prism, and a charge-coupled device (CCD) to generate light, interrogate the active surface at multiple angles, and recover the reflection signal of SPR respectively. Electronic circuitry drives the Spreeta™ SPR transducer, as well as extracts and analyzes the signal it produces to characterize the SPR result and determine whether a compound of interest is present. On the face of the Spreeta™ SPR transducer, a single use functionalized chip bears the plasmon-generating metal as the active, interrogated surface and the self-contained fluidics portion of a chip into which the sample is injected and retained. 
     The SPR technique is readily sensitive to mass changes well below the level of nanograms per milliliter without the benefit of secondary labeling techniques. The technique has the ability to use a mass-labeling technique for secondary amplification if necessary to provide additional signal under the condition of a positive binding event. For example, the system will recognize a bound virus, and generate an additional signaling event, allowing for greater sensitivity, improved likelihood of positive identification, and reduction of the lower detection limit. 
     Though SPR has been developed as a technology over the past 20 years, it is still being advanced through physical and computational means. Faster processing with modern hardware allows for a more compact electronics package, enabling portability, as well as faster sampling rates and resultant smoothness and reliability of signal. 
     In the physical interactions which take place on the active SPR surface, there are other advancements in the form of surface modification, perhaps most notably in the realm of nanoparticle labeling and nanotexturing of surfaces. This incorporation of nanotechnology with SPR is still in a very experimental state, but shows promise to improve the detection limits of the technique. SPR technology has largely been limited to laboratory use, where it provides high-sensitivity, high-specificity molecular identification and is utilized in sample testing for the presence of cells, viruses, biomolecules, nucleic acids and other compounds. 
     The background art is characterized by U.S. Pat. Nos. 3,656,833; 4,931,402; 5,156,976; 5,209,904; 5,607,643; 5,912,456; 6,432,364; 6,611,367; 6,756,223; 6,870,627; 6,956,695; 7,307,730; 7,333,197; 7,554,657; 7,738,934; 7,842,242; and 7,842,247; and U.S. Patent Application Nos. 2003/0003018; 2003/0206708; 2006/0188401; 2007/0222998; 2007/0279634; 2009/0103099; 2009/0303489; 2010/0096561; 2010/0103421; 2010/0150781; 2010/0216975; and 2010/0284012; the disclosures of which patents and patent applications are incorporated by reference as if fully set forth herein. The background art is also characterized by the following patents and patent applications: TW 249768; WO 2008/007115; and WO/2009/022985. 
     BRIEF SUMMARY OF THE INVENTION 
     As used herein, the following terms and variations thereof have the meanings given below, unless a different meaning is clearly intended by the context in which such term is used. 
     “A,” “an” and “the” and similar referents used herein are to be construed to cover both the singular and the plural unless their usage in context indicates otherwise. 
     “About” means within five percent of a recited parameter or measurement, and preferably within one percent of such parameter or measurement. 
     “Comprise” and variations of the term, such as “comprising” and “comprises,” are not intended to exclude other additives, components, integers or steps. 
     “Exemplary,” “illustrative,” and “preferred” mean “another.” 
     “Horizontal” means substantially parallel to the orientation of the card insertion slot. 
     “Vertical” means substantially perpendicular to the orientation of the card insertion slot. 
     In an illustrative embodiment, the invention offers this powerful recognition technology in a standalone platform for sample testing in the field. In this embodiment, the invention provides a solution for detection of chemical and biological agents (e.g., vector-borne viruses) which utilizes a field-portable, SPR-based detector. A battery powered instrument receives a single-use sensor card that is specifically functionalized for the target of interest. These sensor cards are capable of being prepared so that they can test for multiple analytes simultaneously. 
     In an illustrative embodiment, the invention is a sensor comprising: a housing having a card slot, a heat sink opening, a knob opening and a display opening; an integrated surface plasmon resonance transducer comprising a light source and a polarizer for producing polarized light, a light transmissive window for transmitting said polarized light and a reflection of said polarized light, a mirrored surface for directing said reflection, and a detector array for detecting said reflection, said integrated surface plasmon resonance transducer being disposed within said housing; a functionalized card disposed in said card slot, said functionalized card comprising a body having an upper wall having an upper outer surface and a lower wall having lower outer surface and a microfluidic channel having a sample input port that is provided with a septum, a sample input reservoir that is in fluid communication with said sample input port, a test channel and a reference channel that are in fluid communication with said sample input reservoir, a waste reservoir that is in fluid communication with said test channel and said reference channel, a hydrophobic filter that is in fluid communication with said waste reservoir, a vacuum output port that is in fluid communication with said hydrophobic filter and a chip having a metallic test surface that forms an interior wall of said test channel and an inner wall of said reference channel for producing said reflection; an index matching fluid disposed between said light transmissive window and said lower outer surface and adjacent to said metallic test surface; a card holding mechanism for positioning said functionalized card against said integrated surface plasmon resonance transducer so that said test channel and said metallic test surface are exposed to said polarized light and so that said detector array is exposed to said reflection, said card holding mechanism comprising a spring that is operative to urge said functionalized card toward said integrated surface plasmon resonance transducer and a card releasing mechanism comprising a knob that is disposed in said knob opening, a drive shaft that is attached to said knob, a spur gear that is attached to said drive shaft, a rack gear for engaging with said spur gear, a horizontally movable wedge for engaging with said rack gear and a vertically movable wedge for engaging with said horizontally movable wedge and with said card, said card releasing mechanism being operative to reverse the motion urged by said spring when said knob is rotated; a heat storage component that is disposed adjacent to said functionalized card and is in contact with said upper outer surface, said heat storage component having a thermistor well, an insulation layer that is disposed adjacent to said heat storage component, a heat sink that is disposed in said heat sink opening, said heat sink having a cavity that is disposed adjacent to said insulation layer, a heating and cooling device that is disposed adjacent to said heat storage component and in said cavity, and a thermistor that is disposed in said thermistor well; a vacuum pump that is in fluid communication with said vacuum output port that is operative to move a sample that has been injected into said sample input port through said microfluidic channel, said vacuum pump being disposed in said housing; a power source that is disposed within said housing; a plurality of control switches that are mounted on said housing; a control circuit interface board that is disposed within said housing; a computer/controller that is disposed within said housing, said computer/controller being operative to receive signals from said thermistor and control the temperature of said heat storage component and to process signals from said integrated surface plasmon resonance transducer and to produce an output; and a display that is mounted in said display opening and that is operative to receive and display said output. In another embodiment, said heating and cooling device comprises: a Peltier assembly. In another embodiment, said functionalized card further comprises: a top cover layer that provides a top for said sample input reservoir and said waste reservoir and that holds said septum and said filter in said functionalized card; a septum spacer layer that contains said septum; a first adhesive layer that joins said top cover layer and said septum spacer layer; a filter spacer layer that contains said filter; a second adhesive layer that joins said septum spacer layer and said filter spacer layer; a reservoir layer in which sample input reservoir and said waste reservoir are formed; a third adhesive layer that joins said filter spacer layer and said reservoir layer; a bottom cover layer that provides a bottom for said sample input reservoir and said waste reservoir; and a fourth adhesive layer that joins said reservoir layer and said bottom cover layer. In another embodiment, said interior wall is functionalized and said inner wall is not. 
     In another illustrative embodiment, the invention is a surface plasmon resonance analytical kit comprising: means for measuring a surface plasmon resonance comprising an integrated surface plasmon resonance transducer that produces a signal; a functionalized card comprising a microfluidic system having a test channel active surface and a reference channel active surface, said functionalized card being adapted to receive a sample; means for holding said functionalized card in optical communication with said integrated surface plasmon resonance transducer, said means for holding comprising elastic members that exert forces that urge said functionalized card toward said integral surface plasmon resonance transducer; means for releasing said functionalized card comprising a rack and pinion that are operative to overcome said forces and allow said functionalized card to move away from said integral surface plasmon transducer; and means for processing said signal to produce an indication of whether said sample contains an analyte of interest. 
     In another illustrative embodiment, the invention is a method for detecting the presence or amount of an analyte in a sample using a biosensor that comprises a card holding mechanism, a thermal mass and a surface plasmon resonance transducer, said method comprising: introducing the sample into a functionalized card, said functionalized card having a waste reservoir, an exit port, a reference channel having a reference channel active surface, and a test channel that is functionalized to retain the analyte on a test channel active surface; rotating an actuator in a first direction that rotates a pinion that moves a rack and a horizontally movable wedge that moves a vertically movable wedge which moves the card holding mechanism to a card receiving position; applying an index matching fluid or conformal coating to said functionalized card and inserting said functionalized card into the biosensor at a location that is adjacent to the thermal mass; rotating said actuator in a second direction that rotates said pinion that moves said rack and said horizontally movable wedge that moves said vertically movable wedge which moves the card holding mechanism to a card holding position with said functionalized card being held against the surface plasmon resonance transducer and in thermal communication with said thermal mass; controlling the temperature of said functionalized card at a set point by measuring a temperature of said thermal mass and heating or cooling said thermal mass; imposing a vacuum on said exit port to move said sample from said sample input reservoir through said test channel and said reference channel into said waste reservoir; measuring a first surface plasmon resonance signal emitted by said test channel active surface and a second surface plasmon resonance signal emitted by said reference channel active surface to produce a test signal and a control signal; and processing said test signal and said control signal in a processor to produce an indication of the presence of the analyte. In another embodiment, the method further comprises: processing said test signal and said control signal in a processor to produce a concentration of the analyte in the sample. In another embodiment, the method further comprises: imposing a pressure difference is operative to produce a flow rate of 20 to 40 microliters per minute across said functionalized card. In another embodiment, controlling the temperature of said functionalized card step comprises use of a proportional stage, an integrator stage and a differentiator stage. In another embodiment, controlling the temperature of said functionalized card is operative to stabilize the temperature of said functionalized card to within 0.15 degrees C. over a sixty minute period. 
     In a further illustrative embodiment, the invention is a method for detecting the presence or amount of an analyte in a sample using a biosensor that comprises a thermal mass, a card holding mechanism and a surface plasmon resonance transducer, said method comprising: a step for introducing the sample into a functionalized card, said functionalized card having a sample reservoir, a reference channel having a reference channel active surface, a waste reservoir, a hydrophobic filter, an exit port and a test channel that is functionalized to retain the analyte on a test channel active surface; a step for rotating an actuator of the card holding mechanism in a first direction that rotates a pinion that moves a rack to a card receiving position; a step for applying an index matching fluid to said functionalized card and inserting said functionalized card into said biosensor at a location that is adjacent to the thermal mass; a step for rotating said actuator of the card holding mechanism in a second direction that rotates said pinion that moves said rack to a card holding position with said functionalized card being held in optical communication with the surface plasmon resonance transducer and in thermal communication with the thermal mass; a step for controlling the temperature of said functionalized card by measuring a temperature of the thermal mass and heating or cooling the thermal mass; a step for imposing a vacuum on said exit port to move said sample from said sample reservoir through said test channel and into said waste reservoir; a step for measuring a first surface plasmon resonance of said test channel active surface and a second surface plasmon resonance of said control active surface to produce a test signal and a control signal; and a step for processing said test signal and said control signal in a processor to produce an indication of the presence or absence of the analyte. In another embodiment, the method further comprises: a step for processing said test signal and said control signal in a processor to produce a concentration of the analyte in the sample. In another embodiment, said step for imposing a pressure difference is operative to produce a flow rate of 20 to 40 microliters per minute across said functionalized card. In another embodiment, said step for controlling the temperature of said functionalized card comprises use of a proportional stage, an integrator stage and a differentiator stage. In another embodiment said step for controlling the temperature of said functionalized card is operative to stabilize the temperature of said functionalized card to within 0.025 degrees C. over a sixty minute period. 
     In yet another illustrative embodiment, the invention is a system for detecting the presence or amount of an analyte in a sample using a biosensor that comprises a thermal mass, a card holding mechanism and a surface plasmon resonance transducer, said method comprising: means for introducing the sample into a functionalized card, said functionalized card having a sample reservoir, a reference channel having a reference channel active surface, a waste reservoir, a hydroscopic filter, an exit port and a test channel that is functionalized to retain the analyte on a test channel active surface; means for rotating a pinion of said card holding mechanism in a first direction that moves a rack to a card receiving position; means for accepting said functionalized card in a slot that is adjacent to the thermal mass; means for rotating said pinion of said card holding mechanism in a second direction that moves said rack to a card holding position with said functionalized card being held in optical communication with the surface plasmon resonance transducer and in thermal communication with the thermal mass; means for controlling the temperature of said functionalized card by measuring a temperature of the thermal mass and heating or cooling the thermal mass; means for imposing a pressure difference to move the sample from said sample reservoir through said test channel and said reference channel into said waste reservoir; means for measuring a test channel surface plasmon resonance of said test channel active surface and a reference channel surface plasmon resonance of said reference channel active surface to produce a test signal and a control signal; and means for processing said test signal and said control signal in a processor to produce an indication of the presence or absence of the analyte. In another embodiment, the system further comprises: means for processing said test signal and said control signal in a processor to produce a concentration of the analyte in the sample. In another embodiment, said means for imposing a pressure difference is operative to produce a flow rate of 20 to 40 microliters per minute across said functionalized card. In another embodiment, said means for controlling the temperature of said functionalized card comprises a proportional stage, an integrator stage and a differentiator stage. In another embodiment, said means for controlling the temperature of said functionalized card is operative to stabilize the temperature of said functionalized card to within 0.025 degrees C. over a sixty minute period. 
     In yet another illustrative embodiment, the invention is a field deployable surface plasmon resonance based biosensor comprising: a functionalized card comprising a microfluidic system that delivers a sample to a functionalized test surface chip in a test channel and in a reference channel; a card holding mechanism that comprises springs that hold said functionalized card in a card holding position during a test and a knob, pinion, rack and wedges that are operative to compress said springs when said card holding mechanism is in a card releasing position before and after said test; 
     a thermal control system that is operative to control the temperature of said functionalized card indirectly by controlling the temperature of a thermal mass that is disposed adjacent to said functionalized card when the card holding mechanism is in the card holding position; an integrated surface plasmon resonance transducer that is operative be in optical communications with said activated test surface chip and is operative to characterize reflections from said activated test surface chip and produce output signals; a processor that is operative to process said output signals; and a user interface comprising switches and a display that is operative to accept input from a user and to present biosensor results to said user. In another embodiment, said thermal control system comprises a Peltier assembly. 
     Further aspects of the invention will become apparent from consideration of the drawings and the ensuing description of exemplary embodiments of the invention. A person skilled in the art will realize that other embodiments of the invention are possible and that the details of the invention can be modified in a number of respects, all without departing from the concept. Thus, the following drawings and description are to be regarded as illustrative in nature and not restrictive. 
    
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
       The features of the invention will be better understood by reference to the accompanying drawings which illustrate exemplary embodiments of the invention. In the drawings: 
         FIG. 1  is a perspective view of an illustrative embodiment of the invention. 
         FIG. 2  is a plan (front) view of an illustrative embodiment of the invention. 
         FIG. 3  is perspective view of an illustrative embodiment of the invention with the front cover removed. 
         FIG. 4A  is a perspective view of the card holding mechanism of an illustrative embodiment of the invention. 
         FIG. 4B  is an elevation (end) view of the card holding mechanism of an illustrative embodiment of the invention. 
         FIG. 4C  is another perspective view of the card holding mechanism of an illustrative embodiment of the invention. 
         FIG. 5A  is a perspective view of the card of an illustrative embodiment of the invention. 
         FIG. 5B  is an elevation (end) view of the card of an illustrative embodiment of the invention. 
         FIG. 5C  is a plan drawing of the active surface portions of the card of an illustrative embodiment of the invention. 
         FIG. 6A  is a cross sectional view of an illustrative embodiment of the biosensor showing the card holding mechanism in place. 
         FIG. 6B  is a zoomed in version of the cross sectional view of  FIG. 6A . 
         FIG. 7A  is schematic block diagram illustrating a basic temperature control system in accordance with an illustrative embodiment of the invention. 
         FIG. 7B  is schematic block diagram illustrating a digital temperature control system in accordance with an illustrative embodiment of the invention. 
         FIG. 8A  is a plot of thermal stability achieved with an illustrative embodiment of the field deployable biosensor. 
         FIG. 8B  is a detailed plot of the results displayed in  FIG. 8A  to scale permitting analysis of outliers and average results. 
     
    
    
     The following reference numerals are used to indicate the parts and environment of an illustrative embodiment invention on the drawings: 
     
       
         
               
               
             
           
               
                   
               
             
             
               
                 1 
                 top cover/housing 
               
               
                 2 
                 bottom cover/housing 
               
               
                 3 
                 horizontal card insertion slot, card insertion slot, slot 
               
               
                 4 
                 display, screen 
               
               
                 5 
                 actuator, knob. 
               
               
                 6 
                 heat sink 
               
               
                 7 
                 control switches 
               
               
                 8 
                 Peltier assembly, heater/cooler 
               
               
                 9 
                 control circuit interface board 
               
               
                 10 
                 controller, computer 
               
               
                 11 
                 functionalized, single use card, single use card, card 
               
               
                 12 
                 vacuum pump, pump 
               
               
                 13 
                 heat storage component, thermal reservoir, thermal mass 
               
               
                 14 
                 card holding mechanism 
               
               
                 15 
                 Spreeta ™ SPR transducer, SPR transducer, transducer 
               
               
                 16 
                 sample input reservoir, sample reservoir 
               
               
                 17 
                 test region, test channel 
               
               
                 18 
                 waste reservoir 
               
               
                 19 
                 sample input port, injection port, septum 
               
               
                 20 
                 vacuum output port, vacuum port 
               
               
                 21 
                 metallic test surface chip 
               
               
                 22 
                 drive shaft 
               
               
                 23 
                 functionalization port 
               
               
                 24 
                 optical oil interface, index matching fluid 
               
               
                 25 
                 rack gear, rack 
               
               
                 26 
                 spur gear, pinion 
               
               
                 27 
                 horizontally movable wedge 
               
               
                 28 
                 vertically movable wedge 
               
               
                 29 
                 hydrophobic filter, filter 
               
               
                 30 
                 field deployable surface plasmon resonance based biosensor,  
               
               
                   
                 biosensor, device 
               
               
                 31 
                 reference region, reference channel 
               
               
                 32 
                 active surface 
               
               
                 33 
                 duct 
               
               
                 34 
                 component fasteners 
               
               
                 36 
                 linear guides 
               
               
                 38 
                 compression springs, springs 
               
               
                 40  
                 insulation 
               
               
                 42 
                 bolts, posts 
               
               
                 44 
                 thermistor wells 
               
               
                 46 
                 thermal device cavity 
               
               
                 50 
                 top cover layer 
               
               
                 52 
                 first adhesive layer 
               
               
                 54 
                 septum spacer layer 
               
               
                 56 
                 second adhesive layer 
               
               
                 58 
                 filter spacer layer 
               
               
                 60 
                 third adhesive layer 
               
               
                 62 
                 reservoir layer 
               
               
                 64 
                 fourth adhesive layer 
               
               
                 66 
                 bottom cover layer 
               
               
                 70 
                 basic temperature control system 
               
               
                 72 
                 set point 
               
               
                 74 
                 first node 
               
               
                 76 
                 differentiator stage 
               
               
                 77 
                 integrator stage 
               
               
                 78 
                 gain stage, proportional stage 
               
               
                 80 
                 second node 
               
               
                 82 
                 copper propagation delay 
               
               
                 84 
                 temperature sensor 
               
               
                 86 
                 pulse-width modulator 
               
               
                 88 
                 H bridge 
               
               
                 90 
                 analog-to-digital converter 
               
               
                 92 
                 digital temperature control system 
               
               
                   
               
             
          
         
       
     
     DETAILED DESCRIPTION OF THE INVENTION 
     Referring to  FIGS. 1 and 2 , an illustrative embodiment of the invention is presented. In this embodiment, field deployable surface plasmon resonance based biosensor  30  is used to test aqueous samples for analytes of interest. Examples of analytes include proteins, viruses, bacteria, toxins, pathogens, contaminants, chemical compounds, and nucleic acids. 
     In this embodiment, biosensor  30  is surrounded by a compact housing that comprises top cover  1  and bottom cover  2 . Card insertion slot  3  is provided in top cover  1  and display or screen  4 , heat sink  6  and control switches  7  protrude through top cover  1 . Actuator or knob  5  protrudes from the side of the housing and Peltier assembly  8  protrudes through heat sink  6 . 
     Biosensor  30  is field deployable and may be powered by either alternating current (AC) or direct current (DC) power devices. Biosensor  30  may be used to perform any detection test that is predetermined by a single use, functionalized card  11 . In an illustrative embodiment, functionalizing card  11  involves applying an antibody or some other activating agent which is particular to the desired detection test to produce an active surface through a process well known in the art. Functionalization of single use card  11  is preferably accomplished prior to deployment of biosensor  30  to the field. Single use card  11  is placed in device  30  via slot  3 . 
     Referring to  FIGS. 3 ,  4 A,  4 B, and  4 C, after card  11  is inserted in horizontal card insertion slot  3 , the card is pressed down against SPR transducer  15  by turning knob  5  of card holding mechanism  14 . In this embodiment, turning knob  5  rotates spur gear  26  which interfaces with rack gear  25  causing linear motion of rack gear  25 . Rack gear  25  is preferably attached to a wedged shaped component (horizontally movable wedge  27 ) with standard screws. Horizontally movable wedge  27  has an upper surface disposed at a first angle relative to horizontal (as that term is designed above) that is in contact with another wedge (vertically movable wedge  28 ) that has a lower surface that has a second angle that is complementary to the first angle of the upper surface of horizontally movable wedge  27 . The first angle and the second angle are complementary in that they sum to ninety degrees. 
     In this embodiment, vertically movable wedge  28  is vertically movably attached to heat storage component  13 . Heat storage component  13  is preferably made of copper or another substance with a similar thermal capacitance and is fixed to bottom cover  2  by means of component fasteners  34  (e.g., washers) and bolts  42 . Linear guides  36  are fixed to heat storage component  13  that have slots into which posts on vertically movable wedge  28  are vertically movable, which restricts the motion of vertically movable wedge  28  to vertical (as that term is defined above). 
     In this embodiment, compression springs  38  are disposed concentric to bolts  42  beneath component fasteners  34  and exert a downward force on heat storage component  13 . By turning knob  5  clockwise, springs  38  are compressed by the upward motion of heat storage component  13 , allowing the insertion or removal of card  11 . Turning knob  5  counterclockwise allows springs  38  to exert a downward force on heat storage component  13 , causing card  11  to be pressed against SPR transducer  15 . Illustrative embodiments of SPR transducer  15  are disclosed in U.S. Pat. Nos. 5,912,456 (see especially  FIG. 2 ) and 6,870,627 (see especially  FIG. 3 ); the disclosures of which patents are incorporated by reference as if fully set forth herein. In the embodiments disclosed herein, there is no metallic layer or coating on the light transmissive window of the SPR transducer. Rather, the metallic layer or coating is provided on metallic surface chip  21 . 
     This arrangement allows for a constant (from one card insertion to the next) force as supplied by the springs  38  and not from unregulated/uncontrolled force supplied by the operator (by turning knob  5 ). Springs  38  are guided by posts  42  that are arranged vertically in respect to card  11 . Springs  38  exert a force on heat storage component  13  which transfers the force to card  11  and the associated interface between card  11  and SPR transducer  15  which is preferably index matching fluid  24 . 
     In an illustrative embodiment, operation of device  30  is accomplished by manipulation of switches  7  which are mounted on top cover  1 . Responses and user feedback are displayed on screen  4 . Many levels of operation are possible depending on the desired result. In a simple operating mode, a positive or negative indication of detection of an analyte is displayed. In a more advanced operating mode, SPR curves and detection level are displayed. In a preferred embodiment, a positive or negative test result for the analyte targeted by single use card  11  and analyte concentration are displayed on screen  4  along with instrument status and brief instructional prompts. 
     Because the SPR technique is sensitive to shifts in temperature, a temperature control method is employed. In an illustrative embodiment, a Peltier assembly  8  is utilized to heat and cool heat storage component or thermal mass  13 . In an illustrative embodiment, Peltier assembly  8  maintains heat storage component  13  at the desired test temperature, e.g., between 20 and 30 degrees Celsius (° C.), controlled to within desired limits. In an illustrative embodiment, from test to test the temperature is held within +/−5° C. and within a test the temperature is held to within +/−0.1° C. 
     Heat storage component  13  is used to precondition and maintain card  11  and the sample it contains at the desired test temperature. In an alternative embodiment, final and fine temperature control is accomplished by resistively heating the metallic test surface chip  21  to the final required test temperature. 
     Unlike the device disclosed in U.S. Patent Application No. 2010/0284012, in which a card made of silicone is used (which was selected for its insulative properties), a more preferred embodiment disclosed herein uses a card  11  made of polyethylene terephthalate (PET), acrylic and a pressure sensitive adhesive, which were selected because of their thermal conductive natures. As noted above, the amount of material in card  11  is minimized for heat transfer reasons and the sample fluid layer is thin for like reasons. This approach allows the achievement of very fine temperature control that is capable of maintaining the test area at a precise setting. 
     Referring to  FIGS. 5A ,  5 B and  5 C, views of an illustrative embodiment of card  11  comprising a microfluidic system are presented. Testing of a sample involves introducing about 0.5 milliliters (ML) of sample into card  11  by injecting it through septum  19  with a syringe, pipette or similar device. Septum  19  both allows insertion of the sample fluid and retains the fluid. During a test, sample fluid is moved from sample reservoir  16  across a functionalized (activated) surface on metallic test surface chip  21  through test channel  17  on the single use card  11  by means of pump  12 . Pump  12  may either be attached to injection port  19 , if movement of a sample fluid is to be driven by pressure, or to vacuum port  20  if the sample fluid is to be pulled across by vacuum. A preferred embodiment employs a vacuum pump  12  that is optimized to generate the desired flow rate of 20 to 40 microliters per minute (μL/min). The applicants discovered that this rate was rapid enough to enable fluidic movement through channels  16 ,  17  and  18 , while slow enough to avoid undesirable outcomes such as sheer or bubble generation (which would incapacitate sample movement). 
     In either case, the sample is transported from sample reservoir  16 , through test channel  17  over the functionalized test area and into waste reservoir  18 . Test channel  17  is about 0.002 inches in depth by about 0.020 inches in width. Each channel/reservoir  16  and  18  is preferably optimized at the following dimensions: 7 millimeters (mm) long, by 0.55 mm wide by, 0.05 mm thick in a serpentine path across card  11 . At the completion of a test, the sample is maintained in waste reservoir  18  for containment of a potentially hazardous substance or for further testing in an appropriate facility. The sample may be removed from card  11  through septum  19  by either applying pressure to drive it out by using a vacuum to pull it out. Control active surface  32  is a portion of metallic test surface chip  21  that is not exposed to the sample. The test active surface is located at the bottom of a portion of test channel  17  and is exposed to the sample. 
     In an illustrative embodiment, filter  29  is built into card  11  and is in fluid communication with waste reservoir  18  and vacuum port  20 . Filter  29  is preferably fabricated from a cellulose material that has been treated to render it hydrophobic (e.g., Whatman 1PS Phase Separator manufactured by Whatman Inc. of Piscataway, N.J.). Filter  20  retains fluid in card  11  but allows air to pass through vacuum port  20 . 
     Referring to  FIG. 5B , an elevation cross-sectional view at the edge of an illustrative embodiment of card  11  is presented. In this embodiment, the layers of card  11  are preferably fabricating by cutting each layer from a sheet of material using a carbon dioxide laser and then stacked. The layers are adhered together using a pressure sensitive adhesive (PSA) (e.g., 3M8211, silicone PSA manufactured by 3M Corporation, Minnesota). 
     In this embodiment, top cover layer  50  is preferably fabricated from PET and forms the top of the channels  16  and  18 . Top cover layer  50  holds septum  19  and filter  29  in card  11 . First adhesive layer  52  joins top cover layer  50  and septum spacer layer  54 . Septum spacer layer  54  contains septum  19  and spaces the adjacent layers apart and is preferably fabricated from PET. Second adhesive layer  56  joins septum spacer layer  54  and filter spacer layer  58 . Filter spacer layer  58  contains filter  29  and spaces the adjacent layers apart and is preferably fabricated from PET. Third adhesive layer  60  joins filter space layer  58  and reservoir layer  62 . Channels  16  and  18  are formed in reservoir layer  62  which is preferably fabricated from acrylic. Fourth adhesive layer  64  joins reservoir layer  62 , metallic test surface chip  21 , and bottom cover layer  66 . The top surfaces of metallic test surface chip  21  and bottom cover layer  66  are coplanar. Bottom cover layer  66  forms the top of channels  16  and  18  and captures filter  29 . Bottom cover layer  66  is preferably fabricated from PET. 
     Card  11  is preferably configured to hold enough sample fluid to complete an assay (approximately 0.5 mL). Fluid flow across card  11  preferably transfers 0.5 mL from sample reservoir  16  to waste reservoir  18  in 20 minutes. In an illustrative embodiment, sample fluid flows through two channels connected in series: the first channel is a reference channel to be used for comparison, and the second channel is the SPR channel. After the fluid has passed through the two channels, it is held in waste reservoir  18  (approximately 0.5 mL). Past waste reservoir  18 , hydrophobic filter  29  is used to retain the sample in card  11 . Channel design is a balance between cost and pressure drop due to flow restriction. Capillary forces are taken into account as well as viscous friction. In a preferred embodiment, the flow restriction (pressure drop) is limited to that which could be achieved by a small vacuum pump. Vacuum is preferred for its inherent safety benefits (e.g., with leaks occurring into card  11  rather than out from card  11 ). The fluid sample is preferably spread out over as much test surface as possible. In this embodiment, the channels are required to be small in cross-sectional area, which dictates that they be limited to an adhesive layer (removed material in the adhesive layer). Card layers of card  11  are preferably held together with a contact adhesive (substrate free glue). The benefits of a single use card design include: no cleaning is required between assays; the transducer never comes in contact with the sample; each card is pre-functionalized (ready to use); each card is contamination free; each card retains the sample (for further testing back in a laboratory); assay turnaround is rapid; changing from one assay to another is facilitated by using another pre-functionalized card; major time and cost savings are achieved; much greater portability is achieved; and less knowledge is required to operate device  30 . 
     In an illustrative embodiment, metallic test surface chip  21  is fabricated from glass with a gold coating deposited by physical vapor deposition. The area of metallic test surface chip  21  is sufficient to cover the light transmissive window in SPR transducer  15 , which light transmissive window does not have a gold layer or which has had its gold layer removed. Preferred specifications for metallic test surface chip  21  are presented in Table 1. 
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Metallic Test Surface Chip Specifications 
               
             
          
           
               
                   
                 Item 
                 Parameter 
               
               
                   
                   
               
               
                   
                 Material 
                 Glass 
               
               
                   
                 Refractive index 
                 1.590 (+/−0.01) @ 900 nm wavelength 
               
               
                   
                 Thickness 
                 0.31 mm (+/−0.02 mm) 
               
               
                   
                 Flatness 
                 0.001 mm 
               
               
                   
                 Parallel surfaces 
                 &lt;1/3° taper in any direction 
               
               
                   
                 Width 
                 12.5 mm (+/−0.0635 mm) 
               
               
                   
                 Length 
                 18.86 mm (+/−0.0635 mm) 
               
               
                   
                 Coating 
                 Au (gold) 
               
               
                   
                 Quality 
                 99.99% pure 
               
               
                   
                 Thickness 
                 50 nm (+/−2.5 nm) 
               
               
                   
                 Bond layer 
                 Titanium or Chromium 
               
               
                   
                 Bond layer thickness 
                 &lt;10 Å 
               
               
                   
                   
               
             
          
         
       
     
     In an illustrative embodiment, the channels  16 ,  17 ,  18 ,  31  are created by cutting a path into the adhesive to form the sides (only 0.002 thick) and by using the adjoining layers to form the tops and bottoms of the channels. In this embodiment, gold coated metallic test surface chip  21  is much larger than the channels and covers a larger than required area. It is not economically viable to gold coat only the channel areas. It is also preferable that the test channels or any fluid pathway (duct) not cross over a joint between two material layers or components. For this reason, gold coated chip  21  is relatively large. 
     Referring to  FIG. 5C , in an illustrative embodiment, active surface  32  of sensor card  11  is disposed on metallic test surface chip  21 . Two regions bear different surface functionality to serve as monitored regions from which SPR dips are gathered; this includes reference region  31  and sample region  17 . In this embodiment, reference region  31  is covered with a monolayer that prevents non-specific binding, and bears no other functionalization or specificity to a particular analyte of interest. Its role is to characterize the sample solution to provide a relative dip position to which the sample channel is compared. Sample region  17  is also functionalized with a monolayer to prevent non-specific binding, as well as tethers to which biomolecules or chemical receptors are attached, providing functional specificity of the surface to a particular analyte of interest. 
     In that biomolecules are utilized in the detection and quantification of analytes, a preservative is preferably used to maintain their functional state. This preservative is injected into the card at functionalization port  23  and is drawn via a vacuum to the other functionalization port  23 . Following this preservation step, functionalization ports  23  are sealed prior to shipment of card  11 . This liquid-based preservative may be dried in situ or maintained in a fluidic state on card  11  until use. An example of such a preservative is ProClin 300 manufactured by Rohm and Haas of Philadelphia, Pa. 
     In an illustrative operating sequence, a test sample is drawn by vacuum pump  12  from sample reservoir  16  and then into the reference channel  31 . As no functional molecules are present on the active surface of reference channel  31 , this surface examines the bulk refractive index of the test sample. The test sample is then drawn through duct  33  which links reference channel  31  to test channel  17 . Test channel  17 , due to its functional specificity to a desired analyte, binds any such analyte in the test sample, effectively changing the angle at which SPR occurs. The change in SPR angle of test channel  17  relative to the change in SPR angle of reference channel  31  over time is therefore directly proportional to the quantity of bound analyte, providing the means for the instrument to detect and quantify the target analyte. 
     Controller  9  is preferably used to control sample temperature, sample movement, sample data acquisition, sample data analysis and user feedback. In an illustrative embodiment, controller  9  is a microprocessor that has the required speed to achieve all control functions. In a preferred embodiment, three Micro Controller PIC processors are used (one for the Spreeta transducer, one for thermal control, and one for the human machine interface). The processors&#39; speed allows hundreds of thousands of data points to be taken and analyzed per second. In an alternative embodiment, a single, faster processor is used to accomplish all three control functions. 
     Referring to  FIGS. 6A and 6B , cross sectional views of an illustrative embodiment of biosensor  30  are presented with card  11  inserted. In this view, card  11  is held in place between thermal mass  13  and vertically movable wedges  28 . Thermal reservoir  13  is provided with thermister wells  44  in which thermistors  84  (not shown in  FIGS. 6A and 6B ) are installed. Thermistors  84  produce signals that are indicative of the temperature of thermal reservoir  13 , and, hence, of the temperature of adjacent card  11 . In this view, Peltier assembly  8  is not shown in thermal device cavity  46  for clarity. In a preferred embodiment, Peltier assembly  8  is Peltier (TEC) Module 19811-9L31-02CN1 manufactured by Custom Thermoelectric of Bishopville, Md. which is controlled by MAX1978ETM Integrated Temperature Controllers manufactured by Maxim Integrated Products, Inc. of Sunnyvale, Calif. 
     Referring to  FIGS. 7A and 7B , schematic block diagrams illustrating a basic temperature control system in accordance with an illustrative embodiment of the invention are presented. The basic temperature control system is presented in  FIG. 7A . In this embodiment, basic temperature control system  70  comprises set point  72 , first node  74 , differentiator stage  76 , integrator stage  77 , gain stage  78 , second node  80 , heater/cooler  8 , copper propagation delay  82 , and temperature sensor  84 . To implement this system as a digital control system, certain latitude is taken with basic concepts that introduce quantification effects. Digital temperature control system  92  comprises set point  72 , node  74 , differentiator stage  76 , integrator stage  77 , gain stage  78 , node  80 , heater/cooler  8 , copper propagation delay  82 , temperature sensor  84 , pulse-width modulator (PWM)  86 , H bridge  88 , and analog-to-digital converter (ADC)  90 . 
     Integrator stage  76 , differentiator stage  77  and gain stage  78  are preferably implemented as periodic updates. In a pure analog PID loop embodiment, these elements would run continuously within the bandwidth of the circuitry. The scaling (gain) factor for each block would be set with component values (capacitors for the differentiator stage and integrator stage) and resistor ratios for the gain (proportional) stage. When implemented digitally, the “gain” of the integrator and differentiator are set by the frequency of updates and the proportional stage gain is set by a scaling factor. 
     In this embodiment, the performance of heater/cooler  8  is very nonlinear. Heater/cooler  8  creates a temperature differential between two surfaces. The temperature of active surface  32  of metallic test surface chip  21  depends on the temperature of thermal mass  13 , as well as the current, or in this case, the duty cycle of H-bridge  88 . Not shown is a delay in heater/cooler  8 . For simulation purposes, it is assumed that the response of heater/cooler  8  is significantly faster than the propagation of temperature through thermal mass  13 . The goal is to create a uniform temperature in thermal mass  13 . To accomplish this, provision of a temperature sensor  84  on the card side of heat sink  6  is preferred. In an alternative embodiment, multiple temperature sensors  84  are used. In another alternative embodiment, multiple heater/cooler  8  elements operating approximately in parallel are used. 
     In response to a step input change in set point  72  or a reading from thermistor  84 , each of the response stages has a different response. Proportional stage  78  creates a step in the width of the signal from PWM  86  that is proportional to the error voltage. Proportional stage  78  cannot drive the error between set point  72  and measured temperature to zero and the PWM width is proportional to the error. If the error is driven to zero, the PWM width contribution from this stage is zero. 
     The purpose of integration stage  77  is to slowly drive the error to zero. This effect compensates for the inability of proportional stage  78  to do so. Changes in the integration contribution must be slow compared to the delay through the feedback system (that is, compared to copper propagation delay  82 ) to prevent overshoots. Integrator stage  77  has infinite gain at direct current (DC). This drives all poles toward the right hand pole, and a tendency to oscillate wildly can result. 
     The purpose of differentiator stage  76  is to introduce a rapid one-time response to a change in the error signal. A rising error signal causes a pulse in heater control voltage that creates a quick response to a sharp change in the set point or measured temperature. 
     The requirement for precise temperature control makes the integration stage part of the control loop important if the error value is to be driven to zero. The proportional stage  78  is needed to produce a faster response. 
     In an illustrative embodiment, set point  72  is constant. After biosensor  30  is turned on, thermal mass  13  is brought up to temperature and remains there. Thus, system  92  does not have to track set point. 
     It can be appreciated that other components are capable of achieving similar results and the descriptions given herein are to be taken as an example of an embodiment. 
     Working Example 
     Temperature drift studies on an embodiment of the invention disclosed herein showed temperature stability over a sixty minute period within 0.15 degrees C. This has a profound impact on improved sensitivity of the device 
     An experiment to determine temperature drift was performed. The instrument was allowed to equilibrate (rest) in the test location for 30 minutes prior to testing. Next, a test card was loaded with a filtered, deionized water sample. The loaded test card  11  was placed into the instrument as per standard protocol (pressed fully into biosensor  30  and knob  5  was rotated to the test or card holding position). Then biosensor  30  was supplied with power and turned on. Upon startup, biosensor  30  established a thermal set point three degrees above ambient temperature and began heating to that set point. 
     Next, vacuum pump  12  was started and run until a signal was achieved on both channels (reference or control and sample or test), then stopped. Data were taken for 35 minutes under no-flow conditions to determine thermal stability without flow. This process continued while vacuum pump  12  was again started for a period of 20 minutes under flow conditions to determine thermal stability with flow. Finally, flow, data acquisition, and thermal management were all terminated at the end of the test period. Data were taken from the biosensor  30  which were used to produce the plots shown in  FIGS. 8A and 8B . Dip position is an indicator of the refractive index of the test solution, and therefore is used to indicate the presence of any bonded analyte. 
     RU is the preferred unit of measure for biosensor  30 . Thermal drift may be measured in RU, as it directly relates to the measurements already taken by the instrument. A temperature change of 1 degree Celsius has an impact of approximately 100 RU at temperatures near room temperature in which the instrument was operated. RU are called ‘refractive units’ and constitute one millionth of one RIU (refractive index unit, also known as RI when absolute). RI are units more familiar to the layperson. These units are measures of the absolute index of refraction: where the RI of air is 1.0, water is 1.33, glass around 1.52, etc. As the instrument measures changes in refractive index as the means of its sensitivity, it makes sense to express the output in terms of such changes. 
     The plots presented in  FIGS. 8A and 8B  reveal the following: Thermal drift over the course of a sixty minute period was very small (36 RU), meaning that the total drift over the sample period of 20 minutes was 12 RU and 1.5 RU respectively; putting the embodiment on par with laboratory size biosensors that retail for over $100,000. In addition, the embodiment presents relative stability of the signal, translating to stability and reliability of the measurement. 
     Although some embodiments are shown to include certain features or steps, the applicants specifically contemplate that any feature or step disclosed herein may be used together or in combination with any other feature or step on any embodiment of the invention. It is also contemplated that any feature or step may be specifically excluded from any embodiment of the invention.