Abstract:
A surface plasmon resonant device provides practical portable operation through the use of a low power high efficiency LED source and a high-efficiency prism sample cell pre-loaded with probe molecules and sealed for field use. A simple mechanical control allows adjustment of angulation of the light and camera for accurate response outside of the laboratory.

Description:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
   This invention was made with United States Government support awarded by the following agency: DOD ARPA F30602-01-2-0555. The United States has certain rights in this invention. 

   CROSS-REFERENCE TO RELATED APPLICATIONS 
   BACKGROUND OF THE INVENTION 
   The present invention relates to instruments for chemical and biological analyses employing surface plasmon resonance, and in particular, to a portable version of such an instrument suitable for field use. 
   In surface plasmon resonance imaging, a sensor consisting of a thin metallic film is illuminated by polarized light of an appropriate wavelength and angle of incidence on a “reflecting” side of the film. The energy from the light couples to electrons of the metal of the film creating a resonant condition (surface plasmon resonance) that is highly sensitive to surface conditions on a “sensing” side of the film opposite the side that is illuminated. 
   Probe molecules are attached to the sensing side of the metallic film to selectively bind with target molecules in a solution to be analyzed. This binding, through the agency of the electron resonance in the film, causes a drop in reflectance of the reflecting side of the film. Detection of the decrease in reflected light thus provides a sensitive measurement of the binding of target molecules to the probe molecules, in turn providing an indication of the content of the solution being analyzed. 
   By placing a variety of different probe molecules on the sensing surface of the film, many different target molecules may be rapidly assessed. Importantly, the target molecules need not be labeled with fluorescent dye or the like prior to analysis. 
   Current surface plasmon resonance (SPR) equipment is large, complex, and expensive, and normally confined to use in a laboratory environment. A hand-held SPR device that could be easily transported to the field for remote measurements would be extremely valuable in assessing disease and detecting bio-terrorism and a variety of other analytic uses. 
   BRIEF SUMMARY OF THE INVENTION 
   The present inventors have developed a number of innovations that allow a standard SPR machine to be significantly reduced in size, cost, and electrical power consumption so that it may be rendered suitable for field use. Importantly, the inventors have determined that a standard narrow band LED may replace high-powered illumination sources previously used. An integral prism sample cell provides efficient light coupling to the metal film aiding in the use of the more energy efficient, but lower powered source. Construction of an integrated, disposable prism, metal film, and sample flow cell prevent contamination that may be incident to field use. Use of the low power light source together with a digitizing electronic camera allows the entire system to be operated using power and processing of a standard computer, for example, a laptop computer, readily available in or transportable to field locations. 
   Specifically then, the present invention provides a portable surface plasmon resonance imaging system having a sampling cell with a metallic film. The metallic film has probe molecules attached to a first side exposed to material flow through the sampling cell and a transparent support attached to a second side opposite the first side. An electronic camera positioned after a monochromatic filter receives reflected light from the second side of the metallic film originating at a light source constructed of a light emitting diode coupled with a polarizing element. 
   It is thus one object of the invention to provide an SPR device that may use a relatively low power, light-emitting diode (LED). The present inventors have determined that although the total luminance from an LED is far below that provided by white light sources in conventional SPR equipment, the narrow band concentration of the light energy from an LED, especially when used with additional features of the invention that provide improved light coupling, can be sufficient for SPR measurements. 
   The light emitting diode may be an infrared diode. 
   Thus, it is another object of the invention to maximize useable light energy by employing a high output LED emitting light frequencies to which standard electronic cameras are sensitive. 
   The invention may further include a cable connecting the electronic camera and the light source to a general purpose computer. The cable may include power leads communicating power from a power source contained in the computer to the electronic camera and the light source for powering the same. In at least one embodiment, the portable computer may be a laptop computer and the cable may be a universal serial bus (USB) cable. 
   Thus, it is another object of the invention to provide an interface drawing power from, and communicating data to, a standard computer, simplifying the design, improving portability and lowering cost. Use of a computer power supply, especially a laptop battery, is enabled by the low power light source of the LED. 
   The sampling cell may be a plastic prism having the metallic film attached to a first face of the prism. 
   Thus it is another object of the invention to provide a lightweight, disposable sampling system that provides extremely good light coupling so as to make best use of the light from the LED. 
   The prism may be held by a clamp removably holding the disposable prism in the optical path and the clamp may provide a fixed registration surface interfitting with at least two of the faces of the prism to fix the prism at a predefined location within the optical path. 
   Thus it is another object of the invention to provide a simple means for exchanging sample cells in the field making use of a clamp type structure with preset or fixed registration surfaces. 
   The prism may include an integral flow cell portion defining a cavity next to the side of the metallic film having the attached probe molecules for flow of sample material from a flow cell inlet to a flow cell outlet. 
   Thus it is another object of the invention to provide a wholly sealed sample chamber that may be disposed of after use and that does not require a clean environment for assembly, such as all would be difficult to obtain in the field. 
   The light source may be supported within the housing on a first swing arm pivoting in a radius about a point on the surface of the metallic film of the sampling cell to illuminate a region about the point through the transparent support of the sampling cell. Likewise, the electronic camera may be supported within the housing on a second swing arm pivoting in a radius about the point on the surface of the metallic film of the sampling cell and receiving reflective light from the second side of the metallic film. A mechanism between the first and second swing arms my move them simultaneously in symmetrical opposition about normal to the surface of the metallic film. 
   Thus it is another object of the invention to provide for simple and rapid adjustment of the angle of incidence and reflectance of the light beam to maximize sensitivity of the measurement. 
   An operator may have a first end communicating with the mechanism, and a second end accessible outside of the housing may be operated by one hand. 
   Thus it is another object of the invention to provide a system useable by a single individual in the field holding the device in one hand and operating the operator with their free hand. 
   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 perspective view of the portable SPR device of the present invention attached to a laptop computer for fieldwork; 
       FIG. 2  is a front elevational view of the device of  FIG. 1  with one side wall removed to show an internal sample cell, an angulation mechanism holding a solid state camera and LED light source, and a pump; 
       FIG. 3  is an exploded perspective view of the sample cell of  FIG. 2 , such as provides an integrated flow cell, metal film, and prism; 
       FIG. 4  is a fragmentary, front elevational cross section of the sample cell of  FIG. 3  installed in the housing of  FIG. 2 , showing retraction of a clamp holding the sample cell and showing an O-ring seal connecting the sample cell to an interface plate; 
       FIG. 5  is a schematic block diagram of the circuitry of the device of  FIGS. 1 and 2  showing connection of both power and data; 
       FIG. 6  is a simplified display of an image obtained by the camera of  FIG. 2  displayed on the display of the laptop of  FIG. 1  showing sample regions defined by an intersection between strips of probe molecules and a serpentine sample path; 
       FIG. 7  is a plot of percent reflection versus angle of reflection showing adjustment of the angle for maximum contrast between the sample regions of  FIG. 6 ; 
       FIG. 8  is the flowchart showing the principal steps of analyzing sample material using the present invention; and 
       FIG. 9  is an alternative embodiment of the sample cell of  FIGS. 4 and 3 , showing an open area chamber design and the use of a well and integrated interface plate. 
   

   DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
   Referring now to  FIG. 1 , a portable SPR device  10  includes an analyzer unit  12  attached, via a data and power cable  14 , to a conventional laptop computer  16  or other standard computer system. The data and power cable  14  may, for example, be a universal serial bus (USB) cable such as provides a path allowing the analyzer unit  12  to receive power from the batteries or other power supply of the conventional laptop computer  16  and communicate data to the conventional laptop computer  16 . 
   The analyzer unit  12  includes a generally box-shaped housing  18  such as may be comfortably held by an individual in one hand. An angulation knob  20 , to be described in more detail below, extends from one vertical sidewall of the housing  18 . A top wall of the housing  18  provides a sample inlet port  22  into which a sample for testing may be introduced and a sample outlet port  24  which may be connected to a self-contained vacuum port  26 . The housing  18  is preferably of a rugged, opaque material, for example, aluminum or plastic. 
   Referring now to  FIG. 2 , a sample including generally a carrier liquid such as water and molecules to be analyzed, may enter the sample inlet port  22 , introduced by pipette or other instrument. The sample then passes through interface plate  32 , exposed at the upper wall of the housing  18 , to be received by an integrated test cell  28 . From the integrated test cell  28 , the sample passes to the sample outlet port  24  to be drawn through tubing  30  to the vacuum port  26 . The vacuum port  26  communicates with a filter trap  34  trapping the sample and filtering liquid from air, the latter which passes through electric pump  36  to be exhausted via channel  38  through a side wall of the housing  18 . 
   Referring also to  FIGS. 3 and 4 , the integrated test cell  28  is contained within the housing  18  beneath a top wall of the housing  18  to be generally shielded from the environment and ambient light. An upper face of the integrated test cell  28  is held against a lower face of the interface plate  32  so that the sample inlet port  22  attached to the interface plate  32  aligns with a cell inlet port  54  of the integrated test cell  28  and the sample outlet port  24  attached to the interface plate  32  aligns with a cell outlet port  54  of the integrated test cell  28 . O-rings  58 , fitting in shallow toroidal grooves in the interface plate  32 , provide a seal when the integrated test cell  28  is pushed upward against the interface plate  32  as will be described below. 
   Referring now to  FIG. 3 , the integrated test cell  28  includes an optical prism  40  being generally a triangular prism having a base face  42   a  and two side faces  42   b  and  42   c  whose planes together define an isosceles triangular prism. In the preferred embodiment, the apex of the prism  40  representing the junction between faces  42   b  and  42   c  may be flattened or truncated to save material and space. The prism  40  is preferably constructed of a transparent plastic of high refractive index such as polystyrene. 
   A gold film  44  is deposited on the base face  42   a  and forms the metallic film needed for SPR measurement. A series of stripes or patches of probe molecules  48 , for example single-stranded DNA containing a sequence complementary to a sequence of interest, are then deposited on the exposed surface of the gold film  44  (the sensing surface) according to methods well known in the art. 
   A flow cell block  50  may have a serpentine channel  52  cut in a surface facing face  42   a  to attach to face  42   a  to define a serpentine fluid path adjacent to the gold film  44  and crossing the strips of probe molecules  48 . Cell inlet port  56  and cell outlet port  54  are holes in the flow cell block  50  communicating with the serpentine channel  52  at the ends of the serpentine channel  52  and pass through the flow cell block  50  to its upper face removed from the prism  40 . For field use, the flow cell block  50  is preferably permanently attached to the prism  40  by adhesive or mechanical means so as to limit the possibility of contamination of the contained fluid path and probe molecules. Prior to use, an adhesive label (not shown) may be placed on the upper surface of block  50  to prevent contaminants from entering into the cell inlet port  56  and cell outlet port  54 . 
   Preferably, the integrated test cell  28  is disposable and freely replaceable so as to allow multiple tests or tests using different probe molecules  48 . For this reason, in the preferred embodiment, the integrated test cell  28  is releasably held by a clamp  60  attached to a lower surface of an upper wall of the housing  18 . The clamp  60  includes a first set of fixed, sloped, registration jaws  62  attached to the housing and abutting face  42   c  of the integrated test cell  28  to orient the face  42   a  to be parallel the lower surface of the interface plate  32 . A second set of jaws  64 , having a similar slope, are moveable in a horizontal direction  65  by a captive knurled nut  66  acting on a screw  68  attached to the movable jaws  64 . Rotation of the knurled nut  66  advances or retracts the movable jaws  64  toward and away from the integrated test cell  28 . The sloping faces of the registration jaws  62  and movable jaws  64  cause the horizontal compression of the integrated test cell  28  between the registration jaws  62  and movable jaws  64  to yield an upward force compressing the interface between the integrated test cell  28  and interface plate  32 . 
   The upper wall of the housing  18  to which the registration jaws  62 , movable jaws  64 , and interface plate  32  are attached, may hinge upward as indicated by arrow  116  about hinge point  118  to allow easy access to the integrated test cell  28  for changing the integrated test cell  28 . 
   Referring generally to  FIG. 3 , registration jaws  62  and movable jaws  64  (not shown in  FIG. 3 ) are bifurcated, providing a central, unobstructed light path  70  to the faces  42   c  and  42   b  along tipped optical paths  72  and  74  intersecting at a point  76  on the surface of the gold film  44  shown in  FIG. 4 . 
   Referring again to  FIG. 2 , first swing arms  82  attaches at pivot  84  to the front and back of the integrated test cell  28  defining an axis intersecting point  76  (shown in  FIG. 4 ). The swing arms  82  move so that a housing  86  attached at a free end of the swing arms  82  removed from the pivot  84  swings in a radius about point  76 . Housing  86  contains a light emitting diode (LED)  88 , preferably emitting light in the infrared region. The light from the LED  88  is directed through a polarizer  90  along the optical path  72  toward the point  76 . Light from the LED  88  passes through face  42   c  of the prism  40  to strike and illuminate the area of the gold film  44 . The angle of the optical path will be approximately, but not necessarily, exactly perpendicular to the face  42   c  for maximum light transmission into the prism  40  with minimal reflection at face  42   c.    
   Light reflected from the surface of the gold film attached to the prism  40  of the integrated test cell  28  exits along optical path  74  approximately perpendicular to the face  42   b  for maximum light transmission into the air with minimal internal reflection at face  42   b . The light is passed through a monochromatic filter  91  having transmission characteristics centered at the peak emission of the diode  88 . This light is received by a charge couple device (CCD) camera  92  or other similar electronic camera contained within a housing  94  and directed back along the optical path  74 . The camera  92  and housing  94  supporting it, is held by swing arms  96  also attached to pivot  84 . As so attached, the housing  94  and camera  92  swing in a radius about point  76  (shown in  FIG. 4 ) so that the camera  92  may receive an image of the gold film  44  around point  76 . 
   The camera  92  may be moved radially along optical path  74  by means of a slide mount  98  supported for linear motion within the housing  94  and moved by a machine screw  100  whose head is retained by housing  94  and whose threads move the slide mount  98  against the bias of a helical spring  102  captured between the housing  94  and the slide mount  98 . The camera  92  may include a replaceable lens assembly  104  allowing the field of view of the gold film  44  to be changed. The slide mount  98  allows accurate focusing of the camera on the surface of the gold film  44 . 
   The pivots  84  for the swing arms  82  and  96  are attached to side walls of the housing  18  to allow the upper wall of the housing  18  to swing upward. 
   Generally, as will be described now, during movement of the swing arms  82  and  96 , optical paths  72  and  74  are maintained in equal angular relationship with respect to a normal  80  to the surface  42   a  to maximize the reflected light received by the camera  92  from the LED  88 . Within this equality constraint, the angle between each optical path  72  and  74  and the normal  80 , hereafter referred to as θ, may also be adjusted to maximize the sensitivity of the camera  92  to changes in reflected light. 
   Adjustment of the angle θ of optical path  72  and  74  while maintaining them in equal relationship to the normal  80  is provided by means of a gear system including two counter-rotating, inter-engaging gears  106  and  108 . Gear  108  communicates via shaft through a sidewall of the housing  18  with knob  20  to be directly turned by a user while gear  106  turns as driven by gear  108 . 
   Spur gears  109  and  110  are attached coaxially to gears  106  and  108 , respectively, to turn therewith, and engage arcuate racks  112  and  114  having radii centered at pivot  84  and attached to swing arms  82  and  96 , respectively. Rotation of gear  108  causes equal and opposite rotation of gear  106  with corresponding rotations of gears  110  and  109  operating on arcuate racks  112  and  114  to ensure equiangular motion of swing arms  96  and  82 . 
   Referring now to  FIGS. 2 and 5 , camera  92  may communicate through wiring  120  with a camera buffer board  122  also contained within the housing  18 . The wiring  120  is flexible and held loosely in the housing  18  to allow movement of the camera  92  radially and angulation. Likewise, the LED  88  and pump  36  communicate via wiring  124  and  126  with an I/O interface board  130  providing switched power for each according to methods well known in the art. The I/O interface board  130  and camera buffer board  122  in turn through power wiring  132  and data wiring  134  with a USB interface board  136  connected with a standard USB interface cable  138  such as provides a path of data communication of image data from the camera  92  and a source of power for the camera  92 , LED  88  and pump  36 , from the power supply of the attached computer, for example, the battery of the laptop computer  16 , and signals from the computer controlling the pump  36  and LED  88 . Alternatively, the pump  36  and LED  88  may be switched by electrical switches located at the analyzer unit  12 . 
   Referring now to  FIGS. 2 and 6 , when the LED  88  is illuminated, the camera  92  will provide an image  140  of the surface of the gold film  44  adjacent to the prism  40 . This image  140  may be communicated to the standard laptop computer to be displayed during an adjustment after introduction of the sample solution. As shown in  FIG. 6 , the image  140  will reveal one or more regularly spaced regions  142  being intersections of the serpentine channel  52  and the strips of probe molecules  48 . Generally, the probe molecules  48  will include both those that will attach to target molecules in the sample material as well as those that do not attach to target molecules so as to provide further discrimination with respect to the target molecules. In addition, other control regions  142 ′ may be located between the strips of probe molecules  48  within or outside of the serpentine channel  52  to provide control and baseline region. 
   As indicated by the first process block of  FIG. 8 , as indicated by process block  150 , after the sample material has been washed through the integrated test cell  28 , the image  140  may be observed and the contrast between the sample regions  142  (and  142 ′) may be adjusted by changing the angulation of the camera  92  and LED  88  using knob  20 . While the present invention provides a mechanical adjustment, it will be understood that this adjustment can also be done under computer control using an electric motor in place of knob  20 . 
   Referring to  FIG. 7 , the reflection off of the gold film  44  as a function of θ will follow a curve  155  that will remain relatively constant after a critical angle  152  is reached and until a region of plasmon resonance  154 . At this point, interaction between the electron resonance and the material on the opposite side of the gold film  44  causes absorption of some proportion of the reflected light. For a given amount of material on the sensing side of the gold film  44 , for example, amount represented by the attached probe molecules  48 , this reflectance will have a minimum  156  at a particular angle θ. 
   The addition of material to the sensing side of the gold film  44  caused, for example, by binding between the probe molecules  48  with the target molecules, will cause the angular dependence of light reflection to shift left as indicated by curve  153  (dashed line) with a minimum  160 . The removal of material to the sensing side of the gold film  44  caused, for example, by regions  142 ′ having no probe molecules  48 , will cause the reflection of light to shift right as indicated by curve  158  (dashed line). 
   At process block  150 , the angulation of the optical axis may be adjusted to a θ 0  point  161 , for example, at a steep part of the curve  155  at which the reflection is between 100% and the minimum  156  in an area with probe molecules  48  prior to binding of the probe molecules  48  and target molecules. In this case, an increase in binding causing a shifting to curve  158 , will produce a significant increase in reflectance as indicated by point  162  from point  161 . Conversely, regions  142  having neither probe molecules nor target molecules will reveal themselves as regions having no change in reflection. 
   Clearly, a variety of different starting points  161  may be provided on both sides of the slopes leading to the resonance point minima  156  and  160  to obtain contrast that may be measured. Generally, it will be important to approach the resonant point from a consistent direction so as to maintain the proper sense between regions  142  having a build up of molecular material and those relatively free of molecular material. 
   In an alternative embodiment, the range angular values θ may be swept, either manually or with a motor communicating with gear  110 , and using an angular resolver to provide data to the computer  16 , values θ i  for each minima for each region  142  can be determined and these values θ i  used for differentiation. 
   Referring now to process block  163  of  FIG. 8 , reflectance at each of the regions  142  is then compared to control regions, or a previously acquired control image to normalize the measurements. Thresholds are applied to identify each region as binding or non-binding and at process block  164  a set of rules is applied to the region characterizations, being in a simplest case, a Boolean statement with region characterizations as binding vs. non-binding used as arguments. For example, if accumulation of material is obtained on a region  142 , not on a second or third region  142 , this may indicate a particular material in the target sample. 
   Referring now to  FIG. 9  in an alternative embodiment, the interface plate  32  of  FIG. 2  may be incorporated directly into the flow cell block  50  of  FIG. 3  to eliminate an additional element subject to contamination. Sample inlet port  22  and outlet port  24  may be integrally incorporated into the interface plate  32  or as shown, the inlet port  22  may be replaced with a shallow receiving well  168  into which extremely small samples may be placed by pipette or the like. Generally the small samples will preferably be used with the serpentine path of the serpentine channel  52  of  FIG. 3 , however,  FIG. 9  also shows an alternative broad area straight channel  170  such as may be useful in certain circumstances. 
   The features of the present invention combine to provide a low cost and compact unit that may be used with standard computers to provide for SPR measurements in the field. Such a device may be used in a handheld fashion or may be attached to remote devices such as robots or the like for field sampling. Different measurements for different targets may be made by simply replacing the integrated test cell  28 . Alternatively, repeated measurements for the same target over time may be made by use of identical, but new integrated test cells  28 . 
   It is specifically intended that the present invention not be limited to the embodiments and illustrations contained herein, but 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.