Abstract:
A droplet sample holder, especially a sample holder for use in a measuring instrument utilizing surface plasmon resonance. The sample holder reduces or minimizes the measurement distortion result of the droplet “pherpheral concentration effect” by surrounding the analysis zone with a wettable (hydrophilic) zone that captures the periphery of the droplet to keep the pheriphery of the droplet and the increased concentration of the analyte out of the analysis zone. The wettable zone is surrounded by a nonwettable (hydrophobic) zone that restricts the periphery of the droplet to analysis zone and the wettable zone.

Description:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     This invention has been created without the sponsorship or funding of any federally sponsored research or development program. 
     BACKGROUND OF THE INVENTION 
     This present invention relates generally to sample holders for measuring instruments and specifically to sample holders for instruments which utilize surface plasmon resonance (SPR) for measuring chemical or biochemical compositions. Surface plasmon resonance is being used in biosensing, in such areas as immunoassay and nucleic acid detection Basically, surface plasmons are electromagnetic waves created along an interface between a conducting material and a non-conducting material. A common technique for their creation is to direct a beam of electromagnetic radiation into a glass prism with an angle of incidence above the critical angle so that it undergoes total internal reflection. The internal reflection creates an evanescent electromagnetic wave at a region outside of the prism adjacent to the surface. When a thin conductive film is deposited on the surface of the prism, surface plasmons will be formed. 
     Surface plasmon resonance occurs when the momentum (or the wave vector) and energy (i.e. frequency) of the evanescent electromagnetic wave are made to match the momentum and energy of the surface plasmons respectively. It is characterized by a sharp decrease in intensity of the reflected beam as its energy is transferred, because of the resonance, to the surface plasmons. 
     The wave vector K e  of the evanescent wave is defined by the equation:
 
 K   e =(ω/ C ) n  sin Θ,
 
     where ω is the angular frequency of the incident beam, c is the speed of light in vacuum, n is the refractive index of glass and Θ is the angle of incidence. The wave vector of the surface plasmon is defined by the equation:
 
 K   sp =(ω/ c )(1/ε m +1/ε s )−½,
 
     where ε m  is the real part of the dielectric constant of the metal and ε s  is the dielectric constant of the substance under test (or in the absence of any substance, of air) surrounding the metal. 
     At resonance, the wave vector of the evanescent wave is the same as that of the surface plasmons so that there is no electromagnetic wave reflected from the surface. Therefore, occurrence of the surface plasmon resonance is given by the equation:
 
K e =K sp .
 
     If a periodic structure such as a grating or a surface acoustic wave is impressed upon the thin metal layer, the above equation becomes:
 
 K   e   +k=K   sp .
 
     where k is the wave vector due to the periodic structure. 
     The above equation provides a useful tool for measuring differences between the values of ε s  of different materials. It also provides a useful tool for detecting the presence of trace surface chemicals in a substance that alters its ε s  value. By measuring the differences of K e  at resonance, the changes in ε s  can be determined. 
     Surface plasmon resonance measuring instruments typically utilize the above equality condition and measure the differences of K e  by varying Θ and sensing the reflected beam at different values of Θ to detect the resonance. In these surface plasmon resonance measuring instruments, sensing the reflected beam at different values of Θ has been accomplished by various methods. Other surface plasmon resonance measuring instruments utilize the above equality condition and measure the differences of K e  by varying parameters and sensing the reflected beam at different values of the varied parameter. 
     In the typical surface plasmon resonance measuring instrument, the sample to be measured are placed in a pattern of droplets on a gold surface of a glass slide. The gold coated slide is referred to as a chip. 
     The present invention is directed generally to surface Plasmon Resonance technology (SPR) which allows the characterization of bio specific interactions of label-free compounds. The invention is specifically directed to a variation of SPR known as the Kretchmann process and, more specifically, to the substrate or chip that is part of the process. In the Kretchmann process, a collimated beam of light, e.g. laser, is directed through a prism to a chip that is supported on the prism. The chip is glass slide coated with metal such as silver or gold. The light hits the glass gold interface. At a specific angle, the light will be absorbed. The nonabsorbed light is reflected back through the prism and detected. Biological samples to be analyzed are deposited on the gold in an array of droplets. Each droplet has a target biological element bound to a ligand, for example, that will have an effect on the reflected light to the detector which will be indicative of the specific biological element in the droplet. 
     More specifically, the Kretchman process uses a constant wave-length light source, e.g. laser. The laser light is directed through a P-polarizer to pass only the P-polarized light. The light is then directed through a prism. A glass slide coated with gold is positioned fixed on the hypotenuse of the prism with refractive index matching fluid. The light hits the glass gold interface. At a specific angle, the light will be absorbed. The non-absorbed light is reflected and detected using a photomultiplier tube or CCD camera. 
     One of the problems associated with conventional chips is that as the droplet spreads over the gold surface and as the droplet dries, the analyte migrates to the periphery of the droplet, so that there is a higher concentration of biological material at the periphery of the droplet than in the body of the droplet. This non-uniform distribution of analyte in the droplet, or “peripheral concentration effect”, can produce a distortion of the light signal received by the detector, and interferes with the ability of the device to correctly analyze the content of the droplet. 
     What is needed is a sample holder for an instrument that can be used for measuring chemical compositions by measuring the dielectric constants thereof utilizing surface plasmon resonance, said sample holder being designed so that the measurement distorting result of the sample droplet “peripheral concentration effect” is minimized or eliminated. 
     SUMMARY OF THE INVENTION 
     This invention is directed to a droplet sample holder, and especially a droplet sample holder surface for use in a plasmon resonance (SPR) measuring instrument. 
     In one implementation, the measuring instrument comprises a block of material transparent to a beam of electromagnetic radiation. This block of material has a surface for providing internal reflection of an electromagnetic radiation beam and one or more separate electrically conductive spots on the outside of the surface. The instrument has a source projecting a source beam of electromagnetic radiation onto a beam steering device. The beam steering device receives the source beam and transmits it into the block at an adjustable angle of incidence to a reflection point on the block surface. In the case of a single spot, the spot is located at the reflection point. In the case of the plurality of spots, a translation system is provided so that each of the spots can be selectively located at the reflecting point. Detection of the reflected beam from each spot is performed through a detector. 
     The “peripheral concentration effect” problem found in the prior art is overcome by the chip of the present invention in which the continuous coating of gold of the prior art chips is replaced by one or an array of separate electrically conductive spots. The chip of the present invention comprises a glass slide that carries one or an array of separate electrically conductive spots. Each spot can be of any shape, but is preferably round. Each spot is surrounded by a layer of wettable (hydrophilic) material which is, in turn, surrounded by a layer of nonwettable (hydrophobic) material. The wettable layer attracts the periphery of the droplet on the surrounded spot and insures that the edges of the droplet are outside of surrounded spot. This moves the droplet periphery, in which periphery the analyte is concentrated, off the spot, and leaves only the central portion of the droplet, in which the analyte is uniformly concentrated, over the spot. This insures that the distribution of the analyte will be uniform across the spot and across the field of analysis. The nonwettable layer restricts the expansion of the droplet and keeps the droplet across the spot and the wettable layer. 
     It will be understood that the concept of wettable and nonwettable is relative to the composition of the droplet. In the typical case where the droplet is mainly water, the wettable means hydrophilic and nonwettable means hydrophobic. It should be understood, however, that the wettability or nonwettability of a surface is a function of the entire content of the droplet. Thus, the wettability or nonwettability of a surface to an aqueous droplet can be greatly affected if the droplet contains even small amounts of surface active molecules, such as detergents. More generally, the wettable surface has a surface energy higher than the surface energy of the droplet and the nonwettable surface has a surface energy lower than the surface energy of the droplet. 
     For a typical aqueous droplet, wettable surfaces include soda glass, and nonwettable surfaces include low-density polyethylene. 
     The chip is shown supported on the surface of a prism that forms part of the SPR apparatus. The apparatus also includes a source of light and a detector. The light source projects a beam of light through the prism to the electrically conductive spots. Light reflected from each spot is received and analyzed by the detector. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The character of the invention, however, may best be understood by reference to one of its structural forms, as illustrated by the accompanying drawings, in which: 
         FIG. 1  is a diagrammatic front elevation view a surface plasmon resonance (SPR) measuring instrument and droplet holder embodying the principles of the present invention, 
         FIG. 2  is a plan view of a droplet holder embodying the principles of the present invention, 
         FIG. 3  is a close-up diagrammatic view of a small volume droplet on a droplet holder embodying the principles of the present invention, 
         FIG. 4  is a close-up diagrammatic view of a medium volume droplet on a droplet holder embodying the principles of the present invention, and 
         FIG. 5  is a close-up diagrammatic view of a high volume droplet on a droplet holder embodying the principles of the present invention. 
     
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT 
     Referring first to  FIG. 1  in which the general principles of the present invention are shown, the chip  18  is shown supported on and optically connected to the internally reflective surface  11  of a prism  12  that forms part of an SPR apparatus, generally indicated by the reference numeral  10 . The chip  18  includes a glass slide  20 , a plurality of electrically conductive spots  22  on the outside surface  21  of the glass slide  20 , and, over each spot  22 , the chip carries a droplet  28  to be analyzed. The apparatus  10  includes a source of light  14  and a detector  16 . The light source  14  projects a beam of light  30  through the prism  12  to a reflective location  31  on the chip  18  and to an electrically conductive spot  22  on the chip  18  at that reflective location  31 . Light reflected from the location  31  and spot  22 , beam  32 , is received and analyzed by the detector  16 . A translation device  25  is provided to move the chip  18  relative to the prism  12  so that each of the spots  22  can be positioned at the reflective location  31  to reflect the beam  30 . The space  15  between the prism  12  and the slide  20  is filed with an index matching fluid  17 . The prism  12 , the slide  20 , and the fluid  17  all have the same index of refraction so that the space  15  causes no refraction of the beams  30  or  31 . 
     The “peripheral concentration effect” problem found in the prior art is overcome by the chip  18  of the present invention in which the continuous coating of gold of the prior art chips is replaced by one or an array of separate electrically conductive spots  22  as shown in a plan view in  FIG. 2 . The chip  18  comprises a glass slide  20  that carries one or an array of separate electrically conductive spots  22 . Each spot  22  can be of any shape, but is preferably round. Each spot  22  is surrounded by a layer  24  of wettable (hydrophilic) material that is, in turn, surrounded by a layer  26  of nonwettable (hydrophobic) material. The wettable layer  24  attracts the periphery  29  of a droplet  28  (not shown) on the surrounded spot  22  and insures that the periphery  29  of the droplet  28  (not shown) is outside of surrounded spot  22 . This moves the droplet periphery  29 , in which periphery the analyte is concentrated, off the spot, and leaves only the central portion  27  of the droplet  28 , in which the analyte is uniformly concentrated, over the spot  22 . This insures that the distribution of the analyte will be uniform across the spot. The nonwettable layer  26  restricts the expansion of the droplet and keeps the droplet across the spot and the wettable layer  24 . 
       FIG. 3  is a close-up front elevation view of a low volume droplet on the chip  18 . The spot  22  on the slide  20  of the chip  18  is surrounded by a layer  24  of wettable (hydrophilic) material which is, in turn, surrounded by a layer  26  of nonwettable (hydrophobic) material. The wettable layer  24  attracts the periphery  29  of a droplet  28  on the surrounded spot  22 , and draws the periphery of the droplet to the periphery of the wettable layer  23  and insures that the periphery  29  of the droplet  28  is outside of surrounded spot  22 . This moves the droplet periphery  29 , near which periphery the analyte is concentrated, off the spot, and leaves only the central portion  27  of the droplet  28 , in which the analyte is uniformly concentrated, over the spot  28 . This insures that the distribution of the analyte will be uniform across the spot. The periphery of the droplet will expand until it reaches the periphery  23  of the wettable layer and the boundary between the wettable and nonwettable layers, at which it will stop. The contact angle between wettable layer and the droplet surface will depend on the volume of the droplet. For low volume droplets, as represented by  FIG. 3 , the contact angle will be less than 90 degrees. 
     Line  35  is a diagrammatic representation of the concentration of the analyte across the diameter of the droplet  28 . The height of the line  35  over the outer surface of the spot  22  and wettable layer  24  represents the concentration of the analyte in the droplet  28 . The analyte is highly concentrated near the periphery  29  of the droplet and relatively lower and uniform over and across the spot  22  and the central portion  27  of the droplet  28 . This uniformity of concentration over the spot  22  improves the effectiveness of the SPR apparatus  10 . The nonwettable layer  26  restricts the expansion of the droplet and keeps the droplet across the spot and the wettable layer  24 . 
       FIG. 4  shows the effect of increased volume in the droplet  28 . As the volume increases, the contact angle  33  increases.  FIG. 4  shows the contact angle at 90 degrees. 
       FIG. 5  shows the effect of further increased volume in the droplet  28 . As the volume increases, the contact angle  33  increases.  FIG. 5  shows the contact angle at greater than 90 degrees. 
     After a droplet is analyzed, the translation device  25  moves a new stop  22  into the reflective location  31  so that the new spot  22  can be analyzed. 
     It will be understood that the concept of wettable and nonwettable is relative to the composition of the droplet. In the typical case where the droplet is mainly water, the wettable means hydrophilic and nonwettable means hydrophobic. It should be understood, however, that the wettability or nonwettability of a surface is a function of the entire content of the droplet. Thus, the wettability or nonwettability of a surface to an aqueous droplet can be greatly affected if the droplet contains even small amounts of surface-active molecules, such as detergents. More generally, the wettable surface has a surface energy or surface tension higher than the surface energy of the droplet and the nonwettable surface has a surface energy or surface tension lower than the surface energy of the droplet. 
     For a typical aqueous droplet, wettable surfaces include soda glass, and nonwettable surfaces include low-density polyethylene. 
     Although this invention is described with reference to specific parameters and implementations, it will be understood that various modifications can be made thereto without substantive departure from the scope of the invention, which is defined by the following claims.