Abstract:
An optical configuration for measuring a difference in refractive index between a first sample and a second sample comprises partitioned first and second optical interfaces symmetrically illuminated by an illumination beam to provide first and second partial beams defined by the refractive index of the first and second samples, respectively. A linear scanned array is aligned in a meridional plane of the optical configuration for detection purposes, and an optical multiplexor is provided upstream of the linear scanned array for receiving the first and second partial beams and defining first and second optical channels carrying optical signal information corresponding to the first and second partial beams. The optical multiplexor switches between optical channels, such that the linear scanned array detects either the first or second optical channel at a given time. Thus, differential measurements are possible using a single linear array. Embodiments for critical angle and surface plasmon resonance refractive index measurements are disclosed. The disclosure also relates to methods for measuring a difference in refractive index between a first sample and a second sample in accordance with the described optical configuration embodiments.

Description:
FIELD OF THE INVENTION 
     The present invention relates generally to optical instruments for measuring refractive index of a substance, and more particularly to an optical configuration for measuring a difference in refractive index between first and second samples, for instance a test sample and a reference sample. The present invention is applicable to differential refractometers and surface plasmon resonance (SPR) biosensor devices. 
     BACKGROUND OF THE INVENTION 
     Refractometers measure the critical angle of total reflection by directing an obliquely incident non-collimated beam of light at a surface-to-surface boundary between a high refractive index prism and a sample to allow a portion of the light to be observed after interaction at the boundary. In transmitted light refractometers, light that is transmitted through the sample and prism is observed, while in reflected light refractometers, the light that is reflected due to total reflection at the surface-to-surface boundary is observed. In either case, an illuminated region is produced over a portion of a detection field of view, and the location of the shadowline between the illuminated region and an adjacent dark region in the detection field of view allows the sample refractive index to be deduced geometrically. Differential refractometers, for example that disclosed in U.S. Pat. No. 5,157,454, have been developed for measuring a difference in refractive index between a test sample and a known reference sample, whereby variable test conditions effecting the measurement result, such as sample temperature, illumination level, etc., can be “subtracted out” to yield a more accurate and precise measurement result. The prior art differential refractometers known to applicants involve moving parts which malfunction or wear out over time, and/or are restricted to the transmitted light variety so as to prevent measurement of samples having relatively high opacity. 
     Optical biosensor devices designed to analyze binding of analyte molecules to a binding layer by observing changes in internal reflection at a sensing interface are also part of the related prior art. More specifically, U.S. Pat. No. 5,313,264 to Ivarsson et al. describes an optical biosensor system that comprises a plurality of side-by-side sensing surfaces 39A-D illuminated by a streak of light 5 extending transversely across the sensing surfaces, and an anamorphic lens system 6 by which rays of light reflected from the respective sensing surfaces are imaged on corresponding columns of a two-dimensional array 7 of photosensitive elements. Accordingly, the signals from the photosensitive elements can be processed to determine a minimum reflectance associated with the resonance angle at each sensing surface. Although the system described in U.S. Pat. No. 5,313,264 avoids the use of moving parts, it is nevertheless optically complex and requires a two-dimensional array, factors that are accompanied by an increase in cost. 
     Finally, it is noted that one-dimensional (linear) arrays of photosensitive elements cells are commonly used in automatic refractometers designed to take non-differential readings with respect to a single test sample. Examples can be found in U.S. Pat. No. 4,640,616 (Michalik) and U.S. Pat. No. 6,172,746 (Byrne et al.). However, applicants are unaware of any critical angle optical device for differential refractive index measurements that operates using a linear array, despite the recognized economy offered by this type of array. 
     BRIEF SUMMARY OF THE INVENTION 
     Therefore, it is an object of the present invention to provide an optical configuration for differential refractive index measurements wherein a first sample and a second sample are illuminated by a single illuminating beam. 
     It is another object of the present invention to provide an optical configuration for differential refractive index measurements that does not rely on moving parts. 
     It is a further object of the present invention to provide an optical configuration for differential refractive index measurements wherein detected light has been reflected rather than transmitted at an optical interface of the configuration. 
     It is a further object of the present invention to provide an optical configuration for critical angle differential refractive index measurements wherein light interacting at first and second optical interfaces corresponding to a first sample and a second sample is detected by a single linear scanned array of photoelectric cells. 
     It is a further object of the present invention to provide an optical configuration for differential refractive index measurements in accordance with the objects stated above, and which operates based on surface plasmon resonance principles for use in a biosensor device. 
     An optical configuration formed in accordance with a first embodiment of the present invention comprises an optical path defining a meridional plane of the configuration. A high index prism in the optical path includes a sample surface divided by a partition residing in the meridional plane, such that a first sample and a second sample supported by the sample surface are located on opposite sides of the meridional plane to establish a first optical interface associated with the first sample and a second optical interface associated with the second sample. An illumination beam traveling along the optical path illuminates both optical interfaces simultaneously to provide a first partial beam defined by the refractive index of the first sample and a second partial beam defined by the refractive index of the second sample. A collecting lens collimates the first and second partial beams and an optical multiplexor means receives the collimated partial beams and defines first and second optical channels containing optical signal information associated with the first and second partial beams, respectively. A cylinder lens and/or a biprism after the optical multiplexing means deflects the partial beams laterally toward the meridional plane of the system for illuminating a linear scanned array of photoelectric cells aligned in the meridional plane. In a first embodiment, the optical multiplexing means comprises a liquid crystal shutter programmed to alternately transmit one partial beam while blocking the other partial beam such that a given scan of the linear array provides signal information with respect to either the first partial beam or the second partial beam, depending upon the corresponding state of the liquid crystal shutter. In a second embodiment, a similar arrangement is used, however the liquid crystal shutter is programmed to transmit and block predetermined portions of each partial beam in an alternating fashion, such that for a given scan of the linear array some of the array cells will provide signal information relating to the first partial beam and some of the cells will provide signal information relating to the second partial beam. The first partial beam exhibits a feature, such as a shadowline or resonance minimum, on the linear scanned array the location of which is indicative of the refractive index of the first sample, while the second partial beam exhibits a similar feature the location of which is indicative of the refractive index of the second sample. 
     A third embodiment based on the first and second embodiments is an adaptation of the basic configuration in order to observe molecular interactions, particularly specific binding of analyte molecules to a binding layer, using the principles of surface plasmon resonance. In accordance with the third embodiment, a thin metallic film is applied to a slide placed on the sample surface or directly to the sample surface, and the first sample and second sample are brought into contact with the metallic film to define first and second evanescent wave optical interfaces. In this embodiment, the locations of resonance minimums exhibited by the first and second partial beams are detected. 
     The present invention further encompasses methods for measuring a difference in refractive index between a first sample and a second sample based on the specified optical configurations. 
    
    
     BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
     The nature and mode of operation of the present invention will now be more fully described in the following detailed description of the invention taken with the accompanying drawing figures, in which: 
     FIG. 1 is a perspective schematic view of an optical configuration formed in accordance with a first embodiment of the present invention; 
     FIG. 2 is a view taken generally along the line  11 — 11  in FIG. 1; 
     FIG. 3 is a view similar to that of FIG. 1, however showing an optical configuration formed in accordance with a second embodiment of the present invention; 
     FIG. 4 is a perspective view showing an optical interface portion of an optical configuration formed in accordance with a third embodiment of the present invention relating to surface plasmon resonance; and 
     FIG. 5 is a perspective view showing an optical interface portion of an optical configuration formed in accordance with a fourth embodiment of the present invention also relating to surface plasmon resonance. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     An optical configuration formed in accordance with a first embodiment of the present invention will now be described with reference to FIG. 1 of the drawings. The optical configuration of the first embodiment is shown generally at FIG.  1  and is designated by the reference numeral  10 . Optical configuration  10  includes an illumination beam  12  traveling along an optical path OP from the beam&#39;s origin at a light source  11 . Illumination beam  12  travels through a focusing optical system  14  preferably including a collimating lens  16 , a narrow bandpass filter  18  for transmitting a narrow bandwidth of light having a central wavelength of 589 nm, a linear polarizer  19 , and a focusing lens  20 . The convergent illumination beam then passes through a pinhole stop  22  at the focal plane of focusing optical system  14 . The divergent beam  12  is then re-focused by a lens  24  and enters a high refractive index prism  26 , for example a sapphire prism, that includes a light entry surface  26 A, a sample surface  26 B contacted by test sample TS and reference sample RS, and a light exit surface  26 C. Preferably, lens  24  is affixed with optical cement to light entry surface  26 A of prism  26 . The illuminating light is focused at a point within prism  26  just below sample surface  26 B, after which point the beam once again becomes divergent. It is noted that polarizer  19  is provided to enable use of the optical configuration in connection with surface plasmon resonance measurements as will be described in a subsequent portion of this description that makes reference to FIGS. 4 and 5. 
     Illumination beam  12  approaches sample surface  26 B as a beam of non-parallel light rays, in this instance divergent light rays, which are obliquely incident to sample surface  26 B at various angles of incidence within a range of angles. Sample surface  26 B is divided by a partition  27  into a first area for receiving a test sample TS and a second area for receiving a reference sample RS. Partition  27  is coplanar with optical path OP as the optical path approaches sample surface  26 B such that the light rays making up illumination beam  12  are symmetrically apportioned between a first optical interface  30 A associated with the test sample TS and a second optical interface  30 B associated with the reference sample RS. Partition  27  is chosen to provide a fluid seal between test sample TS and reference sample RS to prevent the samples from mixing. A synthetic rubber gasket material, for example room temperature vulcanizing (RTV) silicon rubber or VITON® synthetic rubber composition, will provide a suitable barrier. 
     In the present embodiment, first optical interface  30 A and second optical interface  30 B are critical angle optical interfaces respectively defined by the contact area of test sample TS with sample surface  26 B and by the contact area of reference sample RS with sample surface  26 B. These contact areas can be established by dropping the test sample TS and reference sample RS onto sample surface  26 B on opposite sides of partition  27 , by using a flow cell designed to bring test sample TS and reference sample RS into contact with sample surface  26 B on opposite sides of partition  27 , or by otherwise applying test sample TS and reference sample RS to the respective areas of sample surface  26 B. The portion of illumination beam  12  reaching first optical interface  30 A will interact at such interface in accordance with Snell&#39;s Law, whereby rays incident at an angle greater than or equal to the critical angle will be totally internally reflected from sample surface  26 B, and rays incident at an angle less than the critical angle will be refracted and transmitted through the test sample and out of the optical system. Accordingly, the internally reflected light forms a first partial beam  13 A that is defined by the index of refraction of test sample TS. A similar interaction occurs for the portion of illumination beam  12  reaching second optical interface  30 B, whereby internally reflected light forms a second partial beam  13 B that is defined by the index of refraction of reference sample RS. First partial beam  13 A and second partial beam  13 B then pass through exit surface  26 C and continue through a collecting lens  32  for converting the divergent light rays to parallel light rays. 
     In accordance with the present invention, an optical multiplexing means designated generally as  40  is positioned in optical path OP after collecting lens  32  to receive first partial beam  13 A and second partial beam  13 B. Optical multiplexing means  40  defines a first optical channel containing optical signal information associated with first partial beam  13 A and a second optical channel containing optical signal information associated with second partial beam  13 B. In the embodiment of FIG. 1, optical multiplexing means  40  comprises an electro-optical shutter  42  having a first area  42 A arranged to receive first partial beam  13 A and a second area  42 B arranged to receive second partial beam  42 B. In a preferred arrangement, shutter  42  is centered with respect to optical axis OP, and first and second areas  42 A and  42 B are halves of the shutter on opposite sides of meridional plane MP. 
     As seen in FIG. 1, shutter  42  is connected to a shutter drive circuit  60  that signals shutter  42  to alternate at a predetermined frequency between a condition wherein first area  42 A is transparent and second area  42 B is opaque, and a condition wherein first area  42 A is opaque and second area  42 B is transparent. As a result, optical multiplexing means  40  defines a first optical channel corresponding to the exclusive transmission of first partial beam  13 A and a second optical channel corresponding to the exclusive transmission of second partial beam  13 B. It will be readily apparent to those skilled in the art that optical multiplexing means  40  can comprise two individual optical shutters respectively allocated to first partial beam  13 A and second partial beam  13 B and driven in opposite synchronization to produce the desired definition of optical channels. Electro-optical shutter  42  may be a commercially available liquid crystal shutter. Other types of optical shutters may also be used, including mechanical choppers and shutters, acousto-optical shutters, and magnetic shutters. 
     A biprism  44  and a cylindrical lens  46  are positioned along optical path OP downstream of optical multiplexing means  40  for redirecting partial beams  13 A and  13 B laterally toward meridional plane MP. Biprism  44  and cylinder lens  46  act as an anamorphic system to cause each partial beam to be imaged as a line of light illuminating photoelectric cells of a linear scanned array  50  aligned in meridional plane MP. Depending upon the geometry of optical configuration  10 , it is contemplated to provide only one anamorphic optical element, for example either biprism  44  or cylinder lens  46 , to achieve a line of light at linear scanned array  50 , as this would save the cost of providing and locating an additional optical element. 
     Linear scanned array  50  receives first partial beam  13 A and second partial beam  13 B in alternating succession, such that optical signal information associated with either the first optical channel or the second optical channel is transmitted to and received by the linear scanned array at any given instant in time. The timing and frequency at which scanning electronics  61  scans linear array  50  is synchronized by a timing circuit  62  with the oscillation of multiplexing means  40  between the first and second optical channels, whereby a particular optical channel (first or second) is attributable to each scan of linear array  50 . The signal information provided by linear scanned array  50  is preferably summed over a plurality of scans for each respective optical channel. For example, the frequency at which electro-optical shutter  42  alternates between transmission of the first and second optical channels can be less than the scanning frequency of said linear scanned array to allow signal information from a particular optical channel to be accumulated before switching to the other optical channel. 
     As is well understood in the art of critical angle refractometry, first partial beam  13 A will exhibit a shadowline at a first location on linear scanned array  50  that is indicative of the refractive index of test sample TS. In similar fashion, second partial beam  13 B will exhibit a shadowline on linear scanned array  50  that is indicative of the refractive index of reference sample RS. For example, when test sample TS and reference sample RS have the same index of refraction, their respective shadowlines will appear at the same cell-crossing location on linear scanned array  50 . Consequently, the difference in cell-crossing location between the test sample and reference sample shadowlines on linear scanned array  50  provides an indication of the difference in refractive index between the test sample and reference sample. If the refractive index of the reference sample RS is known for the particular test conditions, the refractive index of the test sample TS can be calculated from the measured difference in shadowline locations. 
     It is noted here that various algorithms are available for determining shadowline location on a linear scanned array, as taught for example by U.S. Pat. Nos. 4,640,616; 5,617,201; and 6,172,746; and by commonly-owned U.S. patent application Ser. No. 09/794,991 filed Feb. 27, 2001, each of these documents being hereby incorporated by reference in the present specification. The analog pulse signals from the cells of linear scanned array  50  are digitized by an analog-to-digital converter  64 , and the digitized array information is processed by a central processing unit  66 . An output device  68 , such as a liquid crystal display, computer monitor, printer, or the like, is connected to central processing unit  66  for reporting computed measurement results. 
     FIG. 2 illustrates an optical configuration formed in accordance with a second embodiment of the present invention and identified by reference numeral  110 . Optical configuration  110  is similar to optical configuration  10  of the first embodiment, with electro-optical shutter  42  having a first area  42 A for receiving a first partial beam  13 A and a second area  42 B for receiving second partial beam  13 B. However, in the second embodiment, each of the first and second areas is subdivided into a plurality of sub areas  52 A and  52 B, respectively. The sub-areas  52 A provide a grid-like pattern of opaque and transparent regions in first area  42 A, while the sub-areas  52 B provide a corresponding but opposite grid of opaque and transparent regions in second area  42 B. The various sub-areas  52 A,  52 B of electro-optical shutter  42  are alternated at a predetermined frequency between opaque and transparent states. As a consequence, each scan of linear scanned array  50  will extract signal information from both the first and second optical channels, but at half the resolution of a single channel scan according to the first embodiment. Thus, in the embodiment of FIG. 2, the first optical channel is defined by pulsed transmission of interlaced portions of the first partial beam in alternating succession, and said second optical channel is defined by pulsed transmission of interlaced portions of said second partial beam in alternating succession. 
     It will be recognized that the basic optical arrangements of FIG. 1 or  3  can be used in connection with evanescent wave optical interfaces rather than critical angle optical interfaces by coupling a glass slide having a thin metallic film to sample surface  26 B, or by directly coating sample surface  26 B with a thin metallic film. In the arrangement shown in FIG. 4, a glass slide  70  is provided with a thin metallic film  72  on an upwardly facing surface thereof. In the present embodiment, metallic film  72  includes a layer of chromium approximately ten angstroms thick for adherence to the glass surface of slide  70 , and a gold layer approximately fifty nanometers thick. A synthetic rubber material, such as RTV silicon, VITON® synthetic rubber composition, or like material is applied to metallic film  72  to provide partition  27 . Metallic film  72  is optically coupled, indirectly, to prism sample surface  26 B through transparent glass slide  70  and a thin layer of transparent oil  74  provided between the underside of glass slide  70  and sample surface  26 B. Of course, metallic film  72  can be optically coupled to sample surface  26 B by applying the film directly to sample surface  26 B, as illustrated in FIG.  5 . Test sample TS and reference sample RS are contacted with metallic coating  72  on opposite sides of partition  27 , such that respective first and second optical interfaces are established. As light from illumination beam  12  reaches metallic film  72  at the first optical interface, certain rays will be incident at a resonance angle determined by the refractive index of test sample TS and energy associated with such rays will be absorbed, while the remainder of the rays will be internally reflected by metallic film  72 . As a result of surface plasmon resonance, first partial beam  13 A exhibits a resonance minimum at a first location on linear scanned array  50  that is indicative of the refractive index of test sample TS. Likewise, second partial beam  13 B will exhibit a resonance minimum at a second location on linear scanned array  50  that is indicative of the refractive index of reference sample RS. It is noted here that for surface plasmon resonance applications, a narrow band-pass filter  18  preferably transmits light having a central wavelength of 780 nm. 
     The embodiments of FIGS. 3 and 4 based on evanescent wave principles find useful application in the observation of molecular interactions, particularly in the analysis of specific binding of analyte molecules to a binding layer. Accordingly, prepared slides having a predetermined, application-specific binding layer applied to metallic film  72  can be produced for use with a variety of analytes.