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. First and second linear scanned arrays are positioned on opposite sides of a meridional plane of the optical configuration for respectively detecting the first and second partial beams. Thus, differential measurements are possible based on signal information from the arrays. 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  
         [0001]    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 a first sample and a second sample. The present invention is applicable to differential refractometers and surface plasmon resonance (SPR) biosensor devices.  
         BACKGROUND OF THE INVENTION  
         [0002]    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 shadow line 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.  
           [0003]    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  39 A-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.  
           [0004]    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. Nos. 4,640,616 (Michalik) and 6,172,746 (Byrne et al.). However, applicants are unaware of any critical angle optical device for differential refractive index measurements that operates using linear arrays, despite the recognized economy offered by this type of array.  
         BRIEF SUMMARY OF THE INVENTION  
         [0005]    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.  
           [0006]    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.  
           [0007]    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.  
           [0008]    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 pair of linear scanned arrays of photoelectric cells.  
           [0009]    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.  
           [0010]    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 substantially collimates the first and second partial beams, and a pair of linear scanned arrays is located on opposite sides of the meridional plane to respectively receive the first and second partial beams. The first partial beam exhibits a feature, such as a shadow line or resonance minimum, on one of the linear scanned arrays, while the second partial beam exhibits a similar feature on the other linear scanned array. The array locations of the exhibited features are determined by analyzing the output signals of the array cells, and are indicative of the refractive index of the first and second samples, respectively.  
           [0011]    An alternative embodiment 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. More specifically, 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.  
           [0012]    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  
       [0013]    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:  
         [0014]    [0014]FIG. 1 is a perspective schematic view of an optical configuration formed in accordance with a first embodiment of the present invention;  
         [0015]    [0015]FIG. 2 is a view taken generally along the line II-II in FIG. 1;  
         [0016]    [0016]FIG. 3 is a side schematic view of an optical configuration formed in accordance with a second embodiment of the present invention;  
         [0017]    [0017]FIG. 4 is a top schematic view of a sample prism of the second embodiment, illustrating a line of illumination light formed by a toric lens of the configuration;  
         [0018]    [0018]FIG. 4A is an enlarged view of part of the sample prism illustrated in FIG. 4;  
         [0019]    [0019]FIG. 5 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,  
         [0020]    [0020]FIG. 6 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  
       [0021]    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 band-pass 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 positive 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. 3 and 4.  
         [0022]    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 material, for example room temperature vulcanizing (RTV) silicon rubber or VITON® synthetic rubber composition, will provide a suitable barrier.  
         [0023]    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.  
         [0024]    A first linear scanned array  46 A and a second linear scanned array  46 B are arranged side-by-side on opposite sides of meridional plane MP for receiving first partial beam  13 A and second partial beam  13 B, respectively. Linear scanned arrays  46 A and  46 B each comprise a plurality of photoelectric cells that provide an output pulse during a scan having an amplitude determined by the amount of illumination of the corresponding cell by incident light. The timing and frequency at which scanning electronics  61  scans linear arrays  46 A and  46 B is controlled by a timing circuit  62 . The signal information provided by first linear scanned array  46 A is preferably summed over a plurality of scans, and signal information from second linear scanned array  46 B is preferably summed in the same manner.  
         [0025]    As is well understood in the art of critical angle refractometry, first partial beam  13 A will exhibit a shadowline at a location on first linear scanned array  46 A that is indicative of the refractive index of test sample TS. In similar fashion, second partial beam  13 B will exhibit a shadowline on second linear scanned array  46 B 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 shadow lines will appear at the same cell-crossing location on linear scanned arrays  46 A and  46 B. Consequently, the difference in cell-crossing location between the test sample and reference sample shadow lines on linear scanned arrays  46 A and  46 B 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 shadow line locations.  
         [0026]    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 arrays  46 A and  46 B 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 display monitor, printer, or other reporting device, is linked to CPU  66  for reporting measurement results in a desired format. For example, reporting can be in a non-differential mode  
         [0027]    [0027]FIG. 3 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 generally similar to optical configuration  10  of the first embodiment. However, in the second embodiment, illumination beam  12  is refracted by a toric lens  124  before it enters prism  26 . Toric lens  124  has a minimum power along a transverse meridian (a line normal to meridional plane MP) and a maximum power in a perpendicular meridian. As a result, illumination beam  12  reaches test sample TS and reference sample RS as a well-defined line of light bridging across meridional plane MP, as illustrated in FIGS. 4 and 4A. Optical configuration  110  of the second embodiment also differs from optical configuration  10  of the first embodiment in that it uses a conditioning lens system after prism  26  sequentially comprising a negative lens  130  and a positive lens  132  to provide approximately collimated light that is scaled to fit linear scanned arrays  46 A and  46 B.  
         [0028]    It will be recognized that the basic optical arrangements of FIGS. 1 and 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. 5, 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. 6. 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 location on first linear scanned array  46 A that is indicative of the refractive index of test sample TS. Likewise, second partial beam  13 B will exhibit a resonance minimum at a location on second linear scanned array  46 B 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.  
         [0029]    The embodiments of FIGS. 5 and 6 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.