Abstract:
Embodiments of devices and methods are provided that permit validation of analyte detection using both surface enhanced Raman spectroscopy (SERS) and surface plasmon resonance (SPR). In specific embodiments, a substrate having a surface suitable for SPR is provided, along with a source of electromagnetic radiation to interact with the surface and thereby elicit surface plasmon resonance characteristic of the analyte under study. In some embodiments, surface enhancing structures are also provided on the substrate, and analytes under study are associated with enhancing structures. Another source of electromagnetic radiation is directed at the analyte on the enhancing structures to produce surface enhanced Raman scattering. In certain embodiments, data obtained by these two methods are compared, thereby providing an internally consistent and self-validating method for analyte detection.

Description:
RELATED APPLICATION  
       [0001]    This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Serial No. 60/323,981, filed Sep. 21, 2001. This application also claims priority to U.S. Provisional Patent Application Serial No. 60/156,195, filed Sep. 27, 1999, now abandoned, and to United States Utility patent application Ser. Nos. 09/670,453, filed Sep. 26, 2000, 09/815,909, filed Mar. 23, 2001, and 09/925,189 filed Aug. 8, 2001. Each of the above patent applications is incorporated herein fully by reference. 
     
    
     
       BACKGROUND  
         [0002]    Detection and quantification of analytes in complex mixtures of substances is a substantial component of medical, environmental and industrial processes. However, detection and quantification remains laborious, time consuming and expensive. Generally, measurements and their methods are designed specifically for the analyte to be measured, processes that can be challenging and expensive. Modem drug discovery is based in part, on high throughput screening (HTS) of candidate molecules. For many prior art methods, labeling of the analyte is required, and for detection of nucleic acids, polymerase chain reaction (PCR) is often used. Unfortunately, labeling and PCR methods are time consuming, expensive and can lead to errors.  
           [0003]    Recently, new methods have been developed that can eliminate one or more of the time consuming steps previously considered to be necessary. For example, Raman spectroscopy can be used to detect analytes directly, without the need for labeling or PCR steps. Examples of Raman methods are described in United States Patent Application titled “Particle Structures with Receptors for Analyte Detection”, Ser. No. 09/670,453 and United States Patent Application titled “Addressable Arrays Using Morphology Dependent Resonance for Analyte Detection”, Ser. No. 09/669,369. Both of these patent applications are herein incorporated fully by reference.  
           [0004]    Spectroscopic methods for analyte detection can exhibit “false positives”, in which a signal is interpreted to be from an analyte of interest, but is actually derived from another species. Decreasing false positives can be accomplished using receptor-mediated analyte binding methods, for example, those in U.S. Patent Ser. Nos. 09/670,453 and 09/669,369.  
         SUMMARY  
         [0005]    An object of this invention is to decrease the frequency of false positive results in spectrographic analysis. In general, methods and devices of this invention can reduce the incidence of false positives by providing verification of results obtained using one method by making measurements of the same sample using a different method.  
           [0006]    This invention includes methods and devices for verifying results obtained using resonance spectroscopic methods. In certain embodiments, a sample is analyzed simultaneously using Surface Enhanced Raman Spectroscopy (SERS) and surface plasmon resonance (SPR). By subjecting the same sample to two different analytical methods, the results obtained using each method can be compared to those results obtained using the other method, thereby providing internal validation of the results obtained. Because SERS and SPR methods have technical features in common, the use of those two methods can be accomplished easily and relatively inexpensively. Moreover, because both SERS and SPR permit direct detection of analytes by optical means, pre-treatment of samples may not be required. Because both SERS and SPR are optical methods, they can be accomplished rapidly and information can be stored for further comparison. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0007]    [0007]FIG. 1 is a drawing depicting a process for analyzing analytes using Raman spectroscopy and surface plasmon resonance.  
         [0008]    [0008]FIG. 2 a  depicts an embodiment of this invention for detecting an analyte using Raman spectroscopy and surface plasmon resonance on different areas of a biochip.  
         [0009]    [0009]FIG. 2 b  depicts an embodiment of this invention for detecting an analyte using Raman spectroscopy and surface plasmon resonance on the same area of a biochip.  
         [0010]    [0010]FIG. 3 depicts an embodiment of this invention for simultaneous SERS and SPR analysis of an analyte on a biochip. 
     
    
     DETAILED DESCRIPTION  
       [0011]    This invention includes methods for verifying SERS and SPR results obtained for the same sample. In some cases, the measurements can be made simultaneously A biochip can be prepared having a metal layer suitable for SPR measurements. Methods for preparing such surfaces are known in the art. In certain aspects of this invention, the metal layer can be applied to a surface of a prism. A light source generates a beam that can enter the prism, interact with the metal layer, and thereby generate surface plasmon resonance in the metal layer. Analytes present near this metal layer can be detected and quantified by the production of spectral features characteristic of the analyte present. To increase the relative amount of a desired analyte present, a receptor can be applied to the metal layer. Analytes that can readily associate with the receptor become relatively concentrated near the metal layer, thereby increasing the intensity of SPR signals. SPR signals can be captured by a light detector, and the relative intensity and angle of the output beam can be converted into signals (e.g., electrical or optical) which can be transmitted to a computer or a storage device for analysis.  
         [0012]    Surface enhanced Raman spectroscopy (SERS) can be carried using methods and enhancing structures can be prepared using methods described in U.S. Utility patent application Ser. Nos. 09/670,453, filed Sep. 26, 2000, 09/815,909, filed Mar. 23, 2001, and 09/925,189 filed Aug. 8, 2001, incorporated herein fully by reference. Alternatively, surface enhancing conditions can be provided using roughened metal surfaces as described in U.S. Pat. No. 5,122,127, incorporated herein fully by reference. In some embodiments, SERS can be carried out on the same sample as used for SPR measurements, either simultaneously (by use of a beam splitter) to divide the source light beam into two beams, one for SPR, and another for SERS measurements. Alternatively, two independent light sources can be used, and in other embodiments, a source beam can first be used to perform SERS measurements, and subsequently, to perform SPR measurements. Of course, one can reverse the order of measurements if desired.  
         [0013]    In some embodiments, analyte receptors can be provided to increase the selectivity of the assay system. Analyte receptors for Raman spectroscopy are described in U.S. patent application Ser. Nos. 09/670,453, filed Sep. 26, 2000, 09/815,909, filed Mar. 23, 2001, and 09/925,189 filed Aug. 8, 2001, incorporated herein fully by reference. Receptors can be attached to the SPR surface, to enhancing structures, or to both SPR surfaces and SERS enhancing structures. Additionally, the selectivity and sensitivity of analyte detection can be improved by the use of a passivating agent, such as mercaptoethanol, mercaptohexanol or other mercaptoalkanol. Passivation methods are described in U.S. patent application Ser. No. 09/925,189, herein incorporated fully by reference.  
         [0014]    Detection can be carried out for a variety of analytes, including by way of example only, proteins, nucleic acids, lipids, carbohydrates, low molecular weight compounds of biomedical significance present in organisms such as mammals, fungi, bacteria and viruses, and cellular organelles from eukaryotic organisms. Moreover, complexes of biomolecules can be analyzed using the verified methods of this invention. Detection can be carried out using either a single detector, or using a number of detectors simultaneously. In certain embodiments, a filter-based spectrographic analysis system can be used. Such systems are described in U.S. patent application Ser. No. 09/939,887, incorporated herein fully by reference.  
         [0015]    The results of SERS and SPR measurements can be stored in a database, computer, or displayed on a computer monitor or a print out. The information obtained can be compared using programs to decrease the incidence of false positive results.  
         [0016]    [0016]FIG. 1 depicts a schematic drawing of a process of some embodiments for direct optical detection, verification and measurement (herein termed a “Diodeverim Process” or “DP”). A sample to be analyzed is applied to a biochip, SERS and SPR signals generated by analytes in the sample are collected and stored. The stored signals are compared with each other, and possibly with data previously stored in memory for either the analytes of interest, or for other, contaminating materials which maybe responsible for false positive results. The previous step is optional. Once comparisons of the results obtained by SERS and SPR are made, a report of the results can be displayed, stored, or further used to process the information.  
         [0017]    [0017]FIGS. 2 a  and  2   b  depict embodiments of this invention. FIG. 2 a  depicts an embodiment  2001  having two different areas, one for SERS measurements, an another for SPR measurements. Biochip  2001  comprising a prism  2004  (only the top part of the prism is shown), and having a metal layer  2008  thereon. Prism  2004  can be produced using methods known in the art or purchased commercially (e.g., from Biocore Inc.). Metal layer  2008  is selected to provide surface plasmon resonance conditions. The surface of the prism is shown being divided into two areas by a separator line  2012 , which, in this case, is an area devoid of metal. Area  2016  is depicted as having no metal layer  2008  thereon. However, area  2016  has particle structures  2032  (e.g., nanoparticles, fractal structures or other enhancing structures) that can provide enhancing conditions for SERS measurements. Receptors  2036  are associated with enhancing structures  2032 , and analytes  2044  are shown associated with or binding to receptors  2036 . Area  2018  is an area having a metal layer  2008  thereon, for SPR measurements. Receptors  2036  are depicted associated with metal layer  2008  of area  2018 , and analytes  2044  are shown associated with receptors  2036  and free in solution in drop  2040 , which is sufficiently large to expose analytes to both areas  2016  and  2018 .  
         [0018]    To use a device as shown in FIG. 2 a , area  2016  is illuminated with an incident beam of electromagnetic radiation sufficient to produce a SERS signal from analytes present near the enhancing structures  2032 . Simultaneously or subsequently, area  2018  is illuminated with an incident beam of electromagnetic radiation sufficient to produce a SPR signal from analytes present near the metal layer  2008 .  
         [0019]    [0019]FIG. 2 b  depicts a device for measuring SERS and SPR signals from the same spot, area  2016  of a biochip. Biochip  2001  comprises prism  2004  (only part of the prism is shown) having a layer of metal  2008  thereon. A portion  2016  of the biochip has enhancing structures  2032  thereon, and receptors  2036  are associated with enhancing structures  2032 . Analyte molecules  2044  are shown associated with receptors  2036  and are also free in solution in drop  2040 . When exposed to SERS and/or SPR conditions, the analytes produce a spectral feature characteristic of the analyte under study. One advantage of the instrumentation, methods and devices of this invention is that the SPR and SERS detectors can be simple in design. Many detector elements are common to both SERS and SPR instruments.  
         [0020]    [0020]FIG. 3 shows a device  3000  for detecting and verifying measurements of analytes using SERS and SPR methods. A layer of metal  3004  is on a prism  3008 . Enhancing structures  3012  are optionally present on surface  3004  and have receptors  3016  attached thereto. If enhancing structures  3012  are present, enhanced Raman signals can be produced. If no enhancing are present, receptors  3016  can be attached directly to the surface of metal layer  3004 . Analyte molecules  3020  are show associated with receptors  3016  and free in solution. Light source  3024  produces beams  3028  and  3032 . Beam  3028  is directed through prism  3008  an impinges on the underside of surface  3004 , generating surface plasmon resonance. Beams of light  3036  leaving surface  3004  have angles θ1 and θ2, which are dependent upon the presence of analytes  3020  associated with surface  3004 . Beams  3036  are detected by SPR detector  3040  and the information obtained is transmitted via signal carrier  3046  to computer  3052 . Beam  3032  is directed toward the upper surface of surface  3004 . Raman signals  3050  produced by analytes  3020  associated with receptors  3016 , particles  3012  on surface  3004  are detected by Raman detector  3054 . Signals from Raman detector  3054  are transmitted via signal carrier  3058  to computer  3052 . The signals produced by SPR detector  3040  and Raman detector  3054  are compared and can be displayed on a screen of computer  3052  or on a printer (not shown) or directed to a data bank (not shown) having trusted computing spaces (not shown).  
       INDUSTRIAL APPLICABILITY  
       [0021]    This invention includes methods for detecting analytes in biological, environmental and industrial samples, and for verifying results obtained by providing two optical detection methods and comparing the results obtained from the optical detection methods.