Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
2000-11-20
2004-06-15
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C436S164000, C436S173000, C436S525000, C436S801000, C436S805000, C436S536000, C435S007950, C435S006120, C435S007940
Reexamination Certificate
active
06750065
ABSTRACT:
TECHNICAL FIELD
The present invention relates to methods and materials for assaying analytes using immunoassays and surface enhanced Raman scattering.
It further relates to methods of preparing materials for use in such assays, and kits and other equipment for performing such methods.
PRIOR ART
Surface enhanced Raman scattering
The invention provides a technique based on the principle of “surface enhanced Raman scattering” (SERS) and on a modification of that principle known as SERRS (surface enhanced resonance Raman scattering). These principles are already known and well documented, and have been used before in the detection and analysis of various target materials.
Briefly, a Raman spectrum arises because light incident on an analyte is scattered due to excitation of electrons in the analyte. “Raman” scattering occurs when an excited electron returns to an energy level other than that from which it came—this results in a change in wavelength of the scattered light and gives rise to a series of spectral lines at both higher and lower frequencies than that of the incident light. The scattered light can be detected orthogonally to the incident beam.
Normal Raman lines are relatively weak and Raman spectroscopy is therefore too insensitive, relative to other available detection methods, to be of use in chemical analysis. Raman spectroscopy is also unsuccessful for fluorescent materials, for which the broad fluorescence emission bands (also detected orthogonally to the incident light) tend to swamp the weaker Raman emissions.
However, a modified form of Raman spectroscopy, based on “surface-enhanced” Raman scattering (SERS), has proved to be more sensitive and hence of more general use. The analyte whose spectrum is being recorded is closely associated with a roughened metal surface. This leads to a large increase in detection sensitivity, the effect being more marked the closer the analyte sits to the “active” surface (the optimum position is in the first molecular layer around the surface, ie, within about 20 nm of the surface).
The theory of this surface enhancement is not yet fully understood, but it is thought that the higher valence electrons of the analyte associate with pools of electrons (known as “plasmons”) in pits on the metal surface. When incident light excites the analyte electrons, the effect is transferred to the plasmons, which are much larger than the electron cloud surrounding the analyte, and this acts to enhance the output signal, often by a factor of more than 10
6
.
A further increase in sensitivity can be obtained by operating at the resonance frequency of the analyte (in this case usually a coloured dye attached to the target of interest, although certain target analytes themselves may have suitable colour characteristics to use with appropriate lasers). Use of a coherent light source, tuned to the absorbance maximum of the dye, gives rise to a 10
3
-10
5
-fold increase in sensitivity. This is termed “resonance Raman scattering” spectroscopy.
When the surface enhancement effect and the resonance effect are combined, to give “surface enhanced resonance Raman scattering” or SERRS, the resultant increase in sensitivity and robustness is more than additive. Moreover, the sensitivity does not seem to depend so critically on the angle of orientation of the analyte to the surface, as is the case with SERS alone. A SERRS signal can be more easily discriminated from contamination and background and tends to be less variable with local conditions (e.g., ionic strength or pH when an analysis is carried out in solution). Fluorescence is also quenched, giving cleaner Raman spectra and allowing fluorescent dyes to be used as detectable analytes. Generally, the signal enhancement means that a much larger range of analytes may be usefully detected than using normal Raman spectroscopy. Furthermore, the enhancement means that a less powerful light source is required to excite the analyte molecules.
With SERRS, detection limits down to one molecule have been achieved for compounds which absorb light in the visible wavelength region or the electromagnetic spectrum (see Emory & Nie (1997) “Near-Field Surface-Enhanced Raman Spectroscopy on Single Silver Nanoparticles”, Anal. Chem. 69: 2631-2635). This technique is therefore more sensitive than fluorescence (see e.g., C Rodger et al,
J. Chem. Soc. Dalton Trans.
(1996), pp791-799) and furthermore, the SERRS spectra obtained contain molecular information which permit compound identification and discrimination.
The present invention herein disclosed may be used in either a SERS or SERRS format, and the abbreviation SER(R)S is used hereinafter to demonstrate this. Generally speaking, owing to its sensitivity advantages, SERRS will be preferred. However the use of SERS, particularly with appropriate surface seeking groups, is also contemplated.
SE(R)RS in nucleic acid detection
WO 97/05280 (University of Strathclyde) discusses the use of SE(R)RS in nucleic acid detection and sequencing.
Immunoassays
EP 0 587 008 (Abbott) discusses the use of SE(R)RS immunoassays. Essentially they propose monitoring the formation of a “complex” comprising analyte, binding member (generally antibody), a SE(R)RS label, and a particulate surface. In other embodiments competitive assays are proposed wherein analyte and labelled-analyte-analog compete to form complexes which are then monitored. Complexes between specific binding members and surfaces are generally formed covalently or by direct absorption. A brief mention of multiple analyte detection is made, but no examples are given. Detection levels in the region of &mgr;g/ml are discussed in relation to single analytes (see e.g. Example 16 and FIG.
10
).
The concept of using SE(R)RS for detecting certain analytes simultaneously has been discussed in the art generally (see C H Munro et al in
Analyst
, April 1995, 120, pp993-1003). This study used a variety of prepared samples to show that up to 20 RR-active dyes could be discriminated. The importance of controlled aggregation and purity of the samples was stressed, and in particular a purifying pretreatment step (such as TLC) was advocated.
DISCLOSURE OF THE INVENTION
The present inventors have now developed a novel SE(R)RS based immunoassay which is broadly based on the displacement of SE(R)RS active (generally labelled) analyte analogs which are modified such as to have particular SE(R)RS surface seeking properties. The use of modified analogs in a displacement format (rather than the labels and direct or competitive formats disclosed in the relevant art) offers the potential for improved sensitivity and selectivity of detection, allowing robust quantitative detection of multiple analytes simultaneously from untreated ‘real world’ samples.
Displacement immunoassays, such as those using capillaries, have been disclosed in the art, but not in relation to SE(R)RS. For instance Narang et al (Anal.Chem.1997 69, 961-964 and 2779-2785) and Whelan (Anal.Chem.1993 65, 3581-3665) both used fluorescence detection.
The present inventors have discovered that one of the main problems in using SE(R)RS for quantitative analysis, particularly in an immunoassay displacement format, has been lack of control of the surface adsorption process of the label onto the SE(R)RS surface. Many ligands dynamically adsorb and desorb to surfaces other than the SE(R)RS surface, making it difficult to obtain usable (preferably linear) calibration graphs.
This difficulty was not appreciated in the prior art. The inventors have further provided a solution to this problem by the provision of attachment groups which can be used to give the SE(R)RS label a specific selectivity for SE(R)RS surfaces, thereby making quantitative determination of labelled ligands possible.
The surface seeking groups are soluble in solution but interact preferentially with the SE(R)RS surface, for instance by forming flexible polymeric structures on it, or by utilising flexible ‘bolus’ type structures i.e. which have a number of complexing groups flexibly arranged around a central core. These interactio
Athmani Salah
Sadler Daran Antony
Smith William Ewen
White Peter Cyril
Cheu Jacob
Dann Dorfman Herrell & Skillman P.C.
Le Long V.
University of Strathclyde
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