Method of detecting and/or quantifying an analyte in a biological sample

Patent metadata

Field	Value
Publication number	US-9518986-B2
Application number	US-201214355765-A
Country	US
Kind code	B2
Filing date	Nov 2, 2012
Priority date	Nov 2, 2011
Publication date	Dec 13, 2016
Grant date	Dec 13, 2016

Abstract

An aptamer-based SERS detection technique that directly monitors an aptamer-analyte capture event by generating spectroscopic information regarding the identity of the analyte that has been bound to the aptamer from a complex biological sample. A reproducible SERS spectrum is measured for an aptamer-analyte complex formed on a metal surface and this spectral information is used directly to identify the specific aptamer-analyte complex and optionally also to quantify the analyte in the sample, thus enabling discrimination between true and false positives in quantitative analyte assays on complex biological samples. In one embodiment the aptamer is attached directly to the metal surface and surrounded by a self-assembled monolayer (SAM) of amphiphilic molecules. In an alternative embodiment the metal surface is coated with a SAM and the aptamer is attached to the amphiphilic molecules of the SAM.

First claim

The invention claimed is:

1. A method of identifying an analyte molecule in a biological sample, the method comprising the steps of:
capturing the analyte molecule onto a nano-structured silver SERS surface by means of an analyte molecule-specific aptamer, resulting in a specific aptamer-analyte molecule complex, which has a different pattern of Raman shifts in a SERS spectrum relative to a pattern for the aptamer or the analyte in isolation, the pattern of Raman shifts being unique and reproducible for the specific aptamer-analyte molecule complex, wherein amphiphilic molecules comprising an alkyl thio chain of at least 11 carbon atoms are attached to the silver surface to form a self-assembled monolayer (SAM), the SAM is coated with a layer of oligoethylene glycol molecules, and the analyte molecule-specific aptamer is attached directly to the SAM or attached directly to the silver surface and surrounded by the SAM,
obtaining SERS spectra at a plurality of different voltages;
measuring SERS signal intensity and unique pattern of Raman shifts for the specific aptamer-analyte molecule complex, and
comparing the measured SERS signal intensity and Raman shifts of a SERS spectrum measured for the specific aptamer-analyte molecule complex to a database of reference SERS spectra of aptamer-analyte molecule complexes wherein the database comprises two or more SERS spectra and wherein each SERS spectrum in said database is unique for a different aptamer-analyte molecule complex, thereby identifying the analyte molecule in the aptamer-analyte molecule complex.

2. The method of claim 1 , further comprising comparing the measured SERS signal intensity of the specific aptamer-analyte molecule complex to a standard curve to quantify the abundance of the captured analyte.

3. The method of claim 1 , further comprising confirming a true positive diagnostic result in a quantitative analyte assay on the biological sample if the measured Raman shifts of the SERS spectrum correspond to a reference spectrum for an aptamer-analyte complex of an analyte which is indicative of a disease or condition being diagnosed; or identifying a false positive result if the measured Raman shifts of the SERS spectrum correspond to a reference spectrum for an aptamer-analyte complex of an analyte which is not indicative of a disease or condition being diagnosed.

4. The method of claim 1 , wherein the aptamer is a DNA aptamer.

5. The method of claim 1 , wherein
the aptamer is attached directly to the silver surface and surrounded by the SAM.

6. The method of claim 5 , wherein the aptamer is
attached directly to the amphiphilic molecules of the SAM.

7. The method of claim 1 , wherein the oligoethylene glycol molecules have exposed termini and are derivatised on the termini by the aptamer.

8. A method for diagnosing or quantifying an infection, disease or medical condition in a subject, comprising performing the method of claim 1 on a sample from the subject.

9. A method for monitoring a subject during anaesthesia, comprising performing the method of claim 1 on a sample from the subject.

Assignees

• Univ Cape Town

Inventors

• Blackburn Jonathan Michael
• Evans Michael
• Krishnan Sriram
• Brosseau Christa Lynn

Classifications

• G01N33/94

  involving narcotics {or drugs or pharmaceuticals, neurotransmitters or associated receptors} · CPC title

• C12N2320/10

  in screening processes · CPC title

• G01N2610/00

  Assays involving self-assembled monolayers [SAMs] · CPC title

• G01N33/54353 Primary

  with ligand attached to the carrier via a chemical coupling agent (coatings G01N33/54393) · CPC title

• C12N2310/16

  Aptamers · CPC title