A Raman Spectroscopy-Based Direct Immunoassay to Detect a Specific Antigen

Published: June 29, 2023

Abstract

Source: Hanson, C., et al. Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform. J. Vis. Exp.(2016).

In this video, we describe the Raman spectroscopy-based direct immunoassay method for detecting specific target antigens. The antibodies, which are adsorbed onto the surface of gold nanoparticles attached to Raman probes, interact with target antigens and produce a specific Raman spectrum, which is subsequently detected.

Protocol

1. Prepare UV-Vis/Raman Probes Prepare bare AuNP solution Prepare a 2 ml solution of AuNPs with a concentration of approximately 1 x 1011 particles per ml. If the AuNPs need to be concentrated, fill low-binding centrifuge tubes with 2,000 μl of stock AuNP and centrifuge at 5,000 x g for 20 min or until the supernatant is clear. Remove the supernatant by pipetting, being careful not to disturb the AuNP pellet. Combine the rem…

Representative Results

Figure 1. Prepared immunoassay plate. Image of a prepared immunoassay plate. Rows A through E are test samples while rows F through H are control samples. Column 1 contains the undiluted nanoparticles and every subsequent column has half the concentration of AuNP probes.

Divulgaciones

The authors have nothing to disclose.

Materials

60 nm Gold Nanoparticle Ted Pella, Inc. 15708-6 These are citrate-capped gold nanoparticles. Please see Discussion for relationship between Raman reporter and AuNP surface charge and its imporance to proper selection of AuNP and/or Raman reporter.
Sodium Bicarbonate Fisher Scientific S233-500
Methanol Pharmco-Aaper 339000000
Tris-Buffered Saline (10x) pH 7.5 Scy Tek TBD999
Bottle Top Filtration Unit VWR 97066-202
Tween 20 (polysorbate 20) Scy Tek TWN500 Used as an emulsifying agent for washing steps.
Phosphate-Buffered Saline 10x Concentrate, pH 7.4 Scy Tek PBD999
Protein LoBind Tube 2.0 ml Eppendorf Tubes 22431102 LoBind tubes prevent binding of proteins and AuNPs to surfaces of the tubes.
Protein LoBind Tube 0.5 ml Eppendorf Tubes 22431064 LoBind tubes prevent binding of proteins and AuNPs to surfaces of the tubes.
Microplate Devices UniSeal GE Healthcare 7704-0001 Used for sealing and storing functionalized plates.
Assay Plate, With Low Evaporation Lid, 96 Well Flat Bottom Costar 3370
HPLC-grade water Sigma Aldrich 270733-4L
3,3′-Diethylthiatricarbocyanine iodide (DTTC) Sigma Aldrich 381306-250MG Raman reporter
mPEG-Thiol, MW 5,000 – 1 gram Laysan Bio, Inc. MPEG-SH-5000-1g
OPSS-PEG-SVA, MW 5,000 – 1 gram Laysan Bio, Inc. OPSS-PEG-SVA-5000-1g OPSS-PEG-SVA has an NHS end.
Mouse IgG, Whole Molecule Control Thermo Fisher Scientific 31903 Antigen
Goat anti-Mouse IgG (H+L) Cross Adsorbed Secondary Antibody Thermo Fisher Scientific 31164 Antibody
Human Serum Albumin Blocking Solution Sigma Aldrich A1887-1G Bovine serum albumin can be used instead.
UV-Vis Spectrophotometer Thermo Scientific Nanodrop 2000c
UV-Vis Spectrophotometer BioTek Synergy 2
In-house built 785 nm inverted Raman microscope unit N/A N/A An inverted Raman microscope is best for proper focusing onto surface of the well plate. Otherwise a very low magnification will be used due to height of the 96-well plate. An in-house built system was used as it was cheaper than buying from a vendor. However, any commercially available inverted Raman microscope system can be used.

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A Raman Spectroscopy-Based Direct Immunoassay to Detect a Specific Antigen. J. Vis. Exp. (Pending Publication), e21470, doi: (2023).

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