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

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

1. Prepare bare AuNP solution
   1. Prepare a 2 ml solution of AuNPs with a concentration of approximately 1 x 1011 particles per ml.
      1. 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.
      2. Combine the remaining AuNP solutions into one tube and estimate the concentration by obtaining a UV-Vis measurement and comparing values to known concentrations as t....

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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.