Name: fabricating a uv-vis and raman spectroscopy immunoassay platform
Uploaded: 2016-11-10T15:00:00
Description: Watch this Scientific Journal Video about Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform at JoVE.com

This work was supported by a Research Catalyst Award from Utah State University. The authors would like to thank Annelise Dykes, Cameron Zabriskie, and Donald Wooley for their contributions.

1. Preparation of Buffers
<ol>
	<li>Phosphate Buffered Saline (PBS)
	<ol>
		<li>Dilute 50 ml of 10x PBS with 450 ml HPLC grade water to make a 1x PBS concentration. Sterile filter the solution with a 0.22 &#181;m filter.</li>
		<li>Store solution at room temperature.</li>
	</ol>
	</li>
	<li>Preparation of Tris Buffered Saline + Tween 20 (TBST)
	<ol>
		<li>Dilute 50 ml of 10x Tris Buffered Saline (TBS) with 450 ml HPLC grade water to make a 1x concentration. Add 250 &#956;l of Tween-20 ...

<ol>
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In the detailed protocol, there are several critical points to address. One issue is the choice of Raman reporter and gold nanoparticle. Although the protocol was written to be adapted for individual use, the Raman reporter DTTC was used as an example. DTTC is a positively charged reporter and binds to negatively charged surfaces such as citrate capped AuNPs. This protocol can be adapted for negatively charged reporters by using gold nanoparticles with a positive surface charge. For example, polyethyleneimine (PEI) cappe...

Typical sandwich immunoassays indirectly detect the presence of an antigen using two antibodies. The capture antibody is bound to a solid surface and forms an antibody-antigen complex when in proximity to an appropriate antigen. A detection antibody is then introduced and binds to the antigen. After washing, the antibody/antigen/antibody complex remains and is detected by the labeled detection antibody as demonstrated in Figure 1A. Typical detection is done by a fluorescent or colorimetric detector, limiting multiplexing to 10 analytes due to broad spectral peaks2,3. In contrast, Raman-based systems have much narrower emission peaks resulting in enhanced multiplexing capabilities with sources claiming simultaneous detection of up to 100 analytes2,3.
Many literature sources are available which cover important aspects related to immunoassays4-6 such as step-by-step details to create personalized ELISA kits. Unfortunately, these protocols are for fluorescent or colorimetric detection, limiting multiplexing capability of customized immunoassays. To address this need, we present a detailed procedure to fabricate the UV-Vis/Raman immunoassay published previously7 for a direct immunoassay as illustrated in Figure 1B.
This protocol includes the fabrication of functionalized gold nanoparticle-based probes, illustrated in Figure 2. The procedure to make the Raman/UV-Vis probes begins by binding Raman reporters to the surface of gold nanoparticles (AuNPs). The AuNPs are then functionalized with antibodies that are associated with polyethylene glycol (PEG). Remaining binding sites on the AuNPs are blocked by binding methoxy polyethylene glycol thiol (mPEG-SH) to AuNPs to prevent subsequent non-specific binding during analysis. The prepared AuNP probes are tested by binding to antigens fixed to the wells of a polystyrene plate as illustrated in Figure 1B. Upon washing the plate, the AuNP probes are detected using UV-Vis spectroscopy while the associated Raman reporters are detected with Raman spectroscopy. Combining UV-Vis and Raman spectral data provides two methods of analyses, enhancing the capabilities of this immunoassay.

<table border="0" cellpadding="0" cellspacing="0" width="1041">
	<tbody>
		<tr height="121">
			<td height="121" style="height:121px;width:405px;">60nm Gold Nanoparticle</td>
			<td style="width:159px;">Ted Pella, Inc.</td>
			<td style="width:153px;">15708-6</td>
			<td style="width:324px;">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.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Bicarbonate</td>
			<td>Fisher Scientific</td>
			<td>S233-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol</td>
			<td>Pharmco-Aaper</td>
			<td>339000000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris Buffered Saline (10X) pH 7.5</td>
			<td>Scy Tek</td>
			<td>TBD999</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bottle Top Filtration Unit</td>
			<td>VWR</td>
			<td>97066-202</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tween 20 (polysorbate 20)</td>
			<td>Scy Tek</td>
			<td>TWN500</td>
			<td>Used as an emulsifying agent for washing steps.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline 10X Concentrate, pH 7.4</td>
			<td>Scy Tek</td>
			<td>PBD999</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Protein LoBind Tube 2.0 mL</td>
			<td>Eppendorf Tubes</td>
			<td>22431102</td>
			<td style="width:324px;">LoBind tubes prevent binding of proteins and AuNPs to surfaces of the tubes.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Protein LoBind Tube 0.5 mL</td>
			<td>Eppendorf Tubes</td>
			<td>22431064</td>
			<td style="width:324px;">LoBind tubes prevent binding of proteins and AuNPs to surfaces of the tubes.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microplate Devices UniSeal</td>
			<td>GE Healthcare</td>
			<td>7704-0001</td>
			<td>Used for sealing and storing functionalized plates.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Assay Plate, With Low Evaporation Lid, 96 Well Flat Bottom</td>
			<td>Costar</td>
			<td>3370</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC grade water</td>
			<td>Sigma Aldrich</td>
			<td>270733-4L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3,3&#8242;-Diethylthiatricarbocyanine iodide (DTTC)</td>
			<td>Sigma Aldrich</td>
			<td>381306-250MG</td>
			<td>Raman reporter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mPEG-Thiol, MW 5,000 - 1 gram</td>
			<td>Laysan Bio, Inc.</td>
			<td>MPEG-SH-5000-1g</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OPSS-PEG-SVA, MW 5,000 - 1 gram</td>
			<td>Laysan Bio, Inc.</td>
			<td>OPSS-PEG-SVA-5000-1g</td>
			<td style="width:324px;">OPSS-PEG-SVA has an NHS end.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse IgG, Whole Molecule Control</td>
			<td>Thermo Fisher Scientific</td>
			<td>31903</td>
			<td>Antigen</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-Mouse IgG (H+L) Cross Adsorbed Secondary Antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>31164</td>
			<td>Antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human Serum Albumin Blocking Solution</td>
			<td>Sigma Aldrich</td>
			<td>A1887-1G</td>
			<td>Bovine serum albumin can be used instead.</td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;">In-house built 785nm inverted Raman microscope unit</td>
			<td>N/A</td>
			<td>N/A</td>
			<td style="width:324px;">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.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mini Centrifuge</td>
			<td>Fisher Schientific</td>
			<td>12-006-900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV-Vis Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>Nanodrop 2000c</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV-Vis Spectrophotometer</td>
			<td>BioTek</td>
			<td>Synergy 2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Desalting Columns</td>
			<td>Thermor Scientific</td>
			<td>87766</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabricating a uv-vis and raman spectroscopy immunoassay platform

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available.
Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags1. Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

In this study, 60 nm gold particles were used for UV-Vis spectroscopy. UV-Vis absorption spectra from 400 to 700 nm were collected and the peak areas for each AuNP concentration were determined using an open source spectral analysis software8. Prior to peak integration, the collected spectra underwent baseline correction using a three-point polynomial fit. Peak areas were used to generate a logarithmic calibration curve as demonstrated in Figure 4. It should be noted that Figures 4 </s...

Watch this Scientific Journal Video about Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform at JoVE.com

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, ...

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