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1. Prepare UV-Vis/Raman Probes
<ol>
	<li>Prepare bare AuNP solution
	<ol>
		<li>Prepare a 2 ml solution of AuNPs with a concentration of approximately 1 x 1011 particles per ml.
		<ol>
			<li>If the AuNPs need to be concentrated, fill low-binding centrifuge tubes with 2,000 &#956;l 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.</li>
			<li>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 this is a linear relationship.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Determine the appropriate Raman reporter labeling ratio
	<ol>
		<li>Prepare a working solution of the Raman reporter dissolved in methanol. This concentration will be dependent on the reporter used. In this work, prepare 3,3&#8242;-diethylthiatricarbocyanine iodide (DTTC) at a working solution of 200 &#956;M.</li>
		<li>Assuming a final volume of 100 &#956;l for each well, add enough of the working reporter solution to each well of the first row of a 96-well plate such that the Raman reporter will range in concentrations from 0.2 &#956;M to 10 &#956;M. Add enough HPLC-grade water to each well such that the volume is 80 &#956;l. Add 20 &#956;l of AuNP to each well making a final volume of 100 &#956;l for each well. An example is provided in Table 1.</li>
		<li>Measure the UV-Vis spectra from 400 to 700 nm using a plate-reading UV-Vis spectrophotometer. The appropriate concentration is the highest concentration with defined peaks for the UV-Vis spectra. Repeat step 2.2.2 at increasing concentrations until the highest concentration ratio of Raman reporters to AuNPs is found. 
		NOTE: The dye and the AuNP shape, size, and manufacturer influence the appropriate concentration. Therefore, the steps listed must be evaluated and altered depending on the components used. This protocol involved the use of a positively charged dye. As such, binding between the AuNP and reporter was improved by using negatively charged AuNPs. This was done by using citrate-capped AuNPs. See the Discussion section for further details.</li>
	</ol>
	</li>
	<li>Binding Raman reporter and PEG-Ab to AuNP
	<ol>
		<li>Prepare two 1.5 ml batches of AuNP and Raman reporter at the previously determined concentration, allowing the Raman reporter to bind to the AuNPs for 30 min at room temperature.</li>
		<li>Add the PEGylated antibody (PEG-Ab) to one batch of the AuNP and Raman reporter solution to create a 200:1 ratio of antibodies to particles. This solution will be for the test samples. In a separate microcentrifuge tube, add the PEGylated antigen to the other batch of the AuNP and Raman reporter solution at a 200:1 ratio of antibody to particles to be used as the control. Incubate the solutions for 30 min at room temperature. 
		NOTE: The ratio of antibodies to particles will be specific to the AuNPs and dye used and should be optimized for each individual case. The objective here is to have the highest ratio of antibodies for the AuNP probes to bind to while preventing aggregation of the particles. Use the following equation to determine the appropriate volumes to add together: 
		<img alt="Equation 1" src="/files/ftp_upload/21470/21470_Equation1.jpg" style="margin: auto;" /> 
		where V is volume, and C is concentration expressed in particles or antibodies per ml. The final volume should be approximately 1.5 ml.</li>
	</ol>
	</li>
	<li>Block remaining sites on the AuNP surface with mPEG-SH.
	<ol>
		<li>Prepare mPEG-SH by dissolving solid methoxy polyethylene glycol thiol to a 200 &#956;M concentration using water. Vortex the solution until mPEG-SH is completely dissolved.</li>
		<li>Add mPEG-SH at a 40,000:1 ratio to the AuNP-PEG-Ab solution made in step 2.3. Incubate the solution at room temperature for 10 min to ensure the remaining sites on the gold nanoparticle are blocked. Use the following equation to determine the appropriate volumes to add together: 
		<img alt="Equation 2" src="/files/ftp_upload/21470/21470_Equation1.jpg" style="margin: auto;" /> 
		where V is volume, and C is concentration expressed in particles or antibodies per ml. The final volume should be approximately 1.5 ml.</li>
	</ol>
	</li>
	<li>Recover functionalized Raman probes.
	<ol>
		<li>Centrifuge particles at 5,000 x g for 20 min in low-bind centrifuge tubes or until the supernatant is clear. Remove the supernatant by pipetting being careful not to disturb the AuNPs.</li>
		<li>Resuspend the particles with 1 ml of 1x PBS solution that was made previously. Estimate the AuNP concentration by taking a UV-Vis measurement of a small volume of solution (3 &#956;l) and compare the results to measurements from a known AuNP concentration. Adjust the volume such that the final solution is at least 1 x 1011 particles per ml.</li>
		<li>Store solutions at 4 &#176;C until it is used for functionalizing the immunoassay plate. Use the solutions within one week.</li>
	</ol>
	</li>
</ol>
Table 1. DTTC dilution example. Various dilutions of DTTC and the associated volumes of stock DTTC, gold nanoparticle solution, and water.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">Volumes to add of each component (ml)</td>
		</tr>
		<tr>
			<td>DTTC final concentration (mM)</td>
			<td>DTTC working solution (200 mM)</td>
			<td>AuNP</td>
			<td>Water</td>
		</tr>
		<tr>
			<td>0.2</td>
			<td>0.1</td>
			<td>20</td>
			<td>79.9</td>
		</tr>
		<tr>
			<td>0.6</td>
			<td>0.3</td>
			<td>20</td>
			<td>79.7</td>
		</tr>
		<tr>
			<td>1</td>
			<td>0.5</td>
			<td>20</td>
			<td>79.5</td>
		</tr>
		<tr>
			<td>2</td>
			<td>1.0</td>
			<td>20</td>
			<td>79</td>
		</tr>
		<tr>
			<td>5</td>
			<td>2.5</td>
			<td>20</td>
			<td>77.5</td>
		</tr>
		<tr>
			<td>7</td>
			<td>3.5</td>
			<td>20</td>
			<td>76.5</td>
		</tr>
		<tr>
			<td>10</td>
			<td>5.0</td>
			<td>20</td>
			<td>75</td>
		</tr>
	</tbody>
</table>
2. Immunoassay Plate Preparation
<ol>
	<li>Bind the desired antigen to the immunoassay plate.
	<ol>
		<li>Prepare enough diluted antigens (50 &#956;g/ml) to fill the polystyrene wells. Vortex the solution, and immediately add the solution to the plate wells. Allow the antigen to bind to the plates for 1 hr at room temperature.</li>
	</ol>
	</li>
	<li>Wash off unbound antigens.
	<ol>
		<li>Remove the excess antigen solution by dumping the solution into a disposal container and hitting the plate against a paper towel-covered tabletop.</li>
		<li>Add TBST to the wells to wash the surface then remove the wash in the same manner as stated previously. Repeat this step two more times.</li>
	</ol>
	</li>
	<li>Block remaining binding sites on the plate to prevent non-specific binding.
	<ol>
		<li>Add 70 &#956;l of HSA-blocking solution to each well of the plate and incubate at room temperature for 30 min.</li>
		<li>Remove and rinse the plate using the same procedure as specified in step 3.2. Cover the plate and store dry at 4 &#176;C until ready for further use.</li>
	</ol>
	</li>
	<li>Functionalize the immunoassay plate.
	<ol>
		<li>Add 70 &#956;l of the probe nanoparticles prepared in Section 2 to the first column of a 96-well plate and dilute subsequent columns using a 1:2 serial dilution. Allow the plate to incubate for at least 1 hr. An example of how to prepare the immunoassay plate is given in Figure 1.</li>
		<li>Wash the plate with TBST five times as detailed in step 3.2, making sure to dispose of the AuNPs appropriately. After the final wash, add 70 &#956;l of 1x PBS to each well and cover with a plate seal. 
		NOTE: The control samples should be clear. If the non-specific binding has occurred, the control samples will have a similar color as the test samples.</li>
	</ol>
	</li>
	<li>Test assay sensitivity by UV-Vis and Raman spectroscopy.
	<ol>
		<li>For each well, measure the UV-Vis spectra ranging from 400 to 700 nm using a plate-reading UV-Vis spectrophotometer.</li>
		<li>Using an inverted Raman microscope, focus the objective onto the surface of the well that has the AuNP probes. Obtain Raman spectra of the well. Collect a spectrum ranging from 1,800 cm-1 to 400 cm-1. Repeat this step for all wells.</li>
		<li>Using appropriate spectral software, perform an 11th-order polynomial baseline correction for the Raman spectra and a 3rd-order polynomial for the UV-Vis spectra.</li>
		<li>Using appropriate spectral software, normalize the Raman and UV-Vis spectra. Set the maximum value to 1 and scale all other values accordingly. To normalize the Raman spectra, select a unique polystyrene peak and set it equal to 1, and scale all other values accordingly.</li>
		<li>Using appropriate spectral software, perform peak integration for each spectrum. For Raman spectra, the peak representing the Raman reporter must be in a region absent of polystyrene peaks. To perform peak integration, specify the integral boundaries for the desired peak and record the desired peak area for all samples including the controls.</li>
		<li>Plot the average peak area of interest as a function of the log of the AuNP concentration with error bars for each point indicating its associated standard deviation. Fit these calibration points to a 4-parameter logistic curve.</li>
		<li>Determine the mean value of the blank by averaging the area of the peak of interest for a blank sample. Determine the standard deviation of these areas; this is the standard deviation of the blank.</li>
		<li>For the same peak analyzed in the previous step, find the standard deviation of that peak area for the lowest concentration.</li>
		<li>Calculate the limit of the blank and lower limit of detection as specified in the Representative results section. Use these values with the 4PL calibration curves to determine the LLOD in terms of AuNP concentration.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>60 nm Gold Nanoparticle</td>
			<td>Ted Pella, Inc.</td>
			<td>15708-6</td>
			<td>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>
			<td>Sodium Bicarbonate</td>
			<td>Fisher Scientific</td>
			<td>S233-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Pharmco-Aaper</td>
			<td>339000000</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Buffered Saline (10x) pH 7.5</td>
			<td>Scy Tek</td>
			<td>TBD999</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle Top Filtration Unit</td>
			<td>VWR</td>
			<td>97066-202</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20 (polysorbate 20)</td>
			<td>Scy Tek</td>
			<td>TWN500</td>
			<td>Used as an emulsifying agent for washing steps.</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline 10x Concentrate, pH 7.4</td>
			<td>Scy Tek</td>
			<td>PBD999</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind Tube 2.0 ml</td>
			<td>Eppendorf Tubes</td>
			<td>22431102</td>
			<td>LoBind tubes prevent binding of proteins and AuNPs to surfaces of the tubes.</td>
		</tr>
		<tr>
			<td>Protein LoBind Tube 0.5 ml</td>
			<td>Eppendorf Tubes</td>
			<td>22431064</td>
			<td>LoBind tubes prevent binding of proteins and AuNPs to surfaces of the tubes.</td>
		</tr>
		<tr>
			<td>Microplate Devices UniSeal</td>
			<td>GE Healthcare</td>
			<td>7704-0001</td>
			<td>Used for sealing and storing functionalized plates.</td>
		</tr>
		<tr>
			<td>Assay Plate, With Low Evaporation Lid, 96 Well Flat Bottom</td>
			<td>Costar</td>
			<td>3370</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC-grade water</td>
			<td>Sigma Aldrich</td>
			<td>270733-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#8242;-Diethylthiatricarbocyanine iodide (DTTC)</td>
			<td>Sigma Aldrich</td>
			<td>381306-250MG</td>
			<td>Raman reporter</td>
		</tr>
		<tr>
			<td>mPEG-Thiol, MW 5,000 - 1 gram</td>
			<td>Laysan Bio, Inc.</td>
			<td>MPEG-SH-5000-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>OPSS-PEG-SVA, MW 5,000 - 1 gram</td>
			<td>Laysan Bio, Inc.</td>
			<td>OPSS-PEG-SVA-5000-1g</td>
			<td>OPSS-PEG-SVA has an NHS end.</td>
		</tr>
		<tr>
			<td>Mouse IgG, Whole Molecule Control</td>
			<td>Thermo Fisher Scientific</td>
			<td>31903</td>
			<td>Antigen</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Cross Adsorbed Secondary Antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>31164</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>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>
			<td>UV-Vis Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>Nanodrop 2000c</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Vis Spectrophotometer</td>
			<td>BioTek</td>
			<td>Synergy 2</td>
			<td></td>
		</tr>
		<tr>
			<td>In-house built 785 nm inverted Raman microscope unit</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>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>
	</tbody>
</table>

a raman spectroscopy-based direct immunoassay to detect a specific antigen

Source: Hanson, C., et al. <a href="https://app.jove.com/v/54795">Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform.</a> 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.

<img alt="Figure 1" src="/files/ftp_upload/21470/21470_Figure1.jpg" /> 
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.

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

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 ...

Begin by incubating Raman reporter dye molecules with gold nanoparticles for reporters to bind to the nanoparticles.
Add polyethylene glycol, PEG-conjugated antibodies specific to the target antigen. The antibodies covalently attach to the nanoparticles through gold-disulfide binding. Add thiol-conjugated PEGs to block the remaining nanoparticle sites and prevent nanoparticle aggregation, forming nanoparticle probes.
Coat immunoassay plate wells with the target antigen. Block the remaining plate sites with a solution containing proteins. Add the nanoparticle probes. Perform serial probe dilution across the wells. Incubate for the antibodies in the probes to bind to the antigens, forming complexes.
Use a Raman microscope to determine the probe&#39;s optical response. The incident monochromatic laser excites the gold nanoparticles&#39; surface electrons and causes their oscillation, creating a high local electromagnetic field on the probe surface.
The incident beam and Raman reporters interact, causing enhanced inelastic light or Raman scattering with different wavelengths and frequencies due to the enhanced electromagnetic field. The inelastically emitted Raman signal is used to generate the Raman spectrum.
Plot the Raman reporter peak&#39;s spectral area against the probe concentration to find the lower limit of probe detection and facilitate more sensitive detection of the antigen.

a-raman-spectroscopy-based-direct-immunoassay-to-detect-specific

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JoVE Encyclopedia of Experiments

Biological Techniques

Spectroscopy Techniques

163 Views. 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. 

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

Resonance Raman Spectroscopy of Extreme Nanowires and Other 1D Systems

This paper briefly describes how nanowires with diameters corresponding to 1 to 5 atoms can be produced by melting a range of inorganic solids in the presence of carbon nanotubes. These nanowires are extreme in the sense that they are the limit of miniaturization of nanowires and their behavior is not always a simple extrapolation of the behavior of larger nanowires as their diameter decreases. The paper then describes the methods required to obtain Raman spectra from extreme nanowires and the fact that due to the van Hove singularities that 1D systems exhibit in their optical density of states, that determining the correct choice of photon excitation energy is critical. It describes the techniques required to determine the photon energy dependence of the resonances observed in Raman spectroscopy of 1D systems and in particular how to obtain measurements of Raman cross-sections with better than 8% noise and measure the variation in the resonance as a function of sample temperature. The paper describes the importance of ensuring that the Raman scattering is linearly proportional to the intensity of the laser excitation intensity. It also describes how to use the polarization dependence of the Raman scattering to separate Raman scattering of the encapsulated 1D systems from those of other extraneous components in any sample.

The paper describes a method for producing extreme nanowires by melt infiltration into carbon nanotubes and how 1D systems may be characterized and investigated using Resonance Raman Spectroscopy to determine vibrational and optical excitation energies.

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A protocol of step-by-step Raman and IR spectroelectrochemical analysis is presented.

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Ultrafast Time-resolved Near-IR Stimulated Raman Measurements of Functional &#960;-conjugate Systems

Femtosecond time-resolved stimulated Raman spectroscopy is a promising method of observing the structural dynamics of short-lived transients with near infrared (near-IR) transitions, because it can overcome the low sensitivity of spontaneous Raman spectrometers in the near-IR region. Here, we describe technical details of a femtosecond time-resolved near-IR multiplex stimulated Raman spectrometer that we have recently developed. A description of signal generation and optimization, measurement, data acquisition, and calibration and correction of recorded data is provided as well. We present an application of our spectrometer to analyze the excited-state dynamics of &#946;-carotene in toluene solution. A C=C stretch band of &#946;-carotene in the second lowest excited singlet (S2) state and the lowest excited singlet (S1) state is clearly observed in the recorded time-resolved stimulated Raman spectra. The femtosecond time-resolved near-IR stimulated Raman spectrometer is applicable to the structural dynamics of &#960;-conjugate systems from simple molecules to complex materials.

Ultrafast Time-resolved Near-IR Stimulated Raman Measurements of Functional &amp;#960;-conjugate Systems

Details of signal generation and optimization, measurement, data acquisition, and data handling for a femtosecond time-resolved near-IR stimulated Raman spectrometer are described. A near infrared stimulated Raman study on the excited-state dynamics of &#946;-carotene in toluene is shown as a representative application.

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

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