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 for a 0.05% (v/v) of Tween-20. Sterile filter the solution with a 0.22 &#956;m filter.</li>
		<li>Store at room temperature.</li>
	</ol>
	</li>
	<li>Preparation of Human Serum Albumin (HSA) Blocking Solution
	<ol>
		<li>Weigh 0.45 g of HSA into 15 ml of sterile filtered 1x PBS to make a 3% w/v HSA solution. Vortex solution until HSA is fully dissolved.</li>
		<li>Store HSA solution at 4 &#176;C. 
		NOTE: Bovine Serum Albumin (BSA) can also be used as a blocking solution.</li>
	</ol>
	</li>
	<li>Preparation of PEGylated antibody (PEG-Ab) solution 
	NOTE: The antibody solution must be free from carrier or stabilizing proteins such as BSA, which would interfere with conjugation reactions by competing for the n-hydroxysulfosuccinimide (NHS) binding sites. If the antibody comes in a Tris or glycine buffer solution, it must undergo a buffer exchange to prevent amines or ammonium salts from interfering with the NHS conjugation reaction. If the antibody is in a lyophilized form, it can be resuspended according to the manufacturer&#39;s recommendation at a concentration of 1-10 mg/ml.
	<ol>
		<li>For antibodies in a Tris or glycine buffer, perform a buffer exchange to 100 mM sodium bicarbonate using a desalting column. Use the 100 mM buffer to raise the pH to approximately 8.5 to speed up the conjugation reaction.</li>
		<li>Hydrate ortho-pyridyl disulfide-PEG-NHS (OPSS-PEG-NHS) with 100 mM sodium bicarbonate to a volume of 1 ml at a concentration of 1 mg/ml or greater. 
		NOTE: OPSS-PEG-NHS should be made fresh and used within approximately 20 min. The NHS group on the OPSS-PEG-NHS has a half-life of approximately 20 min in an aqueous solution at pH 8.5.</li>
		<li>Add OPSS-PEG-NHS to the antibody solution at a 2:1 ratio (PEG: Antibody) conjugation ratio to be used for the test samples. In a separate microcentrifuge tube, add OPSS-PEG-NHS to the antigen solution at a 2:1 conjugation ratio to be used for the control. 
		NOTE: The 2:1 ratio is assuming a 50% conjugation efficiency. The objective is to label each antibody with one PEG chain. In this step, over-labeling is better than under-labeling. Use the following equation to determine the appropriate volumes of OPSS-PEG-NHS and antibody solution: 
		<img alt="Equation" src="/files/ftp_upload/54795/54795eq_2.jpg" /> 
		where V is volume, C is concentration expressed in molecules or antibodies per ml. Subscripts PEG and Ab are OPSS-PEG-NHS and antibody, respectively. The final volume should be approximately 250 &#956;l.</li>
		<li>Incubate PEG-Ab solution at 4 &#176;C for 8 hr or overnight. Store solution in working aliquots of approximately 25 &#956;l at -20 &#176;C to limit the freeze thaw cycles and make sure to use low binding tubes.</li>
	</ol>
	</li>
</ol>
2. 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" src="/files/ftp_upload/54795/54795eq_200.jpg" /> 
		where V is volume, 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" src="/files/ftp_upload/54795/54795eq_40000.jpg" /> 
		where V is volume, 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 of the immunoassay plate. Use the solutions within one week.</li>
	</ol>
	</li>
</ol>
<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>
Table 1. DTTC dilution example. Various dilutions of DTTC and the associated volumes of stock DTTC, gold nanoparticle solution, and water.
3. Immunoassay Plate Preparation
<ol>
	<li>Bind desired antigen to the immunoassay plate.
	<ol>
		<li>Prepare enough diluted antigen (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 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 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 3.</li>
		<li>Wash the plate with TBST five times as detailed in steps 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 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 a 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 an 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 an 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 an 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>

<ol><li>Israelsen, N. D., Hanson, C., Vargis, E. Nanoparticle properties and synthesis effects on surface-enhanced Raman scattering enhancement factor: an introduction. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aIsraelsen%2C+ND+%22Sci.+World+J%22">Sci. World J</a>. , e124582(2015).</li>
<li>Wang, Y., Schlücker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rational+design+and+synthesis+of+SERS+labels%5BTitle%5D)+AND+%22Analyst%22%5BJournal%5D)">Rational design and synthesis of SERS labels.</a> Analyst. 138 (8), 2224-2238 (2013).</li>
<li>Wang, Y., Yan, B., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SERS+tags%3A+novel+optical+nanoprobes+for+bioanalysis%5BTitle%5D)+AND+%22Chem.+Rev%22%5BJournal%5D)">SERS tags: novel optical nanoprobes for bioanalysis.</a> Chem. Rev. 113 (3), 1391-1428 (2013).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22The+Immunoassay+Handbook%3A+Theory+and+applications+of+ligand+binding%2C+ELISA+and+related+techniques%22">The Immunoassay Handbook: Theory and applications of ligand binding, ELISA and related techniques</a>. , Elsevier Science: Oxford. Waltham, MA. (2013).</li>
<li>Cox, K. L., Devanarayan, V., Kriauciunas, A., Manetta, J., Montrose, C., Sittampalam, S. Immunoassay Methods. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCox%2C+KL+%22Assay+Guid.+Man%22">Assay Guid. Man</a>. , [Accessed: 28-Mar-2016] 
 <a target="_blank" href="
	http://www.ncbi.nlm.nih.gov/books/NBK92434/
	">
	http://www.ncbi.nlm.nih.gov/books/NBK92434/
	</a> (2004).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22ELISA+development+guide%22">ELISA development guide</a>. , [Accessed: 28-Mar-2016] 
 <a target="_blank" href="https://resources.rndsystems.com/pdfs/datasheets/edbapril02.pdf">https://resources.rndsystems.com/pdfs/datasheets/edbapril02.pdf</a> (2016).</li>
<li>Israelsen, N. D., Wooley, D., Hanson, C., Vargis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rational+design+of+Raman-labeled+nanoparticles+for+a+dual-modality%2C+light+scattering+immunoassay+on+a+polystyrene+substrate%5BTitle%5D)+AND+%22J.+Biol.+Eng%22%5BJournal%5D)">Rational design of Raman-labeled nanoparticles for a dual-modality, light scattering immunoassay on a polystyrene substrate.</a> J. Biol. Eng. 10, (2016).</li>
<li>Menges, F. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMenges%2C+F+%22Spekwin32+-+optical+spectroscopy+software.+Version+1.72.1%22">Spekwin32 - optical spectroscopy software. Version 1.72.1</a>. , [Accessed: 28-Mar-2016] 
 <a target="_blank" href="http://www.effemm2.de/spekwin/">http://www.effemm2.de/spekwin/</a> (2016).</li>
<li>Findlay, J. W. A., Dillard, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Appropriate+calibration+curve+fitting+in+ligand+binding+assays%5BTitle%5D)+AND+%22AAPS+J%22%5BJournal%5D)">Appropriate calibration curve fitting in ligand binding assays.</a> AAPS J. 9 (2), E260-E267 (2007).</li>
<li>Yu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantifying+the+Antibody+Binding+on+Protein+Microarrays+using+Microarray+Nonlinear+Calibration%5BTitle%5D)+AND+%22BioTechniques%22%5BJournal%5D)">Quantifying the Antibody Binding on Protein Microarrays using Microarray Nonlinear Calibration.</a> BioTechniques. 54, 257-264 (2013).</li>
<li>Armbruster, D. A., Pry, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Limit+of+blank%2C+limit+of+detection+and+limit+of+quantitation%5BTitle%5D)+AND+%22Clin.+Biochem.+Rev%22%5BJournal%5D)">Limit of blank, limit of detection and limit of quantitation.</a> Clin. Biochem. Rev. 29 (Suppl 1), S49-S52 (2008).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22EP17-A2%3A+Evaluation+of+Detection+Capability+for+Clinical+Laboratory+Measurement+Procedures%3B+Approved+Guideline%22">EP17-A2: Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline</a>. 32. No 8, Clinical and Laboratory Standards Institute. Wayne, PA, 19087, USA. 
 <a target="_blank" href="http://shop.clsi.org/method-evaluation-documents/EP17.html">http://shop.clsi.org/method-evaluation-documents/EP17.html</a> (2012).</li>
<li>Leigh, S. Y., Som, M., Liu, J. T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Method+for+assessing+the+reliability+of+molecular+diagnostics+based+on+multiplexed+SERS-coded+nanoparticles%5BTitle%5D)+AND+%22Plos+One%22%5BJournal%5D)">Method for assessing the reliability of molecular diagnostics based on multiplexed SERS-coded nanoparticles.</a> Plos One. 8 (4), e62084(2013).</li>
<li>Sinha, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+the+binding+potential+of+cell-surface+receptors+in+fresh+excised+specimens+via+dual-probe+modeling+of+SERS+nanoparticles%5BTitle%5D)+AND+%22Sci.+Rep%22%5BJournal%5D)">Quantification of the binding potential of cell-surface receptors in fresh excised specimens via dual-probe modeling of SERS nanoparticles.</a> Sci. Rep. 5, 8582(2015).</li>
<li>Shi, W., Paproski, R. J., Moore, R., Zemp, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+circulating+tumor+cells+using+targeted+surface-enhanced+Raman+scattering+nanoparticles+and+magnetic+enrichment%5BTitle%5D)+AND+%22J.+Biomed.+Opt%22%5BJournal%5D)">Detection of circulating tumor cells using targeted surface-enhanced Raman scattering nanoparticles and magnetic enrichment.</a> J. Biomed. Opt. 19, 056014(2014).</li>
<li>Xia, X., Li, W., Zhang, Y., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Silica-coated+dimers+of+silver+nanospheres+as+surface-enhanced+Raman+scattering+tags+for+imaging+cancer+cells%5BTitle%5D)+AND+%22Interface+Focus%22%5BJournal%5D)">Silica-coated dimers of silver nanospheres as surface-enhanced Raman scattering tags for imaging cancer cells.</a> Interface Focus. 3 (3), 20120092(2013).</li>
<li>McLintock, A., Cunha-Matos, C. A., Zagnoni, M., Millington, O. R., Wark, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Universal+surface-enhanced+Raman+tags%3A+individual+nanorods+for+measurements+from+the+visible+to+the+infrared+%28514-1064+nm%29%5BTitle%5D)+AND+%22Acs+Nano%22%5BJournal%5D)">Universal surface-enhanced Raman tags: individual nanorods for measurements from the visible to the infrared (514-1064 nm).</a> Acs Nano. 8 (8), 8600-8609 (2014).</li>
</ol>

The authors declare that they have no competing financial interests.

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><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&amp;nbsp;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&amp;nbsp;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&amp;prime;-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&amp;nbsp;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>Mini Centrifuge</td><td>Fisher Schientific</td><td>12-006-900</td><td></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>Desalting Columns</td><td>Thermor Scientific</td><td>87766</td><td></td></tr><tr><td>In-house built 785&amp;nbsp;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>

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, ...

fabricating-a-uv-vis-and-raman-spectroscopy-immunoassay-platform

Research

JoVE Journal

Biochemistry

10.4K Views.  Utah State University. 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. 

Video: Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Bioengineering

Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules is reported. The entire sequence of relevant experimental steps is described, involving the fabrication of nanostructured substrates using electron beam lithography, immobilization of biomolecules on the substrates, and their characterization utilizing surface-enhanced Raman spectroscopy (SERS). Three different designs of nano-biological systems are employed, including protein A, glucose binding protein, and a dopamine binding DNA aptamer. In the latter two cases, the binding of respective ligands, D-glucose and dopamine, is also included. The three kinds of biomolecules are immobilized on nanostructured substrates by different methods, and the results of SERS imaging are reported. The capabilities of SERS to detect vibrational modes from surface-immobilized proteins, as well as to capture the protein-ligand and aptamer-ligand binding are demonstrated. The results also illustrate the influence of the surface nanostructure geometry, biomolecules immobilization strategy, Raman activity of the molecules and presence or absence of the ligand binding on the SERS spectra acquired.

We describe the fabrication and characterization of nano-biological systems interfacing nanostructured substrates with immobilized proteins and aptamers. The relevant experimental steps involving lithographic fabrication of nanostructured substrates, bio-functionalization, and surface-enhanced Raman spectroscopy (SERS) characterization, are reported. SERS detection of surface-immobilized proteins, and probing of protein-ligand and aptamer-ligand binding is demonstrated.

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules ...

Engineering

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Biology

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 &#181;m in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Microfluidics is a powerful tool for the development of diagnostic tests. However, expensive equipment and materials, as well as laborious fabrication and handling techniques, are often required. Here, we detail the fabrication protocol of an acrylic microfluidic device for magnetic micro- and nanoparticle-based immunoassays in a low-cost and simple-to-use setting.

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Fabrication of Flexible Substrate for SERS Detection of R6G and Thiram

Fabrication of polydimethylsiloxane (PDMS)-Based Flexible Surface-Enhanced Raman Scattering (SERS) Substrate for Ultrasensitive Detection

This article presents a fabrication method for a flexible substrate designed for Surface-Enhanced Raman Scattering (SERS). Silver nanoparticles (AgNPs) were synthesized through a complexation reaction involving silver nitrate (AgNO3) and ammonia, followed by reduction using glucose. The resulting AgNPs exhibited a uniform size distribution ranging from 20 nm to 50 nm. Subsequently, 3-aminopropyl triethoxysilane (APTES) was employed to modify a PDMS substrate that had been surface-treated with oxygen plasma. This process facilitated the self-assembly of AgNPs onto the substrate. A systematic evaluation of the impact of various experimental conditions on substrate performance led to the development of a SERS substrate with excellent performance and an Enhanced Factor (EF). Utilizing this substrate, impressive detection limits of 10-10 M for R6G (Rhodamine 6G) and 10-8 M for Thiram were achieved. The substrate was successfully employed for detecting pesticide residues on apples, yielding highly satisfactory results. The flexible SERS substrate demonstrates great potential for real-world applications, including detection in complex scenarios.

Author Spotlight: Development and Application of SERS Flexible Substrates Using Synthesized AgNPs 

This protocol describes a fabrication method for a flexible substrate for surface-enhanced Raman scattering. This method has been used in the successful detection of low concentrations of R6G and Thiram.

This article presents a fabrication method for a flexible substrate designed for Surface-Enhanced Raman Scattering (SERS). Silver nanoparticles (AgNPs) ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Immunology and Infection

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter and are used as a means of intercellular communication. They are emerging as promising diagnostic and therapeutic tools for a variety of diseases. There are two main biogenesis processes used by cells to produce EVs with differences in size, composition, and content. Due to their high complexity in size, composition, and cell origin, their characterization requires a combination of analytical techniques. This project involves the development of a new generation of multiparametric analytical platforms with increased throughput for the characterization of subpopulations of EVs. To achieve this goal, the work starts from the nanobioanalytical platform (NBA) established by the group, which allows an original investigation of EVs based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy (AFM) of vesicular targets trapped on a microarray biochip. The objective was to complete this EV investigation with a phenotypic and molecular analysis by Raman spectroscopy. These developments enable the proposal of a multimodal and easy-to-use analytical solution for the discrimination of EV subsets in biological fluids with clinical potential

This paper proposes a new generation of multiparametric analytical platforms with increased throughput for the characterization of extracellular vesicle subsets. The method is based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy, coupled with Raman spectroscopy, to qualify vesicular targets trapped on a microarray biochip.

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter ...

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Samenvatting

Meer video's verkennen