Name: design and development of aptamer&#8211;gold nanoparticle based colorimetric assays for in-the-field applications
Uploaded: 2016-06-23T13:00:00
Description: Watch this Scientific Journal Video about Design and Development of AptamerÃ¢â‚¬â€œGold Nanoparticle Based Colorimetric Assays for In-the-field Applications at JoVE.com

This work was partially funded by the Air Force Office of Scientific Research and the Assistant Secretary of Defense for Research and Engineering (Defense Biometrics and Forensics Office). JES participation was supported by a National Research Council Research Associateship Award at Air Force Research Laboratory.

1. Synthesis via Citrate Reduction of Gold Nanoparticles (AuNP) and Characterization
<ol>
	<li>Clean an Erlenmeyer flask (500 ml) and large stir bar with 5 ml concentrated nitric acid and 15 ml concentrated hydrochloric acid in chemical safety hood.
	<ol>
		<li>Wet the entire surface of the flask with the acid wash, rinse the flask with nuclease free water, and allow the flask to dry.</li>
	</ol>
	</li>
	<li>Add 100 ml of 1 mM gold(III) chloride; use a sheet of aluminum foil to cover the top of the acid cleaned Erlenme...

<ol>
	<li>Housecroft, C., Constable, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemistry: an introduction to organic, inorganic, and physical chemistry. , 349-353 (2006).</li><li>Bunka, D., Stockley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamers+come+of+age-at+last.">Aptamers come of age-at last.</a> Nat. Rev. Microbiol. 4 (8), 588-596 (2006).</li><li>Mayer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemical+biology+of+aptamers.">The chemical biology of aptamers.</a> Angew. Chem. 48 (15), 2672-2689 (2009).</li><li>Medley, C., Smith, J., Tang, Z., Wu, Y., Bamrungsap, S., Tan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+Nanoparticle-Based+Colorimetric+Assay+for+the+Direct+Detection+of+Cancerous+Cells.">Gold Nanoparticle-Based Colorimetric Assay for the Direct Detection of Cancerous Cells.</a> Anal. Chem. 80 (4), 1067-1072 (2008).</li><li>Giljohann, D., Seferos, D., Daniel, W., Massich, M., Patel, P., Mirkin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanoparticles+for+biology+and+medicine.">Gold nanoparticles for biology and medicine.</a> Angew. Chem. Int. 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Chem. 83 (12), 4440-4452 (2011).</li><li>Smith, J., Griffin, D., Leny, J., Hagen, J., Ch&#225;vez, J., Kelley-Loughnane, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+detection+with+aptamer-gold+nanoparticle+conjugates+coupled+to+an+android-based+color+analysis+application+for+use+in+the+field.">Colorimetric detection with aptamer-gold nanoparticle conjugates coupled to an android-based color analysis application for use in the field.</a> Talanta. 121, 247-255 (2014).</li><li>Alivasatos, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+nanocrustal+molecules+using+DNA.">Organization of nanocrustal molecules using DNA.</a> Nature. 382, 609-611 (1996).</li><li>Wang, L., Liu, X., Song, S., Fan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unmodified+gold+nanoparticles+as+a+colorimetric+probe+for+potassium+DNA+aptamers.">Unmodified gold nanoparticles as a colorimetric probe for potassium DNA aptamers.</a> Chem. Commun. (36), 3780-3782 (2006).</li><li>Liu, J., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+colorimetric+sensing+of+adenosine+and+cocaine+based+on+a+general+sensor+design+involving+aptamers+and+nanoparticles.">Fast colorimetric sensing of adenosine and cocaine based on a general sensor design involving aptamers and nanoparticles.</a> Angew. Chem. 118 (1), 96-100 (2006).</li><li>Pavlov, V., Xiao, Y., Shlyahovsky, B., Willner, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamer-functionalized+Au+nanoparticles+for+the+amplified+optical+detection+of+thrombin.">Aptamer-functionalized Au nanoparticles for the amplified optical detection of thrombin.</a> J. Am. Chem. Soc. 126 (38), 11768-11769 (2004).</li><li>Mayer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemical+biology+of+aptamers.">The chemical biology of aptamers.</a> Angew. Chem. Int. Ed. 48 (15), 2672-2689 (2009).</li><li>Hermann, T., Patel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+recognition+by+nucleic+acid+aptamers.">Adaptive recognition by nucleic acid aptamers.</a> Science. 287 (5454), 820-825 (2000).</li><li>Lee, J., Stovall, G., Ellington, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamer+therapeutics+advance.">Aptamer therapeutics advance.</a> Curr. Opin. Chem. Biol. 10 (3), 282-289 (2006).</li><li>Song, S., Wang, L., Li, J., Zhao, J., Fan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamer-based+biosensors.">Aptamer-based biosensors.</a> TrAC. 27 (2), 108-117 (2008).</li><li>Wei, H., Li, B., Wang, E., Dong, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+sensitive+aptamer-based+colorimetric+sensing+of+protein+using+unmodified+gold+nanoparticles.">Simple and sensitive aptamer-based colorimetric sensing of protein using unmodified gold nanoparticles.</a> Chem. Commun. (36), 3735-3737 (2007).</li><li>Zheng, Y., Wang, Y., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamer-based+colorimetric+biosensing+of+dopamine+using+unmodified+gold+nanoparticles.">Aptamer-based colorimetric biosensing of dopamine using unmodified gold nanoparticles.</a> Sensors and Actuators B. 156 (1), 95-99 (2011).</li><li>Ch&#225;vez, J., MacCuspie, R., Stone, M., Kelley-Loughnane, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+detection+with+aptamer-gold+nanoparticle+conjugates:+effect+of+aptamer+length+on+response.">Colorimetric detection with aptamer-gold nanoparticle conjugates: effect of aptamer length on response.</a> J. Nanopart. Res. 14 (10), 1-11 (2012).</li><li>Neves, M., Reinstein, O., Johnson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+a+stem+length-dependent+binding+mechanism+for+the+cocaine-binding+aptamer.+A+combined+NMR+and+calorimetry+study.">Defining a stem length-dependent binding mechanism for the cocaine-binding aptamer. A combined NMR and calorimetry study.</a> Biochemistry. 49 (39), 8478-8487 (2010).</li><li>Li, H., Rothberg, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-Free+Colorimetric+Detection+of+Specific+Sequences+in+Genomic+DNA+Amplified+by+the+Polymerase+Chain+Reaction.">Label-Free Colorimetric Detection of Specific Sequences in Genomic DNA Amplified by the Polymerase Chain Reaction.</a> J. Am. Chem. Soc. 126 (35), 10958-10961 (2004).</li><li>Li, H., Rothberg, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+detection+of+DNA+sequences+based+on+electrostatic+interactions+with+unmodified+gold+nanoparticles.">Colorimetric detection of DNA sequences based on electrostatic interactions with unmodified gold nanoparticles.</a> Proc. Natl. Acad. Sci. 101 (39), 14036-14039 (2004).</li><li>Smith, J., Medley, C., Tang, Z., Shangguan, D., Lofton, C., Tan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamer-Conjugated+Nanoparticle+for+the+Collection+and+Detection+of+Multiple+Cancer+Cells.">Aptamer-Conjugated Nanoparticle for the Collection and Detection of Multiple Cancer Cells.</a> Anal. Chem. 79 (8), 3075-3082 (2007).</li><li>Martin, J., Ch&#225;vez, J., Chushak, Y., Chapleau, R., Hagen, J., Kelley-Loughnane, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+stringency+aptamer+selection+and+gold+nanoparticle+assay+for+detection+of+cortisol.">Tunable stringency aptamer selection and gold nanoparticle assay for detection of cortisol.</a> Anal. Bioanal. Chem. 406 (19), 4637-4647 (2014).</li><li>Shen, L., Hagen, J., Papautsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point-of-care+colorimetric+detection+with+a+smartphone.">Point-of-care colorimetric detection with a smartphone.</a> Lab on a Chip. 12 (21), 4240-4243 (2012).</li><li>Choodum, A., Kanatharana, P., Wongniramaikul, W., NicDaeid, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+quantitative+colourimetric+tests+for+trinitrotoluene+(TNT)+in+soil.">Rapid quantitative colourimetric tests for trinitrotoluene (TNT) in soil.</a> Forensic. Sci. Int. 222 (1), 340-345 (2012).</li></ol>

Over the past decade, nanoparticle based colorimetric assays have been developed for the detection of targets include small molecules, DNA, proteins, and cells2-4. Assays that use DNA-aptamers with nanoparticles have been gaining interest. Typically, these colorimetric assays are performed by mixing the DNA-aptamer with analyte molecules followed by addition to AuNPs9-10. However, these assays have been utilized in proof-of-concept demonstrations with controlled laboratory settings and with limited,...

Colorimetry is one of the oldest techniques used in analytical chemistry. For this technique, a qualitative or quantitative determination of the analyte is made based on the production of a colored compound1. Typically, color assays use reagents that experience a color shift in the presence of the analyte species, which results in an observable or detectable color change in the visible light spectrum. Colorimetry has been used in the detection of targets ranging from atoms, ions, and small molecules to complex biological molecules such as deoxyribonucleic acids (DNA), peptides, and proteins2-4. For the past two decades, nanomaterials have revolutionized the field of detection assays, particularly with color based assays5-6. Combining the unique chemical and physical properties of nanomaterials with a target selective recognition element, such as antibodies, oligonucleotide aptamers or peptide aptamers, has led to the resurgence in the design and development of colorimetric detection assays7.
Metal nanoparticles have a demonstrated size-dependent color change property, which has been exploited in the design of numerous colorimetric assays. Gold nanoparticles (AuNPs) are of particular interest due to a distinctive red-to-blue color shift, when the dispersed solution of particles is induced to aggregate8, typically through the precise addition of salt. The ability to control the transition from the dispersed (red) to the aggregated (blue) states has led to the creation of colorimetric sensors for ionic, small molecular, peptide, protein, and cellular targets2-4,9. Many of these sensors employ aptamers as the target recognition motif.
Aptamers are DNA or ribonucleic acid (RNA) molecules selected from a random pool of 1012-1015 different sequences10-11. The selection process identifies target recognition elements with binding affinities in the low nanomolar regime, and the systematic evolution of ligands by exponential enrichment (SELEX) is the most commonly known process12-13. Advantages of oligonucleotide based aptamers for sensing applications include ease of synthesis, controllable chemical modification, and chemical stability14-15.
One approach to creating a colorimetric assay combines nanomaterials with recognition elements, consists of combining these two species through the physical adsorption of DNA-aptamer molecules to AuNP surfaces. Through target-aptamer binding, the aptamer experiences a structural change16-18 that alters the interaction of the aptamer with the AuNP surface, which leads to an inducible red-to-blue color response19 with the addition of salt. This astonishing feature of AuNPs provides an observable colorimetric response mechanism for aptamer-based devices that can be used to design colorimetric assays for different analytes.
Color assays designed using non-covalent, physically adsorbed DNA aptamers on AuNP surfaces have the stigma of being a weak sensor platform due to issues with robustness, a propensity for failure outside of controlled laboratory settings, and the lack of information available for use in practical settings. However, the aptamer-AuNP based colorimetric assay was of interest because of the simplicity of operation and observable color response. The goal of this work is to provide a protocol for the design, development, operation, reduction of surface related false positive response, and long-term storage of DNA-AuNP based colorimetric assays using cocaine as the representative analyte. Furthermore, we proposed this adsorbed aptamer assay approach (Figure 1) as being advantageous due to simplicity and ease of use that resulted in fewer steps than the conventional approach for these aptamer-AuNP assays. For this assay, the aptamer was first added to the AuNPs, which were allowed to adsorb to the surface for an extended period of time. An additional advantage to this approach was the reduction of response to non-target analyte molecules related to AuNP surface interactions. However, the reduction in false positive response was at the expense of assay sensitivity. Therefore a balance between surface protection and analyte accessibility is necessary to maintain proper assay function. Moreover, a major defect of analyzing color assays through means other than with instrumentation is that the results are often subjective and open to interpretation from analyst-to-analyst, particularly when trying to differentiate subtle differences in color. Conversely, there are a number of issues with making laboratory based instrumentation usable outside the lab, such as availability of power, practicality with portability, etc. In this work, a color analysis protocol was developed for more portability and to eliminate some of the guesswork commonly associated with color based assay interpretation20-21. Compared to previous approaches, this effort strived to push these assays to their limits for applications beyond laboratory settings.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Gold(III) chloride hydrate</td>
			<td>Sigma</td>
			<td>254169</td>
			<td>99.999% purity is important and solutions were made fresh every time</td>
		</tr>
		<tr>
			<td>Sodium Citrate Dihydrate</td>
			<td>Sigma</td>
			<td>W302600-1KG-K</td>
			<td>We have found the manufacturer greatly affects AuNP assays, and solutions were made fresh every time</td>
		</tr>
		<tr>
			<td>Synergy</td>
			<td>Bio-TEK</td>
			<td>HT</td>
			<td>Any absorbance spectrometer will work, but a platereader provides multiple sample analysis</td>
		</tr>
		<tr>
			<td>4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) Buffer, 1 M sterilized</td>
			<td>Amresco</td>
			<td>J848</td>
			<td>Any sterilized brand will work</td>
		</tr>
		<tr>
			<td>Corning, 250 mL Filter System, 0.22 &#181;m cellulose acetate</td>
			<td>Fisher</td>
			<td>430767</td>
			<td>Other membranes have been found to remove the AuNPs</td>
		</tr>
		<tr>
			<td>UV Spectrophophotometer</td>
			<td>Varian</td>
			<td>Cary 300&#160;</td>
			<td>Any absorbance spectrometer will work</td>
		</tr>
		<tr>
			<td>Magnesium Chloride Hexahydrate</td>
			<td>Fluka</td>
			<td>63068</td>
			<td>&#8805;98% any brand will work</td>
		</tr>
		<tr>
			<td>DNA</td>
			<td>IDT</td>
			<td>Custom</td>
			<td>DNA was purified with a desalting column, higher purification techniques can be used</td>
		</tr>
		<tr>
			<td>Procaine Hydrochloride</td>
			<td>ACROS</td>
			<td>AC20731-1000</td>
			<td>99% stocks of 1 mg/mL in methanol were prepared</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Fisher</td>
			<td>A144S-500</td>
			<td>36.5-38.0% w/w other brands will work</td>
		</tr>
		<tr>
			<td>Cocaine Hydrochloride</td>
			<td>Lipomed</td>
			<td>COC-156-HC-1LM</td>
			<td>We have found the manufacturer greatly affects AuNP assays</td>
		</tr>
		<tr>
			<td>Nitric Acid</td>
			<td>Fisher</td>
			<td>A509-SK212</td>
			<td>65% w/w other brands will work</td>
		</tr>
		<tr>
			<td>Sodium Chloride Solution, 5 M bioreagent grade</td>
			<td>Sigma</td>
			<td>S5150-1L</td>
			<td>Sterile solutions made from solid will work</td>
		</tr>
		<tr>
			<td>Diethyl Pyrocarbonate</td>
			<td>Sigma</td>
			<td>D5758-25 mL</td>
			<td>&#8805;97% any brand will work</td>
		</tr>
		<tr>
			<td>Ecgoninemethylester Hydrochloride</td>
			<td>Lipomed</td>
			<td>COC-205-HC-1LM</td>
			<td>We obtained the EME control from the same manufacturer as the cocaine target</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes, Axygen Scientific, nonsterile, 1.7mL</td>
			<td>VWR</td>
			<td>10011-722</td>
			<td>We have found the manufacturer greatly affects AuNP assays, and the tubes were autoclaved in house</td>
		</tr>
		<tr>
			<td>nuclease free water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>methanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

design and development of aptamer&#8211;gold nanoparticle based colorimetric assays for in-the-field applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The primary objective of this work was to develop and investigate the stability and robustness of aptamer based AuNP colorimetric assays for use in the field. As highlighted in a previous publication, two distinct strategies for creating the assay were investigated7. The assays were termed the Free Aptamer Assay and the Adsorbed Aptamer Assay. The Adsorbed Aptamer Assay was more appealing for the purposes of a fieldable detection assay (Figure 1).
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Watch this Scientific Journal Video about Design and Development of AptamerÃ¢â‚¬â€œGold Nanoparticle Based Colorimetric Assays for In-the-field Applications at JoVE.com

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

The overall goal is to provide a general approach for the development of an aptamer-gold nanoparticle based colorimetric assay for the detection of target molecules, and to provide approaches to improve long-term storage and reduce false positive response rates. This methods answers key questions in the field of gold nanoparticle aptamer colorimetric assays such as the reproducibility of the assays in the field for long-term storage and reducing false positive response rate. These assays can be prone to issues with shelf-life and false positive responses.In this work, we improve over both of these issues. Clean a 500-milliliter Erlenmeyer flask and large stir bar with five milliliters of concentrated nitric acid and 15 milliliters of concentrated hydrochloric acid in a chemical safety hood. Wet the entire surface of the flask with the acid wash.Then rinse the flask with nuclease-free water and allow the flask to dry. Add 100 milliliters of one millimolar gold three chloride. Use a sheet of aluminum foil to cover the top of the acid-cleaned Erlenmeyer flask and heat with continuous stirring on a hot plate until boiling.Next, add 10 milliliters of 38.8 millimolar sodium citrate. The color will change from clear or gray to dark blue or black, and finally to dark to red over several minutes. Continue stirring with the heat off for 10 minutes.Allow the gold nanoparticle suspension to cool to room temperature and add 110 microliters of DEPC with continuous stirring. Cover the entire flask with aluminum foil and allow the DEPC treatment to incubate overnight. The next day, autoclave the gold nanoparticle suspension.Then cool to room temperature and filter through a 0.22 micron pore cellulose acetate membrane. Store the filtered, autoclaved gold nanoparticle stock solution in the dark at four degrees Celsius. Calculate the gold nanoparticle concentration as described in the text protocol.Here, the concentration was calculated to be 10 nanomolar with a size of 15 nanometers as determined by dynamic light scattering. Purchase or synthesize the cocaine-binding aptamer sequences using standard phosphoramidite chemistry. After purifying the aptamers using standard desalting, reconstitute oligonucleotides in nuclease-free water at either 100 micromolar or one millimolar stock solutions.Aliquoted purified aptamers can be stored at 20 degrees Celsius for several months. Combine the DNA with the 10 nanomolar stock gold nanoparticle solution, varying the volume of gold nanoparticles as desired, to provide enough sample for the test to be performed. Incubate the mixture for three to four hours at room temperature and protect from the light.Add an equal volume of 20 millimolar HEPES, two millimolar magnesium chloride and pH 7.4 buffer, and place the sample at four degrees Celsius in the dark overnight. The initial salt concentration needed to induce the assay color response by salt titration is determined with the assay blank. Add 20 microliters of methanol to 180-microliter aliquots of the aptamer-gold chloride assay in a 96-well plate.A critical step in the performance of these assays is to determine the sodium chloride concentration to destabilize the gold nanoparticles once the target has been added. Titrate the samples with increasing volumes of stock sodium chloride solution and determine the equivalence point. Here, determine the sodium chloride volume needed to cause the slightest color change by visual observation.Optimizing the assay response, add 20 microliters of analyte molecules diluted in methanol to 180-microliter aliquots of aptamer-gold nanoparticle assay in a 96-well plate at room temperature. Immediately, add the sodium chloride concentration determined in the previous step to initiate the assay color response. Obtain the largest color change possible by increasing or decreasing the sodium chloride concentration and comparing the target response to the blank response.Use the sodium chloride concentration that provides the largest response difference. Observe or measure the assay response 150 seconds following sodium chloride addition. Analyze the absorbance at 650 nanometers and 530 nanometers, using a spectrophotometer.Plot the results as the ratio of absorbance obtained at 650 nanometers and 530 nanometers as a function of analyte concentration. Normalize the assay response to the blank signal. Prepare the aptamer-gold nanoparticle assay components as described earlier in the video.Make separate solutions containing one gram per milliliter of trehalose and one gram per milliliter of sucrose in nuclease-free water to make the cryogen solution. So a critical step in freezing these assays is determining how much trehalose and sucrose solutions to add to the samples to ensure total and even freezing. Make a solution that contains 19.2 milligrams per milliliter of trehalose and 4.8 milligrams per milliliter of sucrose with the 60 MN4 DNA per gold nanoparticle assay at a final volume of 200 microliters in 1.5 milliliter microcentrifuge tubes.Final cryogen solution concentrations will vary with DNA coverage. Flash freeze the samples using a 146 degrees Celsius freezer or in liquid nitrogen. Store the samples frozen until use.For this work, leave the samples in the 146 degrees Celsius freezer overnight and then transfer to a 20 degrees Celsius freezer for long-term storage. Shown here are representative results of sodium chloride titration curves for untreated and DNA aptamer-treated gold nanoparticles. The initial sodium chloride concentration used in the assay was determined by titration.Representative results of the aptamer-gold nanoparticle colorimetric assay for cocaine and control molecules are shown here. The graphs reveal the effect that increasing the DNA coverage density has on the false positive response rate of the cocaine assay. Shown here are the calibration curve plots obtained from photoimage analysis of the cocaine colorimetric assay.Photos obtained using smartphones. Representative results of the assay response of aptamer-gold nanoparticle colorimetric assay to cocaine and control molecules are presented here. The data compares samples freshly prepared to samples stored frozen up to four weeks.While attempting this procedure, it is very important to determine the proper sodium chloride concentration to destabilize the gold nanoparticles and promote the typical color change upon addition of the target. So after watching this video, you should have a good understanding of how to develop gold nanoparticle aptamer colorimetric assays for the detection of targets of interest. You can use the approaches outlined here to reduce false positive response rates and keep your assays viable for more than a month.

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Development of New Therapeutic Applications Using Microfluidics

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice.   ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes

Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to ...

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically ...