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. Syntese via Citrate Reduksjon av gull nanopartikler (AuNP) og karakterisering <ol><li> Rense en Erlenmeyer-kolbe (500 ml) og stor rørestav med 5 ml konsentrert salpetersyre og 15 ml konsentrert saltsyre i kjemisk avtrekkshette. <ol><li> Fukte hele overflaten av kolben med syrevaske, skylle kolben med nuklease fritt vann, og la kolben for å tørke. </li></ol></li><li> Legg 100 ml 1 mM gull (III) klorid; bruker et ark av aluminiumsfolie for å dekke toppen av syren renset Erlenmeyer-kolbe og varme under kontinuerli...

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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. 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I løpet av det siste tiåret, har nanopartikkel basert kolorimetriske analyser er utviklet for påvisning av mål inkluderer små molekyler, DNA, proteiner og celler 2-4. Analyser som bruker DNA-aptamerer med nanopartikler har blitt stadig mer interesse. Vanligvis blir disse kolorimetriske analyser utført ved å blande DNA-aptamer med analytt-molekyler, etterfulgt av tilsetning til AuNPs 9-10. Imidlertid har disse analysene er benyttet i proof-of-concept demonstrasjoner med kontrollerte laborator...

Kolorimetri er en av de eldste teknikker som brukes i analytisk kjemi. For denne teknikk blir en kvalitativ eller kvantitativ bestemmelse av analytten gjort basert på fremstilling av en farget forbindelse 1. Vanligvis fargeanalyser bruker reagenser som opplever en fargeforskyvning i nærvær av analytten art, noe som resulterer i en observerbar eller påvisbar fargeendring i det synlige lysspekteret. Kolorimetri har vært brukt i deteksjon av mål som strekker seg fra atomer, ioner og små molekyler til komplekse biologiske molekyler slik som deoksyribonukleinsyrer syrer (DNA), peptider og proteiner 2-4. For de siste to tiårene, har nanomaterialer revolusjonert detekteringsområde analyser, spesielt med farge baserte analyser 5-6. Kombinerer de unike kjemiske og fysiske egenskapene til nanomaterialer med et mål selektiv anerkjennelse element, slik som antistoffer, oligonukleotid aptamerer eller peptid aptamerer, har ført til oppblomstring in design og utvikling av kolorimetriske deteksjon analyser 7. Metall nanopartikler har en størrelse vist avhengig fargeendring egenskap, som har blitt utnyttet i utformingen av mange kolorimetriske assays. Gull nanopartikler (AuNPs) er av spesiell interesse på grunn av en særegen rød-til-blå fargeskift, da den dispergerte oppløsning av partikler blir indusert til å samle 8, typisk gjennom den presise tilsetning av salt. Evnen til å kontrollere overgangen fra spredt (rød) til de aggregerte (blå) landene har ført til opprettelsen av kolorimetriske sensorer for ionisk, små molekylære, peptid, protein og cellulære mål 2-4,9. Mange av disse sensorene anvende aptamerer som målet gjenkjennelse motiv. Aptamerer er DNA eller ribonukleinsyre (RNA) molekyler valgt fra en tilfeldig pool av 10 12 -10 15 forskjellige sekvenser 10-11. Utvelgelsesprosessen identifiserer målet rekognisjon elementer med bindingsaffiniteter i lav nanomolar regimet, og den systematiske utviklingen av ligander ved eksponentiell berikelse (SELEX) er den mest kjente prosess 12-13. Fordeler med oligonukleotid basert aptamerer for sensing bruksområder er enkel syntese, kontrollerbar kjemisk modifisering, og kjemisk stabilitet 14-15. En tilnærming til å skape en kolo analysen kombinerer nanomaterialer med anerkjennelse elementer, består av å kombinere disse to artene gjennom fysisk adsorpsjon av DNA-aptamer molekyler til AuNP overflater. Gjennom target-aptamer binding aptamer opplever en strukturell endring som endrer 16-18 samvirkningen mellom aptamer med AuNP overflate, noe som fører til en induserbar rød-til-blå fargerespons 19 med tilsetning av salt. Denne forbløffende trekk ved AuNPs gir en observerbar kolo respons mekanisme for aptamer-baserte enheter som kan brukes til å designere kolorimetriske analyser for ulike analytter. Farge analyser konstruert ved hjelp av ikke-kovalente, fysisk adsorberte DNA aptamerer på AuNP flater har stigmatisering av å være en svak sensorplattform på grunn av problemer med robusthet, en tilbøyelighet for svikt utenfor kontrollerte laboratorie innstillinger, og mangel på informasjon som er tilgjengelig for bruk i praktisk innstillinger. Men aptamer-AuNP basert kolo analysen var av interesse på grunn av enkelhet av drift og observerbare fargerespons. Målet med dette arbeidet er å gi en protokoll for design, utvikling, drift, reduksjon av overflaterelaterte falsk positiv respons, og langtidslagring av DNA-AuNP basert kolorimetriske analyser ved hjelp av kokain som representant analytten. Videre foreslo vi dette adsorbert aptamer analysen tilnærming (figur 1) som fordelaktig på grunn av enkelhet og brukervennlighet som resulterte i færre trinn enn den konvensjonelle tilnærmingen for disse aptamer-AuNP assays. For denne analysen ble det aptamer først tilsatt til AuNPs, som ble tillatt å adsorbere til overflaten for en lengre periode. En ytterligere fordel med denne fremgangsmåten var reduksjonen av responsen på ikke-målanalytten molekyler relatert til AuNP overflate interaksjoner. Imidlertid er reduksjonen i falsk positiv reaksjon var på bekostning av analysesensitiviteten. Derfor en balanse mellom overflatebeskyttelse og analyse tilgjengelighet er nødvendig for å opprettholde riktig analyse funksjon. Videre er en stor mangel for å analysere fargeanalyser gjennom andre måter enn med instrumentering er at resultatene ofte er subjektiv og åpne for tolkning fra analytiker-til-analytiker, særlig ved forsøk på å skille små forskjeller i farge. Motsatt finnes det en rekke problemer med å gjøre laboratorie basert instrumentering bruk utenfor laboratoriet, for eksempel tilgjengelighet av makt, praktisk med portabilitet, etc. I dette arbeidet ble en fargeanalyse protokoll utviklet for more portabilitet og for å eliminere noen av gjetting vanligvis forbindes med farge basert analysen tolkning 20-21. Sammenlignet med tidligere tilnærminger, dette arbeidet forsøkt å presse disse analysene til sine grenser for applikasjoner utover laboratoriet.

<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

Utforming og utvikling av en aptamer-gull nanopartikkel (AuNP) kolorimetrisk analyse for påvisning av små molekyler for in-the-feltet applikasjoner ble undersøkt. Target selektiv AuNP basert farge analyser har blitt utviklet i kontrollerte laboratorie innstillinger proof-of-concept. Imidlertid har disse ordningene ikke blitt utøvd til et punkt av svikt for å fastslå praktisk bruk utover laboratoriet. Dette arbeidet beskriver en generisk tilnærming til design, utvikle og feilsøke et aptamer-AuNP kolo analyse for små molekyl analytter og bruke analysen for in-the-feltet innstillinger. Analysen er fordelaktig fordi adsorberte aptamerer passivere de nanopartikkel overflatene og gir et middel for å redusere og eliminere falske positive responser til ikke-mål analytter. Overgang dette systemet til praktisk bruk nødvendig å definere ikke bare holdbarheten av aptamer-AuNP analysen, men etablering av metoder og prosedyrer for å forlenge langtidslagring capabilities. Også en av de anerkjente bekymringer med kolo avlesning er belastningen på analytikere til å nøyaktig identifisere ofte subtile endringer i farge. For å minske ansvaret på analytikere i feltet, ble en farge analyse protokoll utviklet for å utføre de fargeidentifikasjons oppgaver uten behov for å utføre denne oppgaven på laboratorie karakter utstyr. Fremgangsmåten for å lage og testing av dataanalyse-protokoll er beskrevet. Men for å forstå og påvirke utformingen av adsorberte aptamer analyser, interaksjoner knyttet til aptamer, mål, og AuNPs krever videre studier. Den kunnskapen kan føre til skreddersøm aptamerer for økt funksjonalitet.

<html> Hovedmålet med dette arbeidet var å utvikle og undersøke stabilitet og robusthet aptamer basert AuNP kolorimetriske analyser for bruk i felten. Som fremhevet i en tidligere publikasjon, ble to forskjellige strategier for å skape analysen undersøkte 7. Analysene ble betegnet Free Aptamer-analysen og de adsorberte Aptamer-analysen. Den Absorbert Aptamer analysen var mer attraktivt i forbindelse med en fieldable gjenkjenning analysen (figur 1). <p ...

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 AptamerÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Å“Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

Utforming og utvikling av en aptamer-gull nanopartikkel kolorimetrisk analyse for påvisning av små molekyler for in-the-feltet applikasjoner ble undersøkt. I tillegg ble en smart-anordning kolorimetrisk applikasjon (app) validert og langtidslagring av analysen ble etablert for bruk i felten.

Design og utvikling av Aptamer-gull nanopartikkel-basert kolo Analyser for In-the-feltet Applications

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.

Research

JoVE Journal

Biology

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

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