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.通过金纳米粒子柠檬酸还原（金纳米粒子）与表征合成<ol><li>清洁的Erlenmeyer烧瓶（500ml）中和大型搅拌棒用5毫升浓硝酸和15毫升浓缩的化学安全罩盐酸。 <ol><li>湿用酸洗涤烧瓶的整个表面，冲洗用不含核酸酶的水的烧瓶中，并允许烧瓶干燥。 </li></ol></li><li>加入100毫升1mM的金（III），氯化;使用铝箔的片材，以覆盖酸清洗Erlenmeyer烧瓶和加热连续搅拌的顶部的热板上直到沸腾。 </li><li>加入10 mL 38.8...

<ol>
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在过去十年中，基于纳米颗粒的比色测 ​​定法已被用于检测的目标开发包括小分子，DNA，蛋白质和细胞2-4。使用DNA适体纳米粒子实验已获得的利益。通常，这些比色测 ​​定法是通过将DNA的适体与分析物分子，随后加入到纳米金9-10进行。但是，这些测定法已经用于证明的概念展示具有受控的实验室设置，以有限的，所选择的控制。最新进展过渡这项技术进入该领域已经进行了...

比色法是在分析化学中使用的最古老的技术之一。对于这种技术，分析物的定性或定量测定由根据生产的有色化合物1。通常情况下，颜色测定法使用体验中的分析物质的存在，这将导致在可见光光谱中观察到的或检测的颜色变化的色移的试剂。比色法已在检测的目标，从原子，离子和小分子到复杂的生物分子，如脱氧核糖核酸（DNA）的肽，和蛋白质2-4这样被使用。在过去的二十年中，纳米材料已经彻底改变了检测分析的领域中，特别是与基于颜色测定5-6。与靶选择性识别元件结合纳米材料的独特的化学和物理性质，例如抗体，寡核苷酸适体或肽适体，导致了再起我n中的设计和比色检测试验7的发展。 金属纳米颗粒具有一个证实大小相关的色彩变化特性，这已在许多比色测定法的设计被利用。金粒子（AuNPs）是特别感兴趣的，由于独特的红至蓝色移，当粒子的分散液被诱导聚合8，典型地通过精确的加成盐。控制从分散（红色）过渡到聚集的（蓝色）状态的能力已经导致建立比色传感器，用于离子，小分子，肽，蛋白质和细胞靶2-4,9的。许多这些传感器采用适体作为目标识别基序。 适体是由10 12 -10 15个不同的序列10-11的无规池选择的DNA或核糖核酸（RNA）分子。选择过程确定的目标重新认知元件在低纳摩尔政权结合亲和性，并通过指数富集（SELEX） ​​的配体的系统进化是最公知的过程12-13。基于寡核苷酸适体用于感测应用的优点包括易于合成的，可控的化学修饰，和化学稳定性14-15。 一种方法来创建一个比色测定结合纳米材料与识别元件，包括通过DNA适配子分子金纳米粒子表面的物理吸附这两个物种结合。通过目标核酸适体结合，适体经受能改变与金纳米粒子表面，从而导致与加成盐的可诱导的红至蓝颜色响应19中的适体的相互作用的结构变化16-18。金纳米粒子的这一惊人的特性提供了一种可观察到的比色反应的机制，可用于脱基于适体的设备签署不同的分析物比色法。 使用上金纳米粒子表面的非共价键，物理吸附的DNA核酸适体设计的彩色测定有被微弱的传感器平台的耻辱由于与稳健性，对未受控实验室环境以外的倾向，以及可用于实际应用的信息缺乏问题设置。然而，基于适体的金纳米粒子比色法是因为操作和观察到的色彩响应的简单的兴趣。这项工作的目的是提供一种用于设计，开发，操作，减少表面的相关的假阳性反应，和长期存储用可卡因为代表分析物DNA的金纳米粒子基于比色测定法提供的协议。此外，我们提出了本吸附适配体测定方法（ 图1）为是有利的，由于使用的简单和易用性，导致比这些适体-金纳米粒子屁股常规方法更少的步骤AYS。对于这个测定法中，适体首先​​被加入到纳米金，这被允许吸附到表面的时间较长。一个附加的优点，以这种方法是响应于与金纳米粒子表面的相互作用的非目标分析物分子的减少。然而，在假阳性反应的减少是在测定灵敏度为代价的。因此，表面保护和辅助分析物之间的平衡是必要保持适当的检测功能。此外，通过对色彩分析的一个重大缺陷是​​指除与仪器是结果往往是主观的和分析师对分析师公开的解释，试图区分颜色的细微差别时尤为如此。反之，有许多与使得基于实验室可用的仪器在实验室外，如电源的可用性，实用性与便携性， 等。在此工作的问题，一个色分析协议是为MOR开发Ë便携性并消除一些通常与基础色法20-21解释相关的猜测。相比于以前的方法，这一努力争取到这些分析推到他们的实验室之外设置的应用程序的限制。

<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

进行了检查适体 - 金纳米粒子（金纳米粒子）比色法检测小分子中的非现场应用的设计与开发。基于目标的选择性金纳米粒子的颜色检测已经发展证明了概念控制的实验室设置。然而，这些方案没有被施加到故障点，以确定其超出实验室设置实际使用。这部作品描述了一个通用的方法来设计，开发和故障排除为小分子分析物和使用试验中，该场设置的适体 - 金纳米粒子比色测定。该测定是有利的，因为吸附适体钝化纳米颗粒表面并提供减少和消除对非靶分析物的假阳性反应的手段。转换该系统的实际应用所需的限定不仅适体 - 金纳米粒子测定的保质期，但是建立延伸长期存储capabil方法和程序伊蒂埃斯。此外，与比色读出公认的问题之一是放置于分析师准确识别颜色往往微妙变化的负担。以减少在现场上分析的责任，颜色分析协议设计为无需进行实验室级装备该任务执行的颜色标识的职责。描述了用于创建和测试数据分析的协议的方法。然而，为了理解和影响吸附的适配体测定法的设计中，与适体，目标相关联的交互，以及金纳米粒子需要进一步研究。所获得的知识可能导致剪裁适体的改进的功能。

<html>这项工作的主要目标是开发和研究基于金纳米粒子比色分析在现场使用的稳定性和适体的稳健性。如在先前的出版物强调，用于创建该测定两种不同的策略进行了调查7。该测定法被称为自由适配体测定和吸附适配体测定。的吸附适配体测定是一个fieldable检测测定（ 图1）的目的更吸引人。 <img alt="图1" src="/files/ftp_upload/54063/5406...

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

进行了检查适体，纳米金比色法检测小分子在得到了实地应用的设计与开发。此外，一个智能设备色度应用（应用程序）的验证，并建立了在该领域使用的测定法的长期贮存。

设计和适体 - 金纳米粒子基于比色法的开发在这领域的应用

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

Development of New Therapeutic Applications Using Microfluidics

使用微流体开发新的治疗应用

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