这项工作是从比尔和梅林达盖茨基金会资助，通过“探索大挑战”倡议，由美国国立卫生研究院GM062958 - 01和2R01EB002046通过赠款，赠款（OPP1015402）。这项工作是在根据合同DE - AC52 - 07NA27344根据美国能源部的主持下的劳伦斯 - 利弗莫尔国家实验室的一部分。

1。设置舞台<ol><li>购买一个自定义的寡核苷酸的合成，如Biosearch科技（诺瓦托，CA）或美联认证（德克萨斯州米德兰，）公司的相关性，化学修饰的探针DNA。在其3&#39; -末端的另外一个C6巯基，并在其5&#39; -末端氧化还原活性的亚甲基蓝的探针合成过程中被修改。 </li><li>探针DNA溶解在磷酸缓冲液pH值7.4至200微米的浓度，并确认其浓度用分光光度计在260 nm处测量其吸光度。由于亚甲基蓝基团的DNA探针上的解决方案应该有一个可见的蓝色色调。 </li><li>刚准备1毫升蒸馏水，去离子水（DI水）的三（2 - 羧乙基）膦TCEP 10毫米的解决方案。根据我们的经验，这些TCEP解决方案仍然在黑暗中保存在4 ° C时的一个星期的新鲜</li><li>为了减少任何可能探针DNA溶液中的二硫键，结合2μL的TCEP溶液1μL的探针DNA原液，用移液器轻轻混匀。孵育1小时在黑暗，冷藏集装箱的混合物。最初蓝色的解决方案应该成为TCEP可逆降低亚甲蓝。如果解决方案不很清楚，重复使用一个新鲜的TCEP解决方案的过程，或者可能的话，在室温下。 </li><li>一小时后，稀释1缓冲液的减少DNA探针解决方案，这将削弱它的200nm的浓度。后来，你会孵化金盘电极在200μL这种稀释探头解决方案部分。 </li><li>刚准备至少2毫升2MM mercaptohexanol磷酸盐缓冲液。 </li></ol> 2。传感器的制备<ol><li>结合0.05微米的精细抛光布水氧化铝粉末。金盘电极（CH仪器，美国德克萨斯州奥斯汀​​）波兰坚决按金表面到湿布，并在数字移动8约3分钟每电极的格局。 </li><li> DI水冲洗打磨电极浸入到洋溢着相同的Eppendorf管。超声五分钟，以除去残留的氧化铝粉。 </li><li>放入一个0.5 M的硫酸溶液中的电极，将它们连接到一个电位随着铂金柜台和银/氯化银参比电极，并运行一系列的伏安氧化，减少和电化学清洁其表面。在此之后执行第二个的0.01 M氯化钾在0.1 M硫酸溶液中的电化学清洗。这些清洗程序的细节，可以发现在我们的本性协议文件 1，在这个视频的补充。 </li><li>安排2毫升的Eppendorf管，在一个机架，并填写每个探针DNA溶液200μL。在此解决方案的探针DNA的浓度将定义与探针DNA的包上的传感器的表面密度。传感器的性能很大程度上取决于探针密度，从一个传感器架构下的最优变密度。因此，应在此步骤中使用的探针浓度优化每个新型传感器 2 。探头我们调查的架构，我们采用探针DNA的浓度在这一步的范围从15纳米到2微米，与200纳米，是一个典型的价值。 </li><li>金盘电极与离子水冲洗，然后一小时，他们沉浸在有关的探头在Eppendorf管中的DNA溶液。此时，探针DNA会附着在金电极表面通过一个巯基金自组装单分子膜的形成。 </li><li>离子水冲洗电极，沉浸在2MM mercaptohexanol在Eppendorf管中，并存储在3小时黑暗的地方，在室温下过夜，以确保完整的自组装单分子膜的形成。这一步采用混合单层的一部分mercaptohexanol，以确保形成一个稳定的单层的。你可能想Eppendorf管中，用封口膜密封电极，以防止蒸发。传感器可以储存数天，如有必要，在此解决方案。 </li><li>当您准备使用的传感器，DI水冲洗，至少10分钟，然后浸泡在缓冲。旨在抗体检测的传感器，还必须在100纳米的有关承认链解决方案之前使用，其次是一个非常快速用缓冲液冲洗，浸泡。 </li></ol> 3。传感器检测，DNA检测<ol><li>在这个协议中，17个核苷酸探针链是贴金电极。它有一个亚甲基蓝在其3&#39; -末端的氧化还原记者（图1）。当探针分子杂交捕获链，通过传感器电路中的电流减小。 </li><li>一个新的传感器与离子水冲洗，沉浸在一片空白样本缺乏目标，以记录它产生的背景信号。将传感器的电位电极导线。放置一个铂电极和一个到溶液中的银/氯化银参比电极。 </li><li>运行幅度为25 mV和一个1mV的步长从0到-0.6 V方波测量。最佳的方波的频率将取决于探头架构的细节 2,3;探头架构，我们聘请的最优值通常是在距离60到600赫兹。您应该看到一个圆润峰值约为-0.35 V时，亚甲蓝的氧化还原电位（峰电位可能会改变您的测试解决方案的精确的pH值略有不同）。这个峰值电流从基线的高度是成正比的效率之间的亚甲基蓝和金电极（图2）电子转移。保存此背景下测量。 </li><li>一个解决方案，包含利益的目标DNA分子的电极移动，平衡（5至120分钟的规模，结构和target4，5浓度而定），并收集了第二个方波voltammagram。在-0.35 V峰值的高度将发生变化，从最初的背景测量。这种变化的幅度与分析物的浓度。这是该传感器的主要输出数据（图3）。 </li><li>测量相对信号的变化，这一比例减少或增加信号的背景峰相对，往往更比电流测量的绝对变化重现性，在电极表面面积的变化纠正。要做到这一点，峰值电流和背景峰值电流之间的差​​异分为背景峰值电流。 </li></ol> 4。传感器再生<ol><li>一旦测量完成后，移动到30秒充满离子水的容器传感器，或喷用DI水源源不断为30秒。用新鲜去离子水的重复这两个次以上。注：一些分析物有抗药性的这种方法，他们试图在6 M盐酸胍或70％的乙醇冲洗进取。 </li><li>把传感器将测量到一个解决方案。一分钟内，峰的高度应该返回到其原始值。这是值得注意的，这些传感器通常表现出他们的第一个测试和再生循环过程中稍大的信号变化，并在随后的 6个周期高度一致的结果。 </li></ol> 5。传感器检测，抗体检测<ol><li>在这个协议中，这些传感器中使用的亚甲基蓝和巯基修饰的DNA探针作为一个“ 锚 ”链7。它是直接连接到金电极。这是第二个，“承认”的DNA链，已共价键结合的相关抗原（图4）杂交。我们有好运气，与商业合成房子，如Biosearch技术和帕纳锦社，所需要的DNA抗原嵌合体的合成。杂交步骤进行，转移到一个预制的Eppendorf管含1小时1​​00纳米的有关识别DNA链在PBS的传感器。 </li><li>将有关的空白溶液中的传感器。附加电位电极导线放置一个铂电极和银/氯化银参比电极的解决方案。 </li><li>如上所述，执行方波伏安法。为特定的探头架构，我们采用最佳的方波的频率是60赫兹。您应该看到一个圆形的高峰围绕-0.35五，保存这样的背景下测量。 </li><li>含有目标分析物的电极传送到一个​​解决方案，培育5至60分钟，并收集了第二个方波voltammagram。如果目标抗体是目前在-0.35 V的峰值将有所下降。这种变化的幅度是相关的抗体浓度。 </li></ol> 6。传感器测试，小分子检测<ol><li>在这种情况下，传感器的表面上的探针分子是适体，DNA或RNA分子，已在体外选择绑定一个特定的分子分析物，改变其结构（折叠）结合后其目标分析8,9（图5）。在这里，我们聘请可卡因结合，DNA Stojanovic 10,11实验室开发的适体。 </li><li>一个新的传感器与离子水冲洗，沉浸在一个空白样本缺乏目标，以记录它产生的背景信号。将传感器的电位电极导线。将铂金计数器和一个银/氯化银参考到该解决方案。 </li><li>如上所述，执行方波伏安法。为特定的探头架构，我们已在这里雇用的最佳方波的频率是200HZ（但是，60赫兹工程）。您应该看到一个圆形的高峰围绕-0.35五，保存这样的背景下测量。 </li><li>含有目标分析物的电极传送到一个​​解决方案，孵化〜5分钟，并收集了第二个方波voltammagram。在-0.35 V的峰值高度会发生变化。这种变化的幅度与目标分析物的浓度。如果你不能获得可卡因样品，普鲁卡因，使用的是不受管制的的，可以作为替代品使用。 </li></ol> 7。代表性的成果： 当使用的架构，用来检测DNA，信号应至少减少60％时，在200 nm目标的平衡。三个简短的去离子水冲洗后，应返回的信号非常接近（0.1-5％内）到其原始值。应该接受抗体检测传感器的信号减少40％至80％。基于核酸适体传感器检测可卡因展览以200％的信号增加他们的频率和表面覆盖而定。可卡因传感器，低表面覆盖是最好的3。 <img src="/files/ftp_upload/2922/2922fig1.jpg" alt="图1" /> 图1。电化学DNA生物传感器的DNA检测。 <img src="/files/ftp_upload/2922/2922fig2.jpg" alt="图2" /> 图2的屏幕截图显示在方波伏安法的E - DNA生物传感器产生的信号。 <img src="/files/ftp_upload/2922/2922fig3.jpg" alt="图3" /> 图3的屏幕截图显示在方波伏安法的E - DNA生物传感器产生的信号，分析物杂交前后。 <img src="/files/ftp_upload/2922/2922fig4.jpg" alt="图4" /> 图4。脚手架传感器抗体的检测。 <img src="/files/ftp_upload/2922/2922fig5.jpg" alt="图5" /> 图5。可卡因或普鲁卡因与电化学适体生物传感器检测。 <table border="1"><tbody><tr><td> 自定义的Oligo </td><td> 序列 </td><td> 评论 </td></tr><tr><td>线性探针DNA（LP17） </td><td> 5&#39; - HS -（CH2）6 - TGGATCGGCGTTTTATT -（CH2）7的NH - MB - 3&#39; </td><td> HPLC纯化，可以订购与SS </td></tr><tr><td>目标分析物的DNA </td><td> AATAAAACGCCGATCCA </td><td>未修改</td></tr><tr><td>识别东街</td><td> 5&#39; -抗原- TEG - CAGTGGCGTTTTATTCTTGTTACTG - 3“ </td><td></td></tr><tr><td>脚手架锚</td><td> 5&#39; - HS -（CH 2）6 - GCAGTAACAAGAATAAAACGC CACTGC -（CH 2）7 MB的</td><td> HPLC纯化，可以订购与SS </td></tr><tr><td> A4可卡因的核酸适体</td><td> 5&#39; - HS - AGACAAGGAAAATCCTTCAATGAAGTGGGTCG MethyleneBlue - 3“ </td><td> HPLC纯化，可以订购与SS </td></tr></tbody></table>表1。探针和目标DNA序列。

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Specificity,+Electrochemical+Sandwich+Assays+Based+on+Single+Aptamer+Sequences+and+Suitable+for+the+Direct+Detection+of+Small-Molecule+Targets+in+Blood+and+Other+Complex+Matrices.">High Specificity, Electrochemical Sandwich Assays Based on Single Aptamer Sequences and Suitable for the Direct Detection of Small-Molecule Targets in Blood and Other Complex Matrices.</a> Journal of the American Chemical Society. 131, 6944-6945 (2009).</li><li>Xia, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Electrochemical+Supersandwich+Assay+for+Sensitive+and+Selective+DNA+Detection+in+Complex+Matrices.">An Electrochemical Supersandwich Assay for Sensitive and Selective DNA Detection in Complex Matrices.</a> Journal of the American Chemical Society. 132, 14346-14348 (2010).</li><li>Patterson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Triplex-Forming+Oligonucleotide+Probes+for+the+Reagentless,+Electrochemical+Detection+of+Double-Stranded+DNA.">Using Triplex-Forming Oligonucleotide Probes for the Reagentless, Electrochemical Detection of Double-Stranded DNA.</a> Analytical Chemistry. 82, 9109-9115 (2010).</li><li>Ferguson, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+Microfluidic+Electrochemical+DNA+Sensor.">Integrated Microfluidic Electrochemical DNA Sensor.</a> Analytical Chemistry. 81, 6503-6508 (2009).</li><li>Swensen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous,+Real-Time+Monitoring+of+Cocaine+in+Undiluted+Blood+Serum+via+a+Microfluidic,+Electrochemical+Aptamer-Based+Sensor.">Continuous, Real-Time Monitoring of Cocaine in Undiluted Blood Serum via a Microfluidic, Electrochemical Aptamer-Based Sensor.</a> Journal of the American Chemical Society. 131, 4262-4266 (2009).</li></ol>

一个重要的注意的是，上述实验都将正常工作电极，除非得到了妥善清理。这里是我们的电化学清洗程序的指南。 CH仪器恒电位工作时，我们使用三个宏程序运行这些清洁步骤。 零阶段（电子商务清洁Ø） 沉浸在0.5MH 2 SO 4电极，将它们连接到工作电极的电位。还重视和浸泡的Ag / AgCl参比和铂电极。开始氧化步骤（5秒2 V），然后减少步长为10秒（0.35 V）。 第一阶段（E清洁1） 在相同的酸性条件下，从0.35至1.5 V（20扫描，扫描速度为4 V / S和0.01 V的采样间隔，四个扫描，扫描速率（0.5MH 2 SO 4）启动氧化和还原扫描0.1 V / S和0.01 V的采样间隔）。 第二阶段（E清洁2） 另一个涉及四个不同的潜在范围（所有扫描速度在0.1 V S - 1和0.01 V的采样间隔为10段（0.01 M KCl/0.1 MH 2 SO 4）在酸性条件下进行电化学氧化和还原扫描）：（一）潜在范围从0.2到0.75 V;（ii）潜在的范围从0.2到1.0 V;（三）潜在的范围从0.2到1.25 V;（四）潜在的范围从0.2至1.5 V。 金电极的种类很多，可以用来进行这些实验。此外，如这里的金盘电极，我们已成功与金表面微加工，金线，并在印刷电路板上的黄金。 随着本文中所描述的传感器，许多其他的电化学DNA生物传感器架构已报告。这包括用假结12传感器，三股13 14，三明治，15超级三文治，或三缸16架构。 在未来，我们预期，这些传感器将被用于点保健医疗诊断。他们已经成功地集成到了17,18微流体装置，光学分析物的检测系统，并提供许多优点。特别是，这些传感器可以在浑浊的功能，光学密集和高度自动荧光的样品。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>金盘电极</td><td> CH仪器</td><td> CHI101 </td><td>可重复使用</td></tr><tr><td>合成探针DNA </td><td> Biosearch技术</td><td>自定义</td><td></td></tr><tr><td>合成的目标DNA </td><td>西格玛Genosys </td><td>自定义</td><td></td></tr><tr><td> Mercaptohexanol </td><td>西格玛爱秩序</td><td> 725226 - 1G </td><td>储存在阴凉黑暗的地方</td></tr><tr><td>铂电极</td><td>巴斯</td><td> MW - 1032 </td><td>可重复使用</td></tr><tr><td>银/氯化银参考</td><td>巴斯</td><td> MF - 2052 </td><td>可重复使用</td></tr><tr><td>抛光布</td><td>比埃勒</td><td> 40-7212 </td><td></td></tr><tr><td>氧化铝波兰</td><td>比埃勒</td><td> 40-6325-016 </td><td></td></tr><tr><td>磷酸盐缓冲液，缓冲液，pH 7.4 </td><td>西格玛爱秩序</td><td> P7059 - 1L </td><td></td></tr><tr><td> CH仪器605A </td><td> CH仪器</td><td> 605A </td><td>使用任何电位</td></tr><tr><td>新生小牛血清</td><td>西格玛爱秩序</td><td> N4637 - 500ML </td><td>冻存</td></tr><tr><td> NanoDrop </td><td> Fisher Scientific则</td><td> ND - 2000 </td><td>使用任何的UV - Vis </td></tr><tr><td> PCR混合物</td><td> Bio - Rad公司</td><td> 170-8862 </td><td>冻存</td></tr><tr><td>可卡因</td><td>西格玛爱秩序</td><td> C5776 </td><td>需要DEA的许可</td></tr><tr><td>普鲁卡因</td><td>西格玛爱秩序</td><td> P9879 </td><td>以可卡因的</td></tr><tr><td>反旗抗体</td><td>西格玛爱秩序</td><td> F1804 - 1毫克</td><td></td></tr></tbody></table>

fabrication of electrochemical-dna biosensors for the reagentless detection of nucleic acids, proteins and small molecules

医药是目前实行的，医生送标本到中心实验室进行测试，因此必须等待数小时或数天来收到的结果。快速，床头的测试，许多病人将得到更好的服务。为此，我们的实验室和其他人开发的一种多功能，无试剂生物传感器的平台，可支持定量，无试剂，核酸（DNA和RNA），蛋白质（包括抗体）和小分子直接在未处理的临床及环境样品的分析物的电化学检测。在这段视频中，我们展示了在这个“E - DNA”类的几种生物传感器的制备和使用。特别是，我们制造和展示在聚合酶链反应混合物中，艾滋病毒特异性抗体和毒品可卡因的目标DNA序列的检测传感器。准备过程只需要三个小时孵育过夜的努力，其使用只需要几分钟。

Watch this Scientific Journal Video about Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules at JoVE.com

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

无试剂，“E - DNA”的传感器，电化学传感器表现良好，即使在血液和其他复杂的矩阵直接挑战时，已适应了广泛的核酸，蛋白质和小分子分析物的检测。在这里，我们介绍了这种传感器的制造和使用的一般程序。

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

fabrication-electrochemical-dna-biosensors-for-reagentless-detection

Research

JoVE Journal

Bioengineering

33.0K 次观看.  University Of California Santa Barbara. 无试剂，“E - DNA”的传感器，电化学传感器表现良好，即使在血液和其他复杂的矩阵直接挑战时，已适应了广泛的核酸，蛋白质和小分子分析物的检测。在这里，我们介绍了这种传感器的制造和使用的一般程序。

视频: Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration1,2 and efficient separation and extraction based on ionic mobility.3 For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation4-8 and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood.9-12
ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.13). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species.
We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation.
We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids.

Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate.

对芯片等速电泳离子的分离和纯化核酸

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

科研

生物工程

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

光学检测<em&gt; E。大肠杆菌</em&gt;由细菌介孔硅生物传感器

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

交货蛋白质，多肽或细胞不可渗透的小分子进入活细胞被孵化与核内体溶解试剂dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

电化学 DNA 生物传感器检测食源性致病菌的研究进展

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

毛细管阵列中多核酸的视觉检测

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior in such restrictive conditions. To address this issue, we have developed growth chambers for tip-growing plant cells that contain a series of narrow, micro-fabricated gaps (~1 &#181;m) in a poly-dimethylsiloxane (PDMS) substrate. This transparent material allows the user to monitor tip elongation processes in individual cells during microgap penetration by time-lapse imaging. Using this experimental platform, we observed morphological changes in pollen tubes as they penetrated the microgap. We captured the dynamic changes in the shape of a fluorescently labeled vegetative nucleus and sperm cells in a pollen tube during this process. Furthermore, we demonstrated the capability of root hairs and moss protonemata to penetrate the 1 &#181;m gap. This in vitro platform can be used to study how individual cells respond to physically constrained spaces and may provide insights into tip-growth mechanisms.

We describe a method to investigate the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to elongate through extremely narrow gaps (~1 &#181;m) in a microfluidic device.

微流控装置在极小空间中研究尖端生长植物细胞的伸长能力

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

用于长期无细胞基因表达传导的多层微流体平台

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated media. In this article, we describe detailed protocols that enable the construction of nanofiber architectures in organic solvent mixtures through the self-assembly of uniquely addressable, single-stranded, gamma-modified peptide nucleic acid (&#947;PNA) tiles. Each single-stranded tile (SST) is a 12-base &#947;PNA oligomer composed of two concatenated modular domains of 6 bases each. Each domain can bind to a mutually complimentary domain present on neighboring strands using programmed complementarity to form nanofibers that can grow to microns in length. The SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers. In contrast with analogous DNA nanostructures, which form diameter-monodisperse structures, these &#947;PNA systems form nanofibers that bundle along their widths during self-assembly in organic solvent mixtures. Self-assembly protocols described here therefore also include a conventional surfactant, Sodium Dodecyl Sulfate (SDS), to reduce bundling effects.

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

将伽马改性肽核酸自组装成有机溶剂混合物中复杂的纳米结构

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

具有透化检测功能的连续流动微电穿孔系统的制造和操作

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...

Experimental and Data Analysis Workflow for Soft Matter Nanoindentation

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft biomaterials and cells. This approach has gained a central role in the fields of mechanobiology, biomaterials design&#160;and tissue engineering, to obtain a proper mechanical characterization of soft materials with a resolution comparable to the size of single cells (&#956;m). The most popular strategy to acquire such experimental data is to employ an atomic force microscope (AFM); while this instrument offers an unprecedented resolution in force (down to pN) and space (sub-nm), its usability is often limited by its complexity that prevents routine measurements of integral indicators of mechanical properties, such as Young&#39;s Modulus (E). A new generation of nanoindenters, such as those based on optical fiber sensing technology, has recently gained popularity for its ease of integration while allowing to apply sub-nN forces with &#181;m spatial resolution, therefore being suitable to probe local mechanical properties of hydrogels and cells.
In this protocol, a step-by-step guide detailing the experimental procedure to acquire nanoindentation data on hydrogels and cells using a commercially available ferrule-top optical fiber sensing nanoindenter is presented. Whereas some steps are specific to the instrument used herein, the proposed protocol can be taken as a guide for other nanoindentation devices, granted some steps are adapted according to the manufacturer&#39;s guidelines. Further, a new open-source Python software equipped with a user-friendly graphical user interface for the analysis of nanoindentation data is presented, which allows for screening of incorrectly acquired curves, data filtering, computation of the contact point through different numerical procedures, the conventional computation of E, as well as a more advanced analysis particularly suited for single-cell nanoindentation data.

The protocol presents a complete workflow for soft material nanoindentation experiments, including hydrogels and cells. First, the experimental steps to acquire force spectroscopy data are detailed; then, the analysis of such data is detailed through a newly developed open-source Python software, which is free to download from GitHub.

软物质纳米压痕的实验和数据分析工作流程

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft ...