The authors have nothing to disclose.

1.可处理合成聚苯胺<ol><li>溶解苯胺（1毫升，11毫摩尔）完全在60毫升氯仿中的250毫升圆底烧瓶中。在搅拌600转5分钟，冷却至0-5℃，有冰冻。这通常需要15-20分钟（ 图2A）。 </li><li>十二烷基苯磺酸钠（NaDBS）（7.44克，21毫摩尔）添加到在圆底烧瓶中的苯胺溶液在600rpm搅拌。 </li><li>溶解过硫酸铵（APS）（3.072克，13.5毫摩尔）在20毫升水中，并添加所有的它逐滴在30分钟内，以避免过热的反应。 </li><li>进行在0-5℃反应24小时，并使其达到室温下再24小时。 </li><li>观察反应混合物最初打开乳白色15分钟后，再暗褐色后2小时，最后以24小时后（ 图2B-F）的深绿色。 </li><li>过滤用布氏漏斗聚苯胺-NaDBS解决方案。用80毫升氯仿和120ml水在作为混合eparation漏斗（ 图2G）。 </li><li>在室温下孵育24小时的溶液，并收集从分离漏斗暗绿色聚苯胺，留下未反应的NaDBS和APS在含水上清液。 </li></ol> 2. PANI探针混合和紫外线照射<ol><li>稀聚苯胺溶液10倍，用氯仿 - 水（1:3体积/体积）并通过温和摇动用于在微量离心管15分钟，混合200微升稀释聚苯胺与探针DNA寡核苷酸的6.4微摩尔。 </li><li>照射在交联剂与1200μJ/ cm 2的紫外线的PANI-DNA溶液2分钟。至关重要的是，紫外线照射被限制为指定量。延长暴露于UV损害在聚苯胺的荧光的变化，可能是由于聚苯胺和DNA的共价交联。 </li><li>沉淀物通过离心在17000×g离心6分钟，并立即用磷酸盐缓冲盐水（PBS）中。再次颗粒，并在PBS再中止。 </li></ol> 3. ^ h聚苯胺探头的ybridization <ol><li>添加8微升100微米的互补DNA寡核苷酸或靶核酸200微升聚苯胺探针复合物。 </li><li>在40摇动溶液混合物15分钟进行杂交 C。 </li><li>沉淀通过离心聚苯胺配合物在17000×g离心6分钟。用PBS洗涤，并在水中重新悬浮。 </li></ol> 4.排放稳态荧光测量<ol><li>从不同处理在250nm处添加聚苯胺到96孔微量培养板，并测量在270-850纳米范围内发射荧光通过激发。对于聚苯胺的排放峰值应该围绕500纳米观察。 </li></ol>杂交复式5.荧光显微镜测量<ol><li>滴涂层聚苯胺上的硼硅玻璃盖玻片并干燥在40℃下48小时。 </li><li>在干燥的聚苯胺薄膜添加探头（8微升100μM），并用紫外线（1200微焦/厘米2照射它</suP&gt;）2分钟。 </li><li>用PBS洗聚苯胺探针膜并干燥，在40℃48小时。 </li><li>通过加入靶核酸15分钟进行杂交。这可能是一个生物样品或控制目标寡核苷酸（8微升100μM的的）。用PBS洗涤跟随。 </li><li>获取荧光图像放大40倍，具有500纳米长通滤波器。 </li></ol>

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The work was supported by the University of Southern Mississippi College of Science and Technology and Mississippi College of Science and technology and Mississippi INBRE program (Award Number P204M103476 from the National Institute of general Medical Science).

核酸基于PANI-传感器需要在水中的聚合物增溶，以与DNA和RNA相互作用。聚苯胺在水中的分散体是使用表面活性剂来完成，形成微胶粒如先前报道8。除了这里使用的其它阴离子表面活性剂一样的4-磺基邻苯二甲酸十二烷基酯的NaDBS，如壬基苯酚乙氧基化物，或类似的溴化十六烷基三甲铵阳离子表面活性剂的非离子表面活性剂也可用于加工的聚苯胺9,10的合成。这里所描述的合成始于在2加入单体苯胺的阴离子表面活性剂NaDBS的固定比例:1的摩尔比。表面活性剂NaDBS具有高的亲水 - 亲油平衡（HLB）使由胶束聚合物油滴和水之间建立平衡在氯仿 - 水中乳化苯胺单体的聚合。氧化剂的APS然后以1加入:1.5的摩尔比，以产生克的微胶粒由于聚苯胺，其中该内芯充当成核剂。这导致前往单体的尾部附件，同时也保持聚苯胺处于动态平衡与周围溶剂11,12。以受控的方式进行反应，直到高分子量聚合物形成13。该协议将生成的范围从50纳米至1500纳米的3粒子。临界建立，其中聚苯胺生长缓慢但仍然可溶的反应是在加入的氧化剂的APS，低温，和缺乏强质子酸的慢。反应物的比例的一些优化可能是必要的，以产生分散良好的聚苯胺。用这种方法合成的聚苯胺为一年以上作为乳液稳定。应避免过分稀释作为分散剂的HLB平衡可能被破坏，从而导致聚苯胺沉淀。高速混合可以帮助保持聚苯胺在溶液中。完美的溶解度是不可取的，因为这会妨碍粒料formati上，这是用于在基于乳液的传感器杂交方法的一个重要标准。 之后产生地分散聚苯胺的DNA探针寡核苷酸被附连到通过静电相互作用的聚合物基质。发生聚苯胺亚胺基团的互动与DNA的骨干磷酸盐油然而生。非特异性的PANI-核酸相互作用复杂复杂生物样品14中的分析物检测。实现聚苯胺和探头的DNA之间的关联，可以从自发的相互作用来区分，复数照射紫外线。通过UV引发的电荷定位诱导极性部分的创建增加了聚合物表面15的润湿性。最佳曝光是约2分钟，并且如果在500nm聚苯胺荧光大致升高5X（ 图3）可以得到证实。过度曝光的复合物的UV可导致聚苯胺和DNA，它不像静电相互作用，美国能源部之间的交联不是引起聚苯胺荧光的正视图。 传感器生成的过程简单快捷。聚合反应在48小时后24小时，以与未反应的氧化剂和NaDBS分离后结束。合成将需要如由本文中的协议生成库存聚苯胺的量很少进行是相对于在传感器中使用的量非常大。在高浓度聚苯胺乳液是稳定的;然而，探头附件期间发生任何稀释可能会导致聚苯胺的沉淀，影响传感器的性能。立即使用聚苯胺探针复合物探测目标会避免此问题。总之，复合物和杂交可以创造一个小时下进行英寸PANI-探针膜是更稳定的，并且可以在长期存储中。本传感器技术的一个限制是，它不是非常适合于复用。每个检测事件必须在一个单独的试管中进行。薄膜可以提供复用更好的格式，不同的探头可以在一部电影的不同区域被发现。样品可以在点阵列被应用，并且在每个点的荧光来评价不同基因的表达。 这里所描述的检测机构超过其他系统提供了若干优点。通过涉及聚苯胺的解离机制检测核酸的报告依赖于连接到探针DNA寡核苷酸5荧光。其他策略使用探头，它未能区分靶核酸16-17单碱基对差异的共价结合。标记或检测在本技术的其它二次模式的省略创造了机会使用电活性聚苯胺的其他属性。传感器的响应因而可以扩展到由传感器的连接 - 分离动力学改变电化学性能的测量。采用这种技术实验室将获得S恩索尔系统具有成本效益，除了快速和简单。

Conjugated polymers provide many options for molecular sensors. This includes fluorescence, electronic, and colorimetric responses1. There have been many efforts to incorporate conjugated polymers in nucleic acid sensors. However, most systems require secondary detection, limiting sensing options2. Recently, we reported a conjugated polymer-based sensor platform built on polyaniline (PANI) that exploits properties of this polymer, creating a label-free system3. PANI is an extensively conjugated electro-active polymer with properties such as fluorescence and resistance that are suitable for measuring biological systems4. The excitons within the structure are not localized leading to mobility of the positive charge between monomeric subunits. This provides a flexible scaffold of positive charges that can interact with the negatively charged backbone of DNA5,6. Importantly, electrostatically attached DNA is orientated such that nitrogenous bases can participate in base pairing. Association with DNA alters the electronic properties of PANI, an effect that can be enhanced by UV irradiation (Figure 1)3. Using this system, oligonucleotides complementary to target nucleic acids can be immobilized on PANI. Multiple studies have demonstrated that upon hybridization electrostatically adsorbed oligonucleotides dissociate from PANI or other cationic matrices due to conformational changes caused by the switch to a double-stranded DNA structure3,5,7.
In a sensor system where probe attachment modulates conjugated polymer properties, hybridization events can be transduced without labels or enzymatic modification of probes or target nucleic acids. Conjugated polymers offer great flexibility in detection methods, one of which is fluorescence. Through monitoring PANI fluorescence, concentrations of target nucleic acids as low as 10-11 M (10 pM) can be detected3. Detection is rapid, occurring within 15 minutes of hybridization, and specific where a single mismatch in a target molecule can be differentiated3. 
Fabrication of PANI-sensors is straightforward. High molecular weight PANI can be generated that is well-dispersed in water using standard synthesis procedures involving aniline monomer, surfactant, and controlled addition of an oxidant. Yield can be very high and unreacted oxidant removed by washing with water, ensuring no further PANI growth. PANI-probe association occurs spontaneously upon mixture, and complex formation is enhanced by mild UV exposure. Hybridization can be carried out immediately, and the changes in PANI fluorescence assayed following a short incubation. The simplicity of this technology makes it highly accessible to many laboratories.

<table><tbody><tr><td>Aniline&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>A7401-500&amp;nbsp;</td><td>ACS, liquid, refrigerated</td></tr><tr><td>Ammonium peroxydisulfate</td><td>Fisher Scientific&amp;nbsp;</td><td>A682-500&amp;nbsp;</td><td>ACS, crystalline</td></tr><tr><td>Sodium dodecylbenzene sulfonate</td><td>Pfaltz &amp;amp; Bauer&amp;nbsp;</td><td>D56340&amp;nbsp;</td><td>95% solid</td></tr><tr><td>Chloroform</td><td>Fisher Scientific&amp;nbsp;</td><td>MCX 10601&amp;nbsp;</td><td>Liquid</td></tr><tr><td>DNA primers</td><td>MWG operon</td><td>n/a</td><td>custom DNA sequence ~20&amp;nbsp;bps</td></tr><tr><td>Microplate&amp;nbsp;</td><td>USA Scientific&amp;nbsp;</td><td>1402-9800&amp;nbsp;</td><td>96 well, polypropylene as it is unreactive to chloroform</td></tr><tr><td>Microplate Adhesive Film</td><td>USA Scientific&amp;nbsp;</td><td>2920-0000&amp;nbsp;</td><td>Reduces well-to-well contamination, sample spillage and evaporation</td></tr><tr><td>Microscope Cover Glass</td><td>Fisher Scientific&amp;nbsp;</td><td>12-544-D&amp;nbsp;</td><td>PANI coated on UV irradiated cover glass</td></tr><tr><td>UV crosslinker&amp;nbsp;</td><td>UVP&amp;nbsp;</td><td>HL-2000&amp;nbsp;</td><td>Energy: X100 &amp;mu;J/cm&lt;sup&gt;2&lt;/sup&gt;; Time: 2 min</td></tr><tr><td>Hybridization Oven</td><td>VWR</td><td>01014705 T</td><td>Temperature: 400&amp;nbsp;&amp;#176;C; with rocking for 15 min</td></tr><tr><td>Glass Apparatus&amp;nbsp;</td><td>Fisher Scientific</td><td></td><td>Three necked round bottom flask for reaction; dropping funnel, stoppers, condenser, separating funnel</td></tr><tr><td>Microscope</td><td>Leica Microsystems&amp;nbsp;</td><td>Leica IMC S80</td><td>Magnification 20X; Pseudo color 536 nm; Exposure 86 msec; Gain 1.0x; Gamma 1.6</td></tr><tr><td>Microplate Reader</td><td>Molecular Devices&amp;nbsp;</td><td>89429-536</td><td></td></tr></tbody></table>

a polyaniline-based sensor of nucleic acids

Detection of nucleic acids is at the center of diagnostic technologies used in research and the clinic. Standard approaches used in these technologies rely on enzymatic modification that can introduce bias and artifacts. A critical element of next generation detection platforms will be direct molecular sensing, thereby avoiding a need for amplification or labels. Advanced nanomaterials may provide the suitable chemical modalities to realize label-free sensors. Conjugated polymers are ideal for biological sensing, possessing properties compatible with biomolecules and exhibit high sensitivity to localized environmental changes. In this article, a method is presented for detecting nucleic acids using the electroconductive polymer polyaniline. Simple DNA "probe" oligonucleotides complementary to target nucleic acids are attached electrostatically to the polymer, creating a sensor system that can differentiate single nucleotide differences in target molecules. Outside the specific and unbiased nature of this technology, it is highly cost effective.

图2A捕捉在聚合过程中， 即 ，APS添加前的开始时的反应设置。胶束的形成是在胶束界面反应中的初始步骤过程聚苯胺合成发生。 图2B示出了5分钟后乳状溶液。 30分钟的APS加到在反应后变成浅棕色颜色。 图2C示出了具有低聚物的形成相关的颜色的变化。 图2D示出了4小时后深棕色，表明短链聚苯胺的高浓度，具有一致的一个非常缓慢的反应。最后48小时后，可观察到高的分子量聚苯胺的深绿色溶液被分散在氯仿-水（ 图2E）。 图2F示出了良好分散的，均匀的聚苯胺在分液漏斗中，没有相分离溶液。 图2）或滴涂膜的表面上（ 图3）。聚苯胺薄膜的基础荧光相对较低（ 图3A）。探针DNA是在薄膜中加入，并在室温下干燥。当预期的DNA聚苯胺复合物的形式，在荧光的激烈的增加，可以观察到的（ 图3B）。从带负电荷的DNA的电子流入导致聚苯胺分子较高的电子密度，导致荧光增加。探针-靶双链体的杂交诱导解离后，聚苯胺荧光返回到基底强度（ 图3C）。离解是通过在杂交时的DNA骨架的妥协聚苯胺 - 探针相互作用的变化而引起的。传感核酸用这种技术也可在乳液（ 图3D）直接进行。使用该乳液的益处<html>基于系统和微孔板检测仪仪器的兼容性。这允许聚苯胺荧光的高度精确的测量，并避免了由在膜包衣凹凸困难。乳液格式基于聚苯胺传感器是非常具体的。引入在目标寡一个错配（毫米）的未能恢复基底聚苯胺荧光（ 图3D）。 <img alt="图1" src="/files/ftp_upload/54590/54590fig1.jpg" /> 图1. 紫外线照射调节探头附件从聚苯胺增加目标脱离核酸结果聚苯胺荧光。的杂交和聚合物基础荧光逆转。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54590/54590fig1large.jpg" target="_blank">请点击此处，查看这个数字的放大版。</a> 尔斯/ ftp_upload / 54590 / 54590fig2.jpg"/&gt; 图2. 合成处理的聚苯胺（A）聚合反应的开始。 （B）的5分钟后，当缓慢加入APS。 （C）30分钟后，APS加法。 （D）1小时后。 （E）经过4小时。 （F）后反应48小时绿色环保产品。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54590/54590fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54590/54590fig3.jpg" /> 图3.聚苯胺荧光的使用，以检测核酸杂交。通过滴涂层上硼硅玻璃在（A）的聚苯胺薄膜，随后在40℃干燥48小时。 （B）探针固定后聚苯胺的荧光。 （C）以下用聚苯胺固定化的探针的靶DNA杂交。在之前与任何互补或错配（毫米）的目标寡核苷酸杂交后聚苯胺乳液为500nm（D）荧光。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54590/54590fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Polyaniline-based Sensor of Nucleic Acids at JoVE.com

A Polyaniline-based Sensor of Nucleic Acids

Nucleic acids are common analytes for assessing biological systems; however, bias from enzymatic manipulation can cause concern. Here a method is described for label-free detection of nucleic acids using polyaniline. This sensitive, cost-effective sensor technology can distinguish single nucleotide differences between molecules.

核酸的聚苯胺传感器

Detection of nucleic acids is at the center of diagnostic technologies used in research and the clinic. Standard approaches used in these technologies ...

a-polyaniline-based-sensor-of-nucleic-acids

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.0K 次观看.  University of Southern Mississippi. Nucleic acids are common analytes for assessing biological systems; however, bias from enzymatic manipulation can cause concern. Here a method is described for label-free detection of nucleic acids using polyaniline. This sensitive, cost-effective sensor technology can distinguish single nucleotide differences between molecules.

视频: A Polyaniline-based Sensor of Nucleic Acids

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

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. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

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. ...

科研

生物工程

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of &#8220;point of care&#8221; techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician&#8217;s office and do so in a timely manner, would revolutionize treatment protocols.1 As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules2, methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocene-cysteine system that can be immobilized on a gold surface.

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

合成方法通过固相树脂为基础的方法不对称二茂铁产生的生物共轭系统

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

化学

Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the rapid growing of congeners. DNA aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward highly hydrophobic small molecule targets, such as PAEs, is rarely reported. This work describes a bead-based method designed to select group-specific DNA aptamers to PAEs. The amino group functionalized dibutyl phthalate (DBP-NH2) as the anchor target was synthesized and immobilized on the epoxy-activated agarose beads, allowing the display of the phthalic ester group at the surface of the immobilization matrix, and therefore the selection of the group-specific binders. We determined the dissociation constants of the aptamer candidates by quantitative polymerization chain reaction coupled with magnetic separation. The relative affinities and selectivity of the aptamers to other PAEs were determined by the competitive assays, where the aptamer candidates were pre-bounded to the DBP-NH2 attached magnetic beads and released to the supernatant upon incubation with the tested PAEs or other potential interfering substances. The competitive assay was applied because it provided a facile affinity comparison among PAEs that had no functional groups for surface immobilization. Finally, we demonstrated the fabrication of an electrochemical aptasensor and used it for ultrasensitive and selective detection of bis(2-ethylhexyl) phthalate. This protocol provides insights for the aptamer discovery of other hydrophobic small molecules.

A protocol for the in vitro selection and characterization of group-specific phthalic acid ester- binding DNA aptamers is presented. The application of the selected aptamer in an electrochemical aptasensor is also included.

邻苯二甲酸酯结合 DNA 核酸适体的选择、表征及其在电化学 Aptasensor 中的应用

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

微流体为基础的电化学生物芯片的无标记的DNA杂交分析

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

工程分子识别与单壁碳纳米管仿生聚合物

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used &#946;-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP &#946;-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the &#946;-lactamase activity. This suggests that the nanobody and &#946;-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid &#946;-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

In this work, we report a new method to study protein-protein interactions using a conductimetric biosensor based on the hybrid &#946;-lactamase technology. This method relies on release of protons upon hydrolysis of &#946;-lactams.

基于β-酶的电导生物传感器检测技术在分子间相互作用中的应用

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

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 ...

<meta charset="utf8"></head><body>用于鉴别性 RNA 和 DNA 分析的精密 DNAzyme 纳米机器

DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their functions include tight analyte binding, highly selective analyte recognition, fluorescent signal amplification by multiple catalytic cleavages of a fluorogenic reporter substrate, and fluorogenic substrate attraction for an increase in sensor response. Functional units are attached to a common DNA scaffold for their cooperative action. The RNA-cleaving 10-23 DNMs feature&#160;improved sensitivity in comparison with non-catalytic hybridization probes. The stability of the DNM and the increased chances of substrate recognition are provided&#160;by a double-stranded DNA fragment,&#160;a tile. DNM can differentiate two analytes with a single nucleotide difference in a folded RNA and a double-stranded DNA and detect analytes at concentrations ~1000 times lower than other protein-free hybridization probes. This article presents the concept behind the diagnostic potential of DNA-nanomachine activity and overviews DNM design, assembly, and application&#160;in nucleic acid detection assays.

<meta charset="utf8"></head><body>作者聚焦:DNA 纳米传感器的进步 – 解决分子检测中的灵敏度和选择性挑战

The DNAzyme-based nanomachines can be used for highly selective and sensitive detection of nucleic acids. This article describes a detailed protocol for the design of DNAzyme-based nanomachines with a 10-23 core using free software and their application in the detection of an Epstein-Barr virus fragment as an example.

DNAzyme 10-23 - 用于核酸识别的基于纳米机器

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their ...

核酸的聚苯胺传感器

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此视频中的章节

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

合成方法通过固相树脂为基础的方法不对称二茂铁产生的生物共轭系统

邻苯二甲酸酯结合 DNA 核酸适体的选择、表征及其在电化学 Aptasensor 中的应用

微流体为基础的电化学生物芯片的无标记的DNA杂交分析

工程分子识别与单壁碳纳米管仿生聚合物

基于β-酶的电导生物传感器检测技术在分子间相互作用中的应用

电化学 DNA 生物传感器检测食源性致病菌的研究进展

毛细管阵列中多核酸的视觉检测

DNAzyme 10-23 - 用于核酸识别的基于纳米机器

DNAzyme 10-23 - 用于核酸识别的基于纳米机器