支持这项研究是由国家过敏和传染病研究所的合作协议奖AI075565（DAH）和温迪和肯红宝石基金在小儿泌尿外科研究卓越。 BMC是在小儿泌尿外科Judith和主席罗伯特·温斯顿。

1。电化学传感器的官能<ol><li>制备巯基化的捕获探针的浓度为0.05μM，300μM1,6 - 己二硫醇（HDT）的10mM的Tris-HCl，1mM EDTA和在黑暗中孵育10分钟，在室温下，pH值8.0，0.3M NaCl的。孵化的巯基化的捕获探针与HDT确保捕获探针的硫醇基团上的减少，导致更一致的结果。 </li><li>氮气流中应用裸金16的传感器阵列芯片（s），持续5秒，以除去水分和/或颗粒物。 </li><li> 6微升的HDT-巯基化的捕获探针混合物应用到所有的16个传感器的传感器阵列的工作电极，并在有盖培养皿中存储的传感器芯片（次），在4℃下过夜。巯捕获探针直接绑定到裸金电极和HDT起到防止装填过量的捕获探针，并让他们在一个伸展的构象，促进杂交与目标。 </li><li>该第二天，洗涤的去离子H 2 O的传感器芯片，具有2〜3秒，干燥氮气流中下持续5秒。 </li><li>适用于6微升的10mM的Tris-HCl，1mM EDTA的0.3M NaCl的pH值为8.0，1 mM的6 - 巯基-1 - 己醇（MCH），所有的16个传感器，并孵育50分钟的工作电极。这和所有随后的传感器芯片孵育在有盖的培养皿中，在室温下进行。 MCH作为封端剂，巯基化的捕获探针或热变形温度是在电极表面上不存在的任何间隙填充。 </li></ol> 2。样品制备<ol><li>吸取1毫升细菌培养的数生长期（OD 600≈0.1）离心管和离心16,000 XG 5分钟。取出培养物上清液。将细菌沉淀可立即处理，或储存于-80℃供以后使用。 </li><li>彻底将细菌沉淀重新悬浮于10μl的1 M NaOH中，通过将移液管尖的机器人TOM离心管吹打向上和向下几次。悬浮液在室温下孵育5分钟。 </li><li>中和细菌裂解液加入50μl的1M磷酸缓冲液，pH值7.2，含2.5％牛血清白蛋白（BSA）和0.25 mM的荧光素改性的探针检测器。孵育10分钟，在室温下中和裂解液。荧光改性探测器探针杂交细菌rRNA的目标分子。 </li></ol> 3。电化学传感器检测<ol><li>从所述传感器芯片与去离子H 2 O洗涤的MCH为2-3秒，干燥氮气流中下，持续5秒。 </li><li>适用于中和细菌裂解液4微升各14个传感器的工作电极上，并孵育15分钟。目标检测探针复合杂交巯基化捕获探针固定。 </li><li>应用4微升1纳米桥寡核苷酸在1M磷酸缓冲液，pH值7.2，含2.5％BSA和0.25＆亩，M荧光改性探测器探头2阳性控制传感器（用于信号正常化）和孵育15分钟。 </li><li>在2-3秒，干燥洗净的传感器芯片与去离子H 2 O下氮气流中，持续5秒。 </li><li>应用在1M磷酸盐缓冲液，pH 7.2，含有0.5％酪蛋白的所有的16个传感器的工作电极上的4微升0.5 U / ml的抗荧光素-HRP孵育15分钟。的抗荧光素-HRP结合到固定化的荧光素改性探测器探头。 </li><li>在2-3秒，干燥洗净的传感器芯片与去离子H 2 O下氮气流中，持续5秒。 </li><li>以及应用的膜，贴纸的表面进入传感器芯片安装在传感器芯片和负载。吸取50μLTMB底物到所有16个传感器和接近传感器芯片安装。 </li><li>获取安培法和循环伏安法测量所有16个传感器使用的Helios芯片读卡器。安培的电流是成比例的汇率的TMB r在传感器表面上的排出（参见图1）。 </li></ol>

<ol>
	<li>Wu, J., Campuzano, S., Halford, C., Haake, D. A., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ternary+Surface+Monolayers+for+Ultrasensitive+(Zeptomole)+Amperometric+Detection+of+Nucleic+Acid+Hybridization+without+Signal+Amplification.">Ternary Surface Monolayers for Ultrasensitive (Zeptomole) Amperometric Detection of Nucleic Acid Hybridization without Signal Amplification.</a> Anal. Chem. 82, 8830-8837 (2010).</li><li>Halford, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Antimicrobial+Susceptibility+Testing+by+Sensitive+Detection+of+Precursor+Ribosomal+RNA+Using+a+Novel+Electrochemical+Biosensing+Platform.">Rapid Antimicrobial Susceptibility Testing by Sensitive Detection of Precursor Ribosomal RNA Using a Novel Electrochemical Biosensing Platform.</a> Antimicrob. Agents Chemother. 56, (2012).</li><li>Campuzano, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ternary+monolayers+as+DNA+recognition+interfaces+for+direct+and+sensitive+electrochemical+detection+in+untreated+clinical+samples.">Ternary monolayers as DNA recognition interfaces for direct and sensitive electrochemical detection in untreated clinical samples.</a> Biosens. Bioelectron. 26, 3577-3584 (2011).</li><li>Gau, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+molecular+analysis+without+nucleic+acid+amplification.">Electrochemical molecular analysis without nucleic acid amplification.</a> Methods. 37, 73-83 (2005).</li><li>Patel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+Specific+Capture+Enhances+Sensitivity+of+Electrochemical+Detection+of+Bacterial+Pathogens.">Target Specific Capture Enhances Sensitivity of Electrochemical Detection of Bacterial Pathogens.</a> J. Clin. Microbiol. 49, 4293-4296 (2011).</li><li>Mastali, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+probe+length+and+target+location+for+electrochemical+detection+of+selected+uropathogens+at+ambient+temperature.">Optimal probe length and target location for electrochemical detection of selected uropathogens at ambient temperature.</a> J. Clin. Microbiol. 46, 2707-2716 (2008).</li><li>Liao, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+electrochemical+DNA+biosensors+for+rapid+molecular+identification+of+uropathogens+in+clinical+urine+specimens.">Use of electrochemical DNA biosensors for rapid molecular identification of uropathogens in clinical urine specimens.</a> J. Clin. Microbiol. 44, 561-570 (2006).</li><li>Liao, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+advanced+electrochemical+DNA+biosensor+for+bacterial+pathogen+detection.">Development of an advanced electrochemical DNA biosensor for bacterial pathogen detection.</a> J. Mol. Diagn. 9, 158-168 (2007).</li><li>Pedrero, M., Campuzano, S., Pingarron, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroanalytical+sensors+and+devices+for+multiplexed+detection+of+foodborne+pathogen+microorganisms.">Electroanalytical sensors and devices for multiplexed detection of foodborne pathogen microorganisms.</a> Sensors (Basel). 9, 5503-5520 (2009).</li><li>Kuralay, F., Campuzano, S., Haake, D. A., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+disposable+nucleic+acid+biosensors+for+direct+bioelectronic+detection+in+raw+biological+samples.">Highly sensitive disposable nucleic acid biosensors for direct bioelectronic detection in raw biological samples.</a> Talanta. 85, 1330-1337 (2011).</li><li>Ecker, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ibis+T5000:+a+universal+biosensor+approach+for+microbiology.">Ibis T5000: a universal biosensor approach for microbiology.</a> Nat. Rev. Microbiol. 6, 553-558 (2008).</li><li>Casalta, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+LightCycler+SeptiFast+test+in+the+rapid+etiologic+diagnostic+of+infectious+endocarditis.">Evaluation of the LightCycler SeptiFast test in the rapid etiologic diagnostic of infectious endocarditis.</a> Eur. J. Clin. Microbiol. Infect. Dis. 28, 569-573 (2009).</li></ol>

所有的作家都描述的方法相关专利的发明者。这些专利之一已授权公司Qvella的。 BMC和DAH权益在Qvella公司。 VG是的总统GeneFluidics，电化学传感器阵列芯片的制造商，芯片的贴装，本文中所描述的芯片读卡器。

这里描述的电化学传感器测定使靶核酸的快速检测。的敏感性和特异性取决于靶 - 探针杂交的自由能，而这又取决于捕获和检测器探针的长度和GC含量的一部分。我们通常执行的的杂交步骤在环境温度（约20℃）5，6。然而，在杂交步骤（3.2和3.3）也可以在杂交烘箱中在较高温度下进行，如果芯片被放置在有盖的腔室，其中包含湿润滤纸，以防止蒸发7，8。获得最佳的信号与检测器?...

使用rRNA基因作为目标分子的细菌检测和鉴定了许多优点。 rRNA基因在细菌细胞中的丰度低至每毫升250个细菌提供了一个灵敏度极限，而不需要对目标扩增1。包含独特的物种特异性细菌rRNA基因序列与DNA探针杂交。因此，电化学传感器的阵列可用于识别未知的细菌，其中每个传感器与不同的种属特异性捕获探针官能。应积极控制传感器包括合成的寡核苷酸的目标，“小桥流水”的捕获和检测探针，建立内部校准信号。 电化学传感器有着广泛的基础和转化研究中的应用。例如，这里所描述的测定法被用于精确测量大肠杆菌的效果大肠杆菌生长阶段RRNA和预rRNA基因的拷贝数，这是极大的兴趣，感兴趣的研究人员在细菌生理学2。电化学传感器测定的灵敏度确定的信号噪声比。已探索各种信号放大和降噪方法。我们发现，改善传感器的表面的化学性质，以减少检测器的探头和/或HRP酶的非特异性结合是关键的。混合单层的alkanedithiols mercaptohexanol特别是，已经发现，以减少背景更完全地覆盖在电极表面，同时保持可访问性的捕获探针的目标杂交3。在这些表面化学处理涉及复杂生物样品的分析是特别重要的。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>6-mercapto-1-hexanol (MCH)</td>
 <td>Sigma</td>
 <td>451088</td>
 <td>Store at room temperature</td>
 </tr>
 <tr>
 <td>1,6-hexanedithiol (HDT)</td>
 <td>Sigma</td>
 <td>H-12005</td>
 <td>Store at room temperature</td>
 </tr>
 <tr>
 <td>Thiolated capture probes</td>
 <td>Operon</td>
 <td>N/A</td>
 <td>Store at 100 &mu;M in 0.1x TE at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Fluorescein-modified detector probes</td>
 <td>Operon</td>
 <td>N/A</td>
 <td>Store at 100 &mu;M in 0.1x TE at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Bridging Oligonucleotide</td>
 <td>Operon</td>
 <td>N/A</td>
 <td>Store at 100 &mu;M in 0.1x TE at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Anti-Fluorescein-HRP, Fab fragments</td>
 <td>Roche</td>
 <td>11 426 346 910</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Helios Chip Reader</td>
 <td>GeneFluidics</td>
 <td>GFR-2009</td>
 <td></td>
 </tr>
 <tr>
 <td>Sensor Chip Mount</td>
 <td>GeneFluidics</td>
 <td>GFR-003</td>
 <td></td>
 </tr>
 <tr>
 <td>Film well sticker</td>
 <td>GeneFluidics</td>
 <td></td>
 <td>Shipped with sensor chips</td>
 </tr>
 <tr>
 <td>Bare gold 16-sensor array chips </td>
 <td>GeneFluidics</td>
 <td>SC1000-16X-B </td>
 <td>Store in 100% N2 at room temperature</td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin </td>
 <td>Sigma</td>
 <td>A7906 </td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>1M Phosphate Buffer, pH 7.2 </td>
 <td></td>
 <td></td>
 <td>0.35M NaH2PO4, 0.65M K2HPO4, adjusted to pH 7.2</td>
 </tr>
 <tr>
 <td>Blocker Casein in PBS </td>
 <td>Pierce</td>
 <td>37528 </td>
 <td>Dilute with an equal volume of 1M Phosphate Buffer, pH 7.2, store at 4 &deg;C</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 1. Reagents and Equipment.</td>
 </tr>
 </tbody>
</table>

bacterial detection &amp; identification using electrochemical sensors

电化学传感器被广泛用于快速，准确地测量血糖，并能适用于各种各样的分析物的检测。电化学传感器操作成一个有用的电信号传导生物识别事件。信号转导的发生了耦合到安培的电极的氧化还原酶的活性。传感器的特异性，使用固有特性的酶，葡萄糖氧化酶的葡萄糖传感器的情况下，或的酶和抗体，该抗体或探针之间的关系的商品。 在这里，我们描述的电化学传感器检测方法直接探测和识别细菌。在每一种情况下，这里所描述的探针DNA的寡核苷酸。此方法是基于夹心杂交的捕获和检测探针与目标的核糖体RNA（rRNA基因）。捕获探针被固定到所述传感器表面，而检测器的探头与步骤horseradish过氧化物酶（HRP）。当基片，例如3,3&#39;，5,5&#39; - 四甲基联苯胺（TMB）被添加到绑定到其表面上的复合物与捕捉目标检测器的电极被氧化，在基板由HRP减少工作电极。此氧化还原循环的结果是穿梭的电子从电极到辣根过氧化物酶的底物，生产电极中电流的流动。

我们描述了同样的结构的双抗体夹心ELISA的电化学检测。在图1中所示，目标核糖体RNA（rRNA基因）杂交，捕获和检测探针开发的催化氧化还原反应的HRP标记的抗荧光素抗体片段结合到探测器上的探针的3&#39;荧光素联动。检测灵敏度的一个重要组成部分是金电极的表面化学性质。我们已经发现，由巯基化的捕获探针，mercaptohexanol，和己二硫醇组成的三元单层显着提高了信号-噪声比通过减...

Watch this Scientific Journal Video about Bacterial Detection & Identification Using Electrochemical Sensors at JoVE.com

Bacterial Detection &amp; Identification Using Electrochemical Sensors

我们描述了一种电化学传感器的测定方法，细菌的快速检测和识别。的测定法涉及官能化的传感器阵列与DNA的核糖体RNA（rRNA基因）的种属特异性序列的寡核苷酸捕捉探针。目标rRNA基因的捕获探针和辣根过氧化物酶相连的DNA寡核苷酸检测探针的夹心杂交产生可测量的安培电流。

使用电化学传感器的细菌检测和鉴定

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

bacterial-detection-identification-using-electrochemical-sensors

Research

JoVE Journal

Bioengineering

28.1K 次观看.  Veterans Affairs Greater Los Angeles Healthcare System. 我们描述了一种电化学传感器的测定方法，细菌的快速检测和识别。的测定法涉及官能化的传感器阵列与DNA的核糖体RNA（rRNA基因）的种属特异性序列的寡核苷酸捕捉探针。目标rRNA基因的捕获探针和辣根过氧化物酶相连的DNA寡核苷酸检测探针的夹心杂交产生可测量的安培电流。

视频: Bacterial Detection & Identification Using Electrochemical Sensors

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for sample pretreatment. The method was first described by Fenn and colleagues1 and has been applied to the detection of illicit drugs2 and explosives3-4, the characterization of skin volatiles5, and the analysis of breath6-7.
SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.
We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization8. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.8 Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

Secondary electrospray ionization mass spectrometry (SESI-MS) enables the detection of volatile organic compounds (VOCs) without the need for any sample pretreatment. This protocol provides instructions for the rapid (within minutes) characterization of bacterial VOCs using SESI-MS.

使用辅助电喷雾质谱（SESI - MS）表征细菌挥发

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

科研

生物工程

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. ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

复透皮电化学传感的空心微针为基础的传感器

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

阻隔组织破坏与有机电化学晶体管传感

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

Glutamine Flux Imaging Using Genetically Encoded Sensors

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories.
One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed.

This article will demonstrate how to monitor glutamine dynamics in live cells using FRET. Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed for genetically encoded glutamine sensors.

谷氨酰胺通量成像使用基因编码的传感器

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for ...

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 ...

Flying Insect Detection and Classification with Inexpensive Sensors

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow for the development of many useful applications in vector and pest control for both medical and agricultural entomology. Given this, the last sixty years have seen many research efforts devoted to this task. To date, however, none of this research has had a lasting impact. In this work, we show that pseudo-acoustic optical sensors can produce superior data; that additional features, both intrinsic and extrinsic to the insect&#8217;s flight behavior, can be exploited to improve insect classification; that a Bayesian classification approach allows to efficiently learn classification models that are very robust to over-fitting, and a general classification framework allows to easily incorporate arbitrary number of features. We demonstrate the findings with large-scale experiments that dwarf all previous works combined, as measured by the number of insects and the number of species considered.

We proposed a system that uses inexpensive, noninvasive pseudo-acoustic optical sensors to automatically and accurately detect, count, and classify flying insects based on their flying sound.

飞行昆虫检测与分类用便宜的传感器

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

无标记单分子检测使用微型环光学谐振器

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

使用多色荧光检测液滴微流控光纤

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

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, ...