本文报道的工作的支持补助金奥本大学AUDFS的和美国空军CRADA 07-277-60MDG-01。作者在这篇文章中所表达的意见，不反映美国的空军，国防部，或美国政府的官方政策或立场。

1。设置舞台<ol><li>获取型菌株S。金黄色葡萄球菌 ATCC 12600，S.金黄色葡萄球菌 ATCC 27690和枯草芽孢杆菌 ATCC 6051。耐甲氧西林菌株的金黄色葡萄球菌 - MRSA1，MRSA，MRSA，MRSA MRSA MRSA 5 13 26 34 45，MRSA，B.福氏志贺氏菌，鼠伤寒沙门氏菌 LT2， 炭疽斯特恩耶尔森氏菌enterocolotica，奇异变形杆菌，肺炎克雷伯菌 13882;裂解噬菌体12600。 </li><li>获得PBP 2a的抗体乳胶颗粒。 </li><li>准备NZY的介质描述21。 </li><li>获得的磷酸盐缓冲盐溶液（PBS）中。 </li><li>准备去离子水为LB膜（LB）单层沉积亚相。 </li></ol> 2。噬菌体繁殖和滴定<ol><li>孵育5毫升过夜培养S.金黄色葡萄球菌 ATCC 12600（3.6×10 8菌落形成单位（CFU）/毫升）与500μl的噬菌体（3×10 9 &lt;/ SUP&gt; PFU /毫升）500毫升的NZY介质在2升长颈瓶摇床培养箱中37℃过夜。 </li><li>离心文化在4,424 XG 10分钟，在4°C。 </li><li>再离心上清在4℃下11,325 xg离心10分钟</li><li>通过0.22μm微孔过滤器过滤上清液。 </li><li>离心滤液在75395×g离心1.5小时。 </li><li>沉淀溶解于100μl蒸馏水中。 </li><li>准备的噬菌体悬浮液的10倍稀释液，在NZY培养基中。 </li><li>板1毫升过夜（ON）的S.文化金黄色葡萄球菌 ATCC 12600到每个2板NZY琼脂，以确保所有的表面被水覆盖。取出多余的文化和30分钟，让表面干燥。 </li><li>现货板表面上适当的噬菌体稀释样品（10微升），并在37℃孵育18-24小时。 </li><li>检查斑块的形成，并计算噬菌体滴度。噬菌体滴度（T）的p的数目的基础上估计的噬菌体悬浮液和稀释倍数（F）的10微升体积（v）条形成的laques（N）的数目。滴度计算公式计算:T = N /（VXF），例如，对于空斑数75在稀释10 -7和10微升的体积（10×10 -3毫升），滴定度等于75 / （10×10 -3×10 -7）= 7.5×10 10噬斑形成单位（PFU）每ml悬液。 </li></ol> 3。黄金固定噬菌体的<ol><li>清洁金涂层的石英片（60毫米2）通过等离子蚀刻10分钟，然后在氩气中的灭菌6小时，在UV光下在无菌柜。 </li><li>加入50μl的1×10 9 PFU / ml的噬菌体悬浮液在无菌培养皿中的每一块在金表面，然后在室温下在潮湿的腔室中孵育过夜。 </li><li>删除及滴度剩下的噬菌体悬液。 </li><li>结合的噬菌体件，5倍，用PBS洗涤除去未结合的噬菌体。 </li></ol> 4。在干燥的表面固定化噬菌体侵染测试<ol><li> 金黄色葡萄球菌 ATCC 12600 O / N的文化传播到NZY琼脂平板上，并让干燥。 </li><li>将黄金固定噬菌体到的板面。 </li><li>观察一个区域的温育12小时后溶解在37℃下，该值指示传染性。 </li><li>使用黄金覆盖件在对照实验中，没有噬菌体。 </li></ol> 5。结合的噬菌体液体催化活性测试<ol><li>添加ON文化S.黄色葡萄球菌 ATCC 12600到10毫升的NZY在每一个四个300毫升侧臂的烧瓶中（6×10 6 CFU / ml的每个烧瓶中）。 </li><li>加在两个烧瓶2×10 6 PFU / ml的游离噬菌体。 </li><li>使用作为对照组的另外两个烧瓶。 </li><li>监控到的时间过程中，S。 570分钟由金黄色葡萄球菌细胞裂解测量光密度（OD 600）在30分钟的时间间隔为所有烧瓶。</li><li>以最终在24小时测量。 </li><li>重复步骤5.1-5.5，但与对照烧瓶没有噬菌体结合的噬菌体，而不是免费的噬菌体，使用金币和金币。 </li></ol> 6。孵化过程中结合的噬菌体亏损<ol><li>添加50μl的1×10 9 PFU / ml的噬菌体无菌金片的表面上，在培养皿中，置于潮湿的腔，在室温下过夜。 </li><li>取下悬挂与未结合的噬菌体的黄金表面和滴度。 </li><li>洗涤5次，用PBS，地点在1毫升的NZY溶液摇床培养箱中在15毫升管结​​合的噬菌体金片在37℃下8小时。 </li><li>确定如2所述的分离的噬菌体的滴度。 </li></ol> 7。噬菌体球体准备<ol><li>用1毫升的小瓶分光级的氯仿的等体积的结合库存噬菌体12600悬浮液（10 11 PFU /毫升）的400微升在室温下。 </li><li>轻轻涡旋噬菌体氯仿暂停一分钟持续时间超过5-6次（间隔5秒）。 </li><li>宠物的悬浮液稳定，持续30秒，然后移液管顶端相，含有单层制备吸球粒。 </li><li>径的球状体的悬浮液，用于传输和扫描电子显微镜几微升。 </li><li>使用剩余的球体悬浮生物传感器制造业。 </li></ol> 8。生物传感器的制备<ol><li>清洁QCM-D传感器在氩气等离子体刻蚀10分钟哈里克PDC-32G等离子清洗机。 </li><li>用己烷冲洗传感器，以消除任何有机杂质。 </li><li>准备和清理的电影中所描述的22平衡低谷。 </li><li>填写LB槽，一个副相解决方案和清洁槽屏障和稳定的低谷，在20±0.1°C </li><li>传播300微升等分的噬菌体12600在水suspensi（10 PFU / 11毫升）到LB副相。 </li><li>准备噬菌体单层到LB副相溶液通过允许300微升噬菌体12600水悬浮液（10 PFU / 11毫升），运行等分可湿性玻璃棒部分浸没入亚相22,23倾斜。 </li><li>稳定10分钟后，制备的单分子层，然后将压缩的速度为30毫米/分钟（45厘米/分），直到一个恒定的压力达到19牛顿/米。 </li><li>执行垂直的膜沉积于垂直定位的QCM-D传感器的速率为4.5毫米/分钟，，依次浸渍传感器中的单层七次。电子显微镜观察沉积的噬菌体单层前暴露于细菌样本显示层是连续的，均匀的和均匀的（数据未示出）。 </li><li>重复步骤4.5-4.8噬菌体球体悬浮准备3。 </li><li> MRSA的检测，电子制造噬菌体生物传感器显微镜和椭圆。 </li></ol> 9。生物传感器的测试，歧视耐甲氧西林敏感的葡萄球菌<ol><li>在这个协议中，生物传感器与未改性和改性的噬菌体和噬菌体球体的准备。采用石英晶体微有四个传感器流量室是用来监测结合的细菌的噬菌体固定在QCM传感器表面。 PBP 2a的抗体乳胶珠利用区分耐甲氧西林（MRSA）和敏感的葡萄球菌。在流模式（50微升/分钟）进行所有的测量都在5 MHz。 </li><li>建立一个基线水（〜30分钟）的QCM传感器的共振频率。 </li><li>绘制活甲氧西林敏感的金黄色葡萄球菌的细菌细胞在水（10 9 CFU / ml）的通过测试细胞的悬浮液中。 </li><li>不断监测变化的传感器共振频率和耗能第一泛音，USI使用Q-Soft软件。 </li><li>当传感器的谐振频率和耗散的变化达到饱和电平，加到PBP抗体共轭乳胶珠（6.77×10 10珠/毫升）的悬浮液的流动过程中连续监测频率和耗散。 </li><li>重复步骤9.2-9.5耐甲氧西林金黄色葡萄球菌（MRSA）的细菌细胞。 </li><li>通过大规模的变化，由于细菌结合Sauerbrey方程24,25ΔF=ΔMXN / C，其中C是质量灵敏度常数（C = 17.7纳克为x cm -2 X赫兹-1在5 MHz定义的频率变化） 中，m是质量，n是谐波数（n = 1时）。 </li><li>定义了一个能量耗散的变化（ΔD）从记录的指数衰减振荡（频率和振幅衰减，这使得定量消耗的能量和存储在一个周期内的振荡ê 消散 和电子存储 ，分别为:ΔD =Ë 消散 / 2 存储 πE。 ΔD是测量消散单元（DU），DU = 10 -6相对单位）。 </li></ol>

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G. M., Tufenkji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Capture+Efficiency+and+Antimicrobial+Activity+of+Phage-Functionalized+Model+Surfaces.">Bacterial Capture Efficiency and Antimicrobial Activity of Phage-Functionalized Model Surfaces.</a> Langmuir. 27, 5472-5480 (2011).</li><li>Schofield, D. A., Molineux, I. J., Westwater, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent'+Reporter+Phage+for+the+Detection+of+Category+A+Bacterial+Pathogens.">Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens.</a> J. Vis. Exp. (53), e2740 (2011).</li><li>Voinova, M. V., Jonson, M., Kasemo, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Missing+mass"+effect+in+biosensor's+QCM+applications.">Missing mass" effect in biosensor's QCM applications.</a> Biosens. Bioelectron. 17, 835-841 (2002).</li><li>Gervals, L., Gel, 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=Immobilization+of+biotinylated+bacteriophages+on+biosensor+surfaces.">Immobilization of biotinylated bacteriophages on biosensor surfaces.</a> Sensors and Actuators. 125, 615-621 (2007).</li><li>Nanduri, V., Sorokulova, I. B., Samoylov, A. M., Simonian, A. L., Petrenko, V. A., Vodyanoy, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+as+a+molecular+recognition+element+in+biosensors+immobilized+by+physical+adsorption.">Phage as a molecular recognition element in biosensors immobilized by physical adsorption.</a> Biosens. Bioelectron. 22, 986-992 (2007).</li><li>Sorokulova, I., Watt, 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=Natural+biopolymer+for+preservation+of+microorganisms+during+sampling+and+storage.">Natural biopolymer for preservation of microorganisms during sampling and storage.</a> J. Microbiol. Methods. 88, 140-146 (2012).</li><li>Sanders, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+Laboratory+Techniques:+Plating+Methods.">Aseptic Laboratory Techniques: Plating Methods.</a> J. Vis. Exp. (63), e3064 (2012).</li></ol>

作者宣称，他们有没有竞争经济利益。

这是众所周知的，细菌病原体的生物传感器探针28可以用作噬菌体。这是表明在这项工作中，可以被用来解决的老问题:快速鉴别抗生素抗性及敏感菌株的噬菌体一起与PBP 2a的抗体。 发现，但那些正常的未改性的金黄色葡萄球菌噬菌体细菌检测与QCM装置​​是不适合的，尽管它们绑定细菌。噬菌体尾部是如此之长，声波不能"达到"约束的细菌噬菌体尾结束。这种情况会?...

耐甲氧西林金黄色葡萄球菌已被建议作为必不可少的一个因素，感染及院内爆发4-8。甲氧西林耐药的认可，常见的方式，如纸片扩散法苯唑西林琼脂屏幕测试，或肉汤稀释量身定制的培养条件下，依靠增强表达的阻力。改建包括利用苯唑西林，潜伏期为30或35°C，而不是37℃，并加入氯化钠中的生长培养基。此外，为了正确检测这些类型的技术，一个很长的潜伏期为24小时，而不是16至18小时。快速的技术鉴定耐甲氧西林的灵敏度（&gt; 96％）的水平适当的技术，如维特克GPS-SA卡，快速ATB金黄色葡萄球菌系统，快速迈思肯面板系统包括自动稀释3-11小时后产生的结果9-11。 CRYSTAL MRSA ID系统是一种快速的方法，根据承认生长的影响黄色葡萄球菌的存在下，2％NaCl和4毫克每升苯唑西林具有对氧敏感的荧光传感器。声称灵敏度范围91至100％之间，4小时后孵化12-14。这些表型的方法在其精度是有限的流行株表达异构性的影响。因此，甲氧西林耐药的确认最好的被广泛接受的方法是PCR或DNA杂交mecA基因15。然而，这种技术需要纯化的DNA，是极其敏感的各种混合物（杂质），其中包括细胞碎片16。 此外，这些技术需要很长的时间来执行。策略，以确认mecA基因的产品，蛋白PBP 2a中，可以利用，以确定电阻，并可能更可靠的标准测试技术17。 1,2,18甲氧西林耐药性的金黄色葡萄球菌菌株。在这项工作中，我们提出了一种新的技术，例如在特定的识别和检测MRSA细菌一起构象的MRSA实时确认。对于这个特定的目的S.金黄色葡萄球菌噬菌体结合蛋白单克隆抗体的广泛的主机（包括MRSA菌株）（PBP 2a的）已被使用。 PBP 2a是细胞壁蛋白，它是MRSA的抗生素的电阻率的原因。然而PBP 2a的抗体是不特定为S金黄色葡萄球菌，因为一些其他细菌的抗生素结合蛋白序列相似PBP 2a的19,20。因此在这项工作中，S.金黄色葡萄球菌噬菌体和抗PBP 2a的蛋白已被使用。为了能够开发出一种生物传感器，具体新增Cally MRSA的检测和识别设备已动用两个步骤的行动。最初的步骤使用了S。金黄色葡萄球菌噬菌体单层作为传感器探头，而第二个步骤中使用PBP 2a的特异性抗体。因此，加强一眼便认出S。金黄色葡萄球菌的细菌，其他人会敏感抗生素结合蛋白。当从两个步骤中接收到的信号是正的，则表示的特定检测MRSA。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		 </tr>
		 <tr>
			<td>Phosphate buffered saline (PBS) </td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>P4417 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>spectrophotometric-grade chloroform </td>
			<td>Sigma-Aldrich, 
				St. Louis, MO </td>
			<td>154733 </td>
			<td>(99.8% A.C.S.) </td>
		 </tr>
		 <tr>
			<td>Hexane-Anhydrous </td>
			<td>Sigma-Aldrich, 
				St. Louis, MO</td>
			<td>29609-0</td>
			<td>(95%)</td>
		 </tr>
		 <tr>
			<td>Ethyl Alcohol </td>
			<td>Pharmco products Inc. Brookfield, CT </td>
			<td>64-17-5</td>
			<td>190 Proof</td>
		 </tr>	
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>PBP 2a antibody conjugated latex beads</td>
			<td>Denka Seiken Co., Ltd, Tokyo, Japan</td>
			<td>The MRSA-Screen test</td>
			<td></td>
		 </tr>
		 <tr>
			<td>S. aureus ATCC 12600, S. aureus ATCC 27690 and Bacillus subtilis ATCC 6051 from </td>
			<td>American Type Culture Collection (Manassas, VA);</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>MRSA1, MRSA 2, MRSA 5, MRSA 13, MRSA 26, MRSA 34, MRSA 45, B. anthracis Sterne, Salmonella typhimurium LT2, Shigella flexneri, Yersinia enterocolotica, Proteus mirabilis, Klebsiella pneumoniae 13882; The lytic phage 12600 </td>
			<td>The culture collection of Auburn University, Auburn, AL</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Centrifuge </td>
			<td>Beckman Coulter</td>
			<td>Optima L-90K
				Ultra Centrifuge</td>
			<td></td>
		 </tr>
		 <tr>
			<td>KSV 2200 LB film balance</td>
			<td>KSV Chemicals, Finland</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Light microscope optical system </td>
			<td>CitoViva Technology Inc., Auburn, AL </td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>QCM-D</td>
			<td>Q-Sense AB, V&auml;stra Fr&ouml;lunda, Sweden </td>
			<td>E4</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Scanning electron microscope (SEM)</td>
			<td>JEOL USA Inc., Peabody, MA</td>
			<td>JEOL-7000F SEM </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Transmitting electron microscopy (TEM)</td>
			<td>JEOL USA Inc., Peabody, MA</td>
			<td>JEOL, JEM 2010 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Stericup, Presterilized</td>
			<td>Millipore Corporation, Billerica, MA</td>
			<td>SCGPU05RE</td>
			<td>0.22 &mu;m, GP Express PLUS membrane</td>
		 </tr>
		 <tr>
			<td>Bio-Assay dish</td>
			<td>NUNC A/S, Denmark</td>
			<td>240835</td>
			<td>Dimensions(mm), 245 x 245 x 25</td>
		 </tr>
		 <tr>
			<td>Pipettes</td>
			<td>Gilson, Pipetman, France</td>
			<td>P100, P200, P1000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>C24 Incubator Shaker</td>
			<td>New Brunswick Scientific, CT</td>
			<td>Classic C24</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gold-coated quartz pieces</td>
			<td>Auburn University, AL</td>
			<td></td>
			<td>Homemade</td>
		 </tr>
		 <tr>
			<td>Petri dishes</td>
			<td>Fisher Brand, USA</td>
			<td>0875713</td>
			<td>100 mmX15 mm </td>
		 </tr>
		 <tr>
			<td>SterilGard III Advance</td>
			<td>The Baker Company, ME</td>
			<td>SG403</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Culture Growing Flasks</td>
			<td>Corning Incorporated, NY</td>
			<td>4995</td>
			<td>PYREX 250 ml Erlenmeyer flasks </td>
		 </tr>
		 <tr>
			<td>Optical Spectrometer</td>
			<td>Genesys 20. Thermo Spectronic, USA.</td>
			<td>4001</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Plasma Cleaner</td>
			<td>Harrick Plasma, USA</td>
			<td>PDC-32G</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Millipore water purification system</td>
			<td>Millipore</td>
			<td>Direct-Q</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Imaging Ellipsometer</td>
			<td>Accurion, USA</td>
			<td>nanofilm_ep3se
			</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software </td>
			<td>Q-Sense AB, Sweden</td>
			<td>QSoft, QTools</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

biosensor for detection of antibiotic resistant staphylococcus bacteria

甲结构转化的裂解的噬菌体具有广泛的宿主范围的金黄色葡萄球菌菌株和青霉素结合蛋白（PBP 2a）的抗体的乳胶珠已被用于创建的生物传感器设计（MRSA）和耐甲氧西林敏感（MSSA）S歧视。金黄色葡萄球菌种1,2。裂解噬菌体噬菌体球体已转换成通过用氯仿接口接触。生物传感器表面上已经移到噬菌体球体单层LB膜技术（LB）3。创建的生物传感器已经由耗散跟踪石英晶体微天平（QCM-D），以评估细菌噬菌体的相互作用的研究。细菌的旋转椭球体的相互作用导致降低了谐振频率和两个MRSA和MSSA菌株中耗散的能量增加。细菌绑定后，这些传感器被进一步暴露的青霉素结合蛋白的抗体乳胶珠秒。分析与MRSA的传感器响应PBP 2a的抗体珠;，虽然传感器与MSSA检查没有任何反应。这个实验的区别确定一个明确的耐甲氧西林敏感的学之间的歧视金黄色葡萄球菌菌株。同样绑定和未绑定噬菌体表面上抑制细菌的生长，并在水中悬浮。一旦裂解噬菌体被改变成球体，他们保留自己的强有力的催化活性和大量细菌捕获能力。噬菌体和噬菌体的球状体，可以利用抗生素抗性的微生物进行测试和灭菌。其它应用可能包括利用噬菌体治疗和抗菌表面。

对所有测试菌株的噬菌体展示催化活性金黄色葡萄球菌 ，MRSA菌株，所指示的噬菌体斑点试验。斑块的大小一般从5毫米到15毫米不等。没有对其他测试（ 表1）文化活动被发现。 一个正常的生长的影响ATCC 12600 金黄色葡萄球菌在37℃摇床培养箱在的NZY介质上显示如图1A（曲线标记的空圈）。细菌的数量增加至3.2×10 6〜4.0×10...

Watch this Scientific Journal Video about Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria at JoVE.com

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

溶解性噬菌体生物传感器和抗体有孔玻璃珠，能够区分耐甲氧西林（MRSA）和敏感的金黄色葡萄球菌细菌。在噬菌体一个石英晶体微天平传感器的表面上固定由LB膜法，担任广泛葡萄球菌探针。抗体珠认识MRSA。

生物传感器检测抗生素耐药金黄色葡萄球菌细菌

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

biosensor-for-detection-antibiotic-resistant-staphylococcus

Research

JoVE Journal

Bioengineering

24.0K 次观看.  Auburn University. 溶解性噬菌体生物传感器和抗体有孔玻璃珠，能够区分耐甲氧西林（MRSA）和敏感的金黄色葡萄球菌细菌。在噬菌体一个石英晶体微天平传感器的表面上固定由LB膜法，担任广泛葡萄球菌探针。抗体珠认识MRSA。

视频: Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

荧光检测方法，为微流体液滴平台

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

科研

生物工程

Fabrication of Carbon Nanotube High-Frequency Nanoelectronic Biosensor for Sensing in High Ionic Strength Solutions

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them good candidates for high sensitivity biosensors. When a charged molecule binds to such a sensor surface, it alters the carrier density5 in the sensor, resulting in changes in its DC conductance. However, in an ionic solution a charged surface also attracts counter-ions from the solution, forming an electrical double layer (EDL). This EDL effectively screens off the charge, and in physiologically relevant conditions ~100 millimolar (mM), the characteristic charge screening length (Debye length) is less than a nanometer (nm). Thus, in high ionic strength solutions, charge based (DC) detection is fundamentally impeded6-8.
We overcome charge screening effects by detecting molecular dipoles rather than charges at high frequency, by operating carbon nanotube field effect transistors as high frequency mixers9-11. At high frequencies, the AC drive force can no longer overcome the solution drag and the ions in solution do not have sufficient time to form the EDL. Further, frequency mixing technique allows us to operate at frequencies high enough to overcome ionic screening, and yet detect the sensing signals at lower frequencies11-12. Also, the high transconductance of SWNT transistors provides an internal gain for the sensing signal, which obviates the need for external signal amplifier.
Here, we describe the protocol to (a) fabricate SWNT transistors, (b) functionalize biomolecules to the nanotube13, (c) design and stamp a poly-dimethylsiloxane (PDMS) micro-fluidic chamber14 onto the device, and (d) carry out high frequency sensing in different ionic strength solutions11.

We describe the device fabrication and measurement protocol for carbon nanotube based high frequency biosensors. The high frequency sensing technique mitigates the fundamental ionic (Debye) screening effect and allows nanotube biosensor to be operated in high ionic strength solutions where conventional electronic biosensors fail. Our technology provides a unique platform for point-of-care (POC) electronic biosensors operating in physiologically relevant conditions.

制备碳纳米管高频纳电子生物传感器的传感高离子强度的解决方案

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them ...

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 ...

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

芯片上的压力诱导抗生素易感性测试

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

与FRET流式细胞仪灵敏地检测Proteopathic播种活动

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

医疗器械细菌高通量鉴定驱蚊聚合物

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

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 ...