这项研究是由赠款全球前沿项目（2011-0031944）和KRIBB研究计划项目的智能合成生物学中心的支持。

1.生物传感器的制备<ol><li>按照先前建立的协议2 CFP-MBP-YFP-的His6 -构建质粒pET21a（+）。 </li><li>接种5毫升的Luria肉汤（LB）的与大肠杆菌 DE3菌株的单个菌落，并在37°C孵育16小时振荡。 </li><li>传输1毫升的O / N培养物在含有100ml LB和在振荡培养箱中于37℃孵育直到在600nm（OD 600）的光密度达到0.5（约3小时）的500ml烧瓶中。 </li><li>在1,000×g下收获在50ml的锥形管中的细胞通过离心在4℃下20分钟。 </li><li>用50毫升冰冷的蒸馏水（DW）和离心机快速重悬沉淀在每个管中，在1000×g下在4℃下20分钟。 </li><li>通过轻轻摇动直到溶液（电感受态细胞），用10％（V / V）甘油悬浮在50μl冰冷的DW粒料达到的OD 600 </suB&gt; 100。 </li><li>放置电感受态细胞的混合物（50微升细胞以OD的100 600）和质粒pET21a（+），10纳克- CFP-MBP-YFP-的His6在冰冷电穿孔管在电穿孔设备和电穿孔该混合物（18千伏/厘米，25μF）。 </li><li>迅速1毫升SOC培养基添加到反应杯和重悬轻轻细胞，然后回收在37℃下1小时，在15毫升的圆底管轻轻摇动。 </li><li>涂布在含有100微克/毫升氨苄青霉素的LB板中的细胞，并在37℃孵育12小时。 </li><li>使用循环隔离单菌落并接种菌落在10ml含有LB，在37℃100微克/ ml氨苄青霉素的摇动12小时的。 </li><li>将5ml种子培养添加到500毫升的LB含有100μg/ ml氨苄青霉素和孵育培养在37℃振荡培养箱中。 </li><li>添加0.5mM的异丙基β-D-硫代半乳糖苷（IPTG）的外径600到达时0.5孵育在37℃振荡培养箱中培养24小时。 </li><li>离心机的细胞在4500×g的20分钟（4℃），并轻轻取出上清。 </li><li>重新悬浮于5mL的结合缓冲液（20mM的Tris-HCl，pH值8.0，1mM的PMSF，0.5mM的EDTA，和1mM DTT）沉淀。 </li><li>超声处理上有六个10秒连发冰细胞在200-300瓦，下面的每个突发冷却10秒。 </li><li>离心裂解物在10,000× 克30分钟，在4℃以沉淀细胞碎片。转移上清液（可溶性蛋白）到一个新的收集管。 </li><li>为了实现FRET传感器蛋白质的亲和纯化，负载4毫升澄清的细胞裂解物上样到Ni-NTA亲和柱（5毫升体积），用快速蛋白液相层析（FPLC）18执行色谱测定。 </li><li>用洗涤缓冲液五个柱体积洗一次列I（50mM磷酸盐缓冲液，300毫氯化钠，10mM咪唑，pH 7.0）中。 </li><li>重复该洗涤步骤用洗涤缓冲液II五个柱体积（50mM磷酸盐缓冲液，300毫氯化钠，的20mM咪唑，pH 7.0）中。 </li><li>用洗脱缓冲液五个柱体积（50mM磷酸盐缓冲液，300毫氯化钠，500mM咪唑，pH 7.0）中洗脱传感器蛋白。 </li><li>浓缩和脱盐洗脱样品，填充高达20样品和离心机毫升浓缩器（10,000兆瓦膜尺寸），用于在3000×g下10分钟。重新填充用0.8％的磷酸盐缓冲盐水（PBS）浓缩。重复此步骤两次，第一次填充聚光用20ml样品，然后用PBS再填充。 </li><li>回收浓缩和脱盐传感器蛋白并将其储存在-80℃。 </li></ol> 2.使用FRET分析糖含量的测定注:FRET分析仪结构的细节在我们以前的工作7进行了描述。 <ol><li>制备含0.2 0.8％PBS的检测解决方案传感器蛋白质微米。 </li><li>打开FRET分析仪。按"向上"按钮2秒来校准最佳温度。用"UP"和"DOWN"按钮，将温度设置为53℃，并按下"SET"键。 </li><li>对于校准，请同时按住"UP"和"DOWN"按钮2秒。确认LED面板显示"CALIB"，然后按"SET"键。 </li><li>将只含PBS缓冲液进入分析仪的试管架，然后按"SET"键12.5×12.5×45毫米（长×宽×高）的长方体容器（试管）。 </li><li>以仅包含检测溶液（见2.1）不加糖（麦芽糖/蔗糖）更换试管，然后按"SET"键来校准基准。 </li><li>更换含10毫米糖检测解决方案，一个比色皿，并按下"SET"按钮。 </li><li>以确定一个饮料样品中的糖含量，把1毫升的饮料样品中的1.5毫升微量离心管中并离心16,000×g的1分钟。 注意:基于传感器FRET荧光测定具有不需要特殊的预处理样品的样品，因为只有1％（V / V）的优点包括在总体积。但是，我们建议去除可能影响荧光测定（ 例如，细胞，不溶性微粒，脂质，脂肪，或具有自发荧光的任何材料）的任何材料。此外，如果一强酸，强碱，清洁剂（洗涤剂），或乳化剂（乳化剂）的存在以高浓度并可能影响FRET生物传感器的特性，应当使用有机溶剂或除去中和剂。例如，当乳品脂肪和乳化剂选自冷冻点心消除，将样品以16,000×g离心以离心管30分钟，液体between中的底部沉积物和乳品脂肪顶层被提取。然后己烷等量加入，然后离心以15,000×g的30分钟，以消除脂质。 </li><li>用1-ml注射器除去上清液，并通过注射器过滤器（孔径为0.2μm）进行过滤。 </li><li>放置0.1毫升过滤饮料样品中含有0.9毫升PBS中，并涡旋轻轻1.5毫升离心管中。 注:这是正确稀释饮料样品的关键。在这种情况下，进行1000倍稀释，以使糖浓度将落在该装置的动态范围内。我们建议通过参照在饮料的标签的糖含量预先估计目标糖浓度。 </li><li>加入5微升稀释饮料样品（1％，V / V）的含有0.495毫升检测溶液的反应杯中。 </li><li>放置反应杯在FRET分析仪的反应杯保持器和预热样品溶液至53℃。 </li><li>按"SET"键来测量糖含量。 注意可以通过在五百三十五分之四百八十八毫微米7,8-读取比率评价使用配备有用于温度控制的珀尔帖装置的多标记平板读数器或荧光分光光度计FRET的测量。对于蔗糖的检测，请按照1.1的步骤2.12与CSY-LH传感器2。 </li></ol>

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该协议允许饮料样品中的糖含量的快速和有效的量化，使用定做的FRET分析器7在用于FRET传感器的最佳温度。分析器被设计用最近开发的，廉价的405纳米波段紫外LED作为光源和两个光检测器与硅光电二极管。这个装置是更符合成本效益比其它可比的荧光计。该器件显示高检测灵敏度，在最佳的温度范围为FRET传感器测量两个发射波长带（530纳米和480纳米）的比率特别时。它的灵敏度和强度检测各种糖均优于荧光分光光度计装置7。 该协议的主要目标是支持与定制FRET分析仪基于FRET的传感器的广泛适用性。虽然分析仪间接测量通过FRET传感器糖度，我t是清楚的是，设备结合了一些FRET传感器，包括广泛延伸遗传工程配体特异性，模块化设计，传感器浓度无关的信号，和亚细胞的小分子的精确定位的好处。 FRET传感器实际用于检测多种小分子，包括离子9，血红素10，以及其他的。此外，超过20种FRET构造可以很容易找到，并通过非营利性的存款AddGene 11篇。 尽管FRET分析仪的适用性广，有与该装置的动作的两个主要问题。首先，由于该装置的操作比较简单，样品的预处理是影响检测的质量，除了在设备发生故障的情况下的关键步骤。在这个协议中，一个步骤（样品稀释）足以处理该显然透明液体样品和不含不溶杂色克莱斯。然而，其他的样品，可能需要额外的处理以除去不溶性材料，例如蜂窝式或脂质组分。可以影响的FRET信号的任何自身荧光颗粒应当也被除去，如所指出以下步骤2.7。其次，质量控制和连接与医院信息系统的接口需要与所有类型的床边血糖检测（POCT）工具12来加以解决。由于FRET分析仪的信号质量在很大程度上取决于FRET传感器的质量和在预处理步骤，需要定期质量控制检查，以确保测量保持定期质量控制数据分析的标准信号的范围内。该FRET传感器的稳定性和贮存期，这两者都是用于进一步可靠的应用必需的，应在质量控制检查进行调查。建立指导方针和发展适当的软件也可以解决的连接限制。当前版本FRET分析仪均配备了RS232连接远程命令行控制，但无线通信可能是分析仪的下一个版本，这将对医院信息系统改进的界面的功能。 然而，FRET传感器已经专为底物特异性，一种方法，通常允许更广泛的特异性2。因此，FRET信号可能遇到来自其它成分，包括其他类型的商业饮料糖的意外干扰。进一步的研究应该探索式传感器如何FRET各种糖混合物应对精确量化适量白糖。与生产饮料的公司合作将有助于确认含糖量，以校准FRET分析仪。 可以预期，与各种FRET传感器所提出的便携FRET设备将在POCT应用中使用。 POCT用于评估妊娠，血液血糖水平，生物标记物的蛋白质，传染性细菌，传染性病毒。 POCT方法具有快速的周转时间，并且通常表现出由于少数的处理步骤的低错误率。这些都是POCT在中心实验室检测方法的重要的优点。手持式便携POCT设备，如本文中所描述的装置中，已经吸引了越来越多，因为它们在食物的评估和血糖监测潜在的应用的关注。特别是，在糖尿病患者的血液样本的血糖监测需要一个快速，准确，和具有成本效益的POCT方法13。埃姆斯研究团队开发的第一血糖试纸后，在1965年（使用含有葡萄糖氧化酶条），几种技术都提出了血糖监测的目的12。 FRET的分析仪也可用于检测血液样品中的葡萄糖的血液和周质葡萄糖结合protei适当的预处理N（MglB）14基于FRET蛋白质。 需要对食品质量评估的简单，快速的方法。含糖饮料的消耗与多种疾病和综合症，如在儿童期15体重指数增加，小儿肥胖16和中风17的风险有关。理解这种连接就必须在饮料的糖组分的精确测量。因此，饮料的葡萄糖和果糖的浓度是感兴趣的涉及人体健康的科学家。这个协议说明了FRET分析仪具有最佳的温度控制的高度敏感的性能。该装置可与各种FRET传感器可用于检测各种小molecules-包括葡萄糖和果糖14,15。我们的便携式和可再充电装置，其具有10的电池寿命 - 20小时，这取决于加热协议，适用于POCT。其简单的操作方案MAKES设备易于使用，省去了复杂的工作人员培训。随着技术的改进，包括减少设备规模，预处理步骤最小化，并为现场使用实际要求鉴定，该设备将促进小规模的实验室环境基于FRET的研究开发。

荧光共振能量转移（FRET）已被广泛地用作生物统计传感器来检测小分子如糖，钙离子，氨基酸1-4。 FRET生物传感器含有荧光蛋白，青色荧光蛋白（国家重点计划）和黄色荧光蛋白（YFPs），其稠合到周质结合蛋白（的PBPs）的两端。糖类结合到位于FRET传感器的中间的PBPs，从而导致其随后改变在的PBPs的一端的两个荧光蛋白的距离和跃迁偶极取向传感器的结构变化。这种改变，通过测量EYFP（530纳米），ECFP（480纳米）的发光波长的比率使得能够糖含量的定量分析。由于高灵敏度，特异性，实时监测能力，和FRET生物传感器的响应时间快，这些传感器被广泛应用于环境，工业和医疗应用5。此外，比率式使用FRET生物传感器etric测量具有重要的实际的好处，因为它可以用来测量复杂生物样品，其中所述传感器的浓度不能被容易地控制和背景荧光总是存在于组件。 尽管基于FRET的传感器，用于定量可视化的这些优点，不完整的域名运动转移到荧光蛋白微小的结构性变化产生固有的弱信号强度。这个弱信号限制基于FRET的传感器，用于在体外或体内分析6的应用程序。因此，大多数FRET生物传感器需要使用昂贵的，高度敏感的设备。先前，我们开发了一种廉价和便携式FRET分析仪类似于现有荧光分析仪7的功能。在该装置中，廉价的405纳米波段紫外发光二极管（LED）作为光源，使第激发Ë荧光信号，更换昂贵的灯泡或激光。分析仪的检测系统的散热荧光信号高效率地聚焦到两个光电检测器与硅光电二极管。在最近的研究中，我们发现，在50检测温度的优化- 55°C可能显著放大比例FRET信号8。这个特定的温度信号增强，与定做的FRET分析仪一起使得能够快速且高灵敏度更一般诊断应用中的使用的FRET传感器。 在这个协议中，我们通过量化市售饮料的糖含量表现出最佳的FRET的温度条件下的FRET分析仪的通用性。这个协议提供了FRET设备操作的细节，以及传感器和样品制备的简要说明。我们预计，该报告将推动便携式的潜在应用分析器在小规模的实验室环境，并提供与基于FRET的生物传感器的廉价的现场诊断装置的进一步发展奠定了基础。

<table><tbody><tr><td>LB</td><td>BD</td><td>#244620</td><td></td></tr><tr><td>isopropyl &amp;beta;-D-thiogalactoside (IPTG)</td><td>Sigma</td><td>I6758</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma</td><td>A9518</td><td></td></tr><tr><td>Tri-HCl</td><td>Bioneer</td><td>C-9006-1</td><td></td></tr><tr><td>PMSF</td><td>Sigma</td><td>78830</td><td></td></tr><tr><td>EDTA</td><td>Bioneer</td><td>C-9007</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>D0632</td><td></td></tr><tr><td>NaCl</td><td>Junsei</td><td>19015-0350</td><td></td></tr><tr><td>phosphate-buffered saline (PBS)</td><td>Gibco</td><td>70011-044</td><td>0.8% NaCl, 0.02% KCl, 0.0144% Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 0.024% KH&lt;sub&gt;2&lt;/sub&gt;OP&lt;sub&gt;4, &lt;/sub&gt;pH 7.4</td></tr><tr><td>SOC</td><td></td><td></td><td>2% tryptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MGCl&lt;sub&gt;2&lt;/sub&gt;, 20 mM Glucose</td></tr><tr><td>Resource Q</td><td>Amersham Biosciences</td><td>17-1177-01</td><td>6 &amp;times; 30 mm anion-exchange chromatography column&amp;nbsp;</td></tr><tr><td>HisTrap HP1</td><td>Amersham Biosciences</td><td>29-0510-21</td><td></td></tr><tr><td>Quartz cuvette</td><td>Sigma</td><td>Z802875</td><td></td></tr><tr><td>AK&amp;Auml;KTAFPLC</td><td>Amersham Biosciences</td><td>18-1900-26</td><td>a fast protein liquid chromatography (FPLC)</td></tr><tr><td>Cary Eclipse</td><td>VarianInc</td><td></td><td>a fluorescence spectrophotometer</td></tr><tr><td>VICTOR&amp;nbsp;&amp;nbsp;</td><td>PerkinElmer</td><td>2030-0050</td><td>a multilabel plate reader</td></tr><tr><td>&lt;i&gt;E. coli&lt;/i&gt; JM109 (DE3)</td><td>Promega</td><td></td><td>Electrocompetent cells</td></tr><tr><td>A (Beverage)</td><td>Korea Yakult Co. (Korea)</td><td>Birak</td><td>Fermented drinks</td></tr><tr><td>B (Beverage)</td><td>Lotte Foods (Korea)</td><td>Epro</td><td>Soft drink</td></tr><tr><td>C (Beverage)</td><td>Lotte Foods (Korea)</td><td>Getoray</td><td>Sports drink</td></tr></tbody></table>

highly sensitive and rapid fluorescence detection with a portable fret analyzer

在荧光共振能量转移最近的改进（FRET）传感器已经使它们的使用，以检测各种小分子，包括离子和氨基酸。然而，FRET传感器的先天弱信号强度，以防止在各个领域的应用并使得使用昂贵的，高端的荧光计必要的一个重大挑战。先前，我们构建可以特异性测量两个发射波长带（530和480纳米）的比率，以实现高的检测灵敏度具有成本效益的，高性能的FRET分析仪。最近，人们发现FRET传感器与细菌周质结合蛋白检测用在临界温度范围的50最高灵敏度的配体 - 55℃。这份报告描述了使用我们的便携式FRET分析仪具有特定的温度-FRET传感器市售饮料样品评估中含糖量的协议。我们的结果表明，附加的预热FRET的传感器的过程显著增加的FRET比率信号，以使糖含量更精确的测量。定做的FRET分析仪和传感器成功地应用到量化糖含量在三种类型的商业饮料。我们预计，设备的尺寸进一步降低和性能的提高将有利于环境中使用手持式分析仪，其中高端装备不可用。

以执行使用FRET分析糖含量的定量分析，必须建立一个拟合曲线推定从观察的FRET比率目标糖浓度。设r定义CFP的发光强度的比在480nm和在530nm（式1）中产生的YFP的发光强度。 <img alt="式（1）" src="/files/ftp_upload/54144/54144equation1.jpg" />可以通过观察FRET比r，在不同的糖浓度来生成（在53℃的CMY-BII）的FRET生物传感器的剂量-反应曲线。然后该曲线可以表示为S形S形曲线如下: <img alt="公式（2）" src="/files/ftp_upload/54144/54144equation2.jpg" />r max和R 最小值代表分别为0的糖浓度和饱和（1000μM）中，信号比; X 0表示，在50％的反应糖浓度; p表示的反应，这是接近1或-1的斜率。在本研究中，R 最大 ，R min时 ，X 0和p分别是4.256，2.672，71.779，和1。在模型拟合所用浓度范围为1微米至1000微米。 使用方程1和2，市售的饮料的糖含量是用FRET分析仪定量。两个麦芽糖FRET传感器进行了检查，以测试信号r，取决于各种温度2,8。第一FRET传感器，CMY-0，是由CFP，麦芽糖结合蛋白（MBP），和YFP的一个基本的基于FRET的传感器，其中nØ接头肽。第二传感器，CMY-BII，具有MBP和两个荧光蛋白2之间的丝氨酸-精氨酸接头。 如图1A示，CMY-0不在测定温度50℃以下观察到的，因为有0和1mM麦芽糖浓度之间没有信号差。两者的信号差异FRET传感器分别50和55℃之间最大（ 图1）8。为了量化三种市售饮料的糖含量，产生在53-℃下将CMY-BII传感器的剂量-响应曲线（ 图2A）和三个样品的麦芽糖含量是确定的转换的FRET比率进麦芽糖浓度。 为样品A是由谷物如水稻和大麦，这是重要的麦芽糖来源的，将样品预期含有相对高麦芽糖含量（平均11.892克/ 235毫升）（ <st荣&gt;图2A）。与此相反，样品C是一个运动饮料，有三种饮料中的最低麦芽糖（0.29ml克/ 250毫升）。这些结果表明，FRET的分析仪可以在最佳的温度下使用，以最大限度地提高的糖含量的定量，省去了昂贵的高端FRET的检测装置的需要。 <img alt="图1" src="/files/ftp_upload/54144/54144fig1.jpg" /> 图1.的FRET信号差介于0和1mM麦芽糖使用FRET分析仪在不同温度（A）的CMY-0传感器表明在温度低于50℃，在不同的麦芽糖浓度无信号差异。（B）中的CMY-BII传感器能区分在一个宽的温度范围在0和1毫麦芽糖之间的FRET信号差。 （ - 55℃50）在这两种情况下，该信号差异显着在特定的温度范围内增加。电子商务RROR条表示标准偏差。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54144/54144fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54144/54144fig2.jpg" /> 图2.麦芽糖三市售的饮料。（A）剂量反应曲线量化的内容为CMY-BII（B）三种饮料样品麦芽糖含量进行定量。请注意，"总糖"表示所有的糖（包括麦芽糖）由饮料标签上的饮料制造商报告的数量。误差条表示标准偏差。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54144/54144fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer at JoVE.com

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

这个协议描述了使用定制便携式FRET分析器荧光共振能量转移（FRET）的传感器数据的快速且高灵敏度定量。该装置被用于最大化的检测灵敏度，有利的糖含量的实际和有效的评估的临界温度范围内检测麦芽糖。

高度敏感和快速荧光检测用便携式分析仪FRET

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

highly-sensitive-rapid-fluorescence-detection-with-portable-fret

Nanyang Technological University

Research

JoVE Journal

Biochemistry

9.0K 次观看.  Korea Research Institute of Bioscience and Biotechnology. 这个协议描述了使用定制便携式FRET分析器荧光共振能量转移（FRET）的传感器数据的快速且高灵敏度定量。该装置被用于最大化的检测灵敏度，有利的糖含量的实际和有效的评估的临界温度范围内检测麦芽糖。 

视频: Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

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

Single-Molecule F&ouml;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule F&#246;rster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems.

Single-Molecule F&#246;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Presented here is a protocol for performing single-molecule F&#246;rster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.

单分子Fürster共振能量传递方法，用于GEN1对霍利迪结分辨率进行实时调查

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the ...

遗传学

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

表面固定生物分子的单分子荧光检测的自动化系统

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

生物学

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 ...

A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis of the intrinsic photothermal response of hemoglobin molecules, the sensor enables high-sensitivity, chemical-free measurement of [Hb]. [Hb] detection capability with a limit of 0.12 g/dl over the range of 0.35 - 17.9 g/dl has been demonstrated previously. The method can be readily implemented using inexpensive consumer electronic devices such as a laser pointer and a webcam. The use of a micro-capillary tube as a blood container also enables the hemoglobin assay with a nanoliter-scale blood volume and a low operating cost. Here, detailed instructions for the PT-AS optical setup and signal processing procedures are presented. Experimental protocols and representative results for blood samples in anemic conditions ([Hb] = 5.3, 7.5, and 9.9 g/dl) are also provided, and the measurements are compared with those from a hematology analyzer. Its simplicity in implementation and operation should enable its wide adoption in clinical laboratories and resource-limited settings.

A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

一种快速和无农药血红蛋白检测法与光热角光散射

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis ...

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

荧光技术结合测定活细胞中的力敏感蛋白动力学

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

实时监控的配体 - 受体相互作用的荧光共振能量转移

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. It has been used in a wide range of biophysical and molecular biological experiments, including the measurement of molecular forces, characterization of conformational dynamics of biomolecules, observation of intracellular colocalization of proteins, and determination of receptor-ligand interaction times. In a widefield microscopy configuration, experiments are typically performed using surface-immobilized probes. Here, a method combining single-molecule tracking with alternating excitation (ALEX) smFRET experiments is presented, permitting the acquisition of smFRET time traces of surface-bound, yet mobile probes in plasma membranes or glass-supported lipid bilayers. For the analysis of recorded data, an automated, open-source software collection was developed supporting (i) the localization of fluorescent signals, (ii) single-particle tracking, (iii) determination of FRET-related quantities including correction factors, (iv) stringent verification of smFRET traces, and (v) intuitive presentation of the results. The generated data can conveniently be used as input for further exploration via specialized software, e.g., for the assessment of the diffusional behavior of probes or the investigation of FRET transitions.

This article presents a method for spatiotemporal analysis of mobile, single-molecule F&#246;rster resonance energy transfer (smFRET)-based probes using widefield fluorescence microscopy. The newly developed software toolkit allows the determination of smFRET time traces of moving probes, including the correct FRET efficiency and the molecular positions, as functions of time.

移动式单分子FRET探头的自动化二维时空分析

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

开源高内涵分析利用自动荧光寿命成像显微镜

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...