Support for this work was provided by the Bill and Melinda Gates Foundation Grand Challenges in Global Health: Develop Technologies That Allow Assessment of Multiple Conditions and Pathogens at Point-of-Care. K.M.D. was supported by an NSF Graduate Research Fellowship (2012095464).

1.铱的合成（Ⅲ） <ol><li>称取53.6毫克（50微摩尔）物[Ir（PPY）2 -Cl〕2和溶解在5ml二氯甲烷中。添加这种溶液到50ml Erlenmeyer烧瓶装有搅拌棒。 </li><li>称出AgOTf 26.2毫克（100微摩尔）和溶解在5ml甲醇中。 </li><li>添加溶解AgOTf到物[Ir（PPY）2 -Cl〕2溶液并搅拌1小时。 注:乳白色浆液应该产生。 </li><li> 1小时后，过滤通过硅胶浆液成25×95毫米兰小瓶并用旋转蒸发器（5-10分钟）蒸发掉剩余的溶剂。 </li><li>一旦大部分溶剂被除去，检查油状黄色残留物仍保留在小瓶中。向该残余物中加入1毫升甲醇，以重新溶解产物和冻干1-2天，得到黄色固体产物。 </li><li>用H表征的Ir（PPY）2（H 2 O）2 +产品（IR1）&lt;SUP&gt; -1 NMR（CDCl 3中），ESI和紫外可见如前所述。23 </li></ol> 2.互动IR1与各种氨基酸<ol><li>制备IR1的甲醇2mM的溶液（MW537.10克/摩尔）。涡旋，以确保固体完全溶解。 </li><li>准备以下氨基酸和生物分子HEPES 100微米的解决方案缓冲液（HBS，100毫米的HEPES，137毫米氯化钠，pH值7.4）丙氨酸，天门冬氨酸，半胱氨酸，他的，异亮氨酸，赖氨酸，丝氨酸，尝试，缬氨酸。 </li><li>吸取100μl的每种氨基酸的成黑色96孔板中。加入100微升的HBS到板以用作空白。 </li><li>加入2.5微升2mM的IR1溶液到各样品和摇动板在板振荡器上10分钟。 </li><li> 10分钟后，用96孔板读数器（365前/ 400-700的EM）取得的样品的发射光谱。 <ol><li>将板件仪器，打开平板阅读器软件来建立一个新的实验。 </li><li>设置新的EXPeriment读取荧光扫描用365nm的激发波长和9处的狭缝宽度与光学在顶部位置。 </li><li>读从300-700纳米的发光带的5nm的步长大小。 </li><li>导出的数据分析的数据。 </li></ol></li><li>转移样品到一个明确的96孔板和图像使用UV透照的发射。 </li></ol> 3.滴定IR1与BNT-II <ol><li>制备BNT-II（MW8233.6克/摩尔）在HBS的1mM储备溶液以及IR1的甲醇2mM的溶液。 </li><li>从该股的解决方案，准备1毫升100μMBNT-II在哈佛商学院。涡流离心管，以确保样品混合。 </li><li>在HBS串联稀释100μM的BNT-Ⅱ一半到1.56μM的BNT-II的最终浓度。 </li><li>加入100微升每种稀释液一式三份，用HBS作为空白，在96孔板的孔中。向每个样品中加入2.5微升2mM的IR1。 </li><li>嘘刹车的板在板振荡器上10分钟。 </li><li>振荡后，读出的发光强度在使用96孔板读数器510纳米（激发在365nm处）。 <ol><li>将板件仪器，打开平板阅读器软件来建立一个新的实验。 </li><li>设置新的实验来读取荧光端点365 nm，发射510 nm的波长的激发波长。设置狭缝宽度为两个波长为9nm，与光学器件在顶部位置。 </li><li>阅读板块和导出数据进行分析。 </li></ol></li><li>转移样品到一个明确的96孔板和图像使用UV透射仪滴定。 </li></ol>在IR1 / BNT-II系统使用生物层干涉4.实时动力学分析<ol><li>加入200μl动力学缓冲液（KB; 1×磷酸盐缓冲盐水0.02％吐温-20），以8个孔在黑色96孔板的第一列并插入板插入塑料传感器持有人。小心在塑料夹持器，使得尖端悬浮于缓冲液的孔中转移8的Ni（II）的NTA生物传感器的第一列。放置塑料支架在干涉仪的左侧。 </li><li>准备2毫升以KB为0.5μm的解决方案BNT-Ⅱ。 </li><li>准备500微升IR1的KB 10微米的解决方案，并通过连续半稀下降到0.156微米KB。 </li><li>到一个新的黑色96孔板，吸管200微升KB的所有8个孔列A和C. </li><li>在相同的板，吸管200微升0.5μMBNT-II溶液到孔在列B的</li><li>吸取200微升每个稀释度IR1到D列，以KB为开始的空白以及D1的。添加稀释，以使井IR1浓度下列增加。 </li><li>将此板在仪器中包含预润湿传感器板的右侧。 </li><li>在生物层干涉测量软件，建立一个基本KINET集成电路实验通过定义板，测定法，和传感器的提示。 <ol><li>要定义板，在屏幕上选择列，单击右键，并选择井的适当定义。选择"缓冲区"为列A和C，"加载"为B列，而"样本"列D. </li><li>通过添加以下步骤定义在接下来的选项卡中的检测（点击"添加"）:平衡（自定义），60秒;载入中，120秒;基线，60秒;协会，120秒;与解离，300秒。选择平衡步骤，并双击A列重复加载（B列），基准线（C列），社区（D列），而解离（C列）。选择的Ni（II）NTA传感器。 </li><li>移动到下一个选项卡，并确认传感器在列A塑料传感器支架上。 </li><li>确认实验概述正确，插入一个文件名，确保"延迟实验"被选中，并按下"开始"。 ñOTE:由于仪器的自动化性质，一旦动力学实验概述，会发生的步骤进行编程。 </li></ol></li><li>一旦动力运行完成后，处理的规定处理软件中的数据。 <ol><li>与在左侧面板的底部感兴趣的文件名的文件夹上双击。然后，在面板的顶部下双击"动力学"对应的文件夹。移动到处理选项卡。 </li><li>检查左侧的减法框以打开传感器选择屏幕。右键单击以及A4和改变井型参考嘛。 </li><li>检查框旁边对齐Y轴，并确保所选择的步骤就是基线。指定时间范围是过去五年秒基线（约55-60秒）。 </li><li>检查跨一步修正框并选择对齐到解离。确保Savitzky-Golay滤波复选框被选中，然后按"过程数据！̶1; </li><li>移动分析选项卡。确保"协会解离"是步骤来分析和模型是1的选择:1。选择本地，全面配合和按"飞度曲线！" </li><li>选中表格中的所有曲线，然后右键单击设置所有曲线为一种颜色。下一步，选择一个全球（全部）配合，通过色彩，而Rmax未连接的传感器所分组。按"飞度曲线！"获得全球动力学数据。 </li></ol></li></ol> 5.在珠检测BNT-II和rcHRP-II与IR1 <ol><li>制备各1ml的1μM的溶液的BNT-II和rcHRP-Ⅱ在HBS吐温（HBST; HBS用0.025％吐温20）。 </li><li>串联一半在HBST稀释每种蛋白至15.6纳米的最终浓度。 </li><li>吸取100μl的每种稀释液一式三份，变成白色96孔板中，用稀释液，以从最低到最高浓度下的柱。 </li><li>移取10微升50微米的Ni（II）NTA磁性琼脂糖颗粒进入每个稀释度良好。 <ol><li> VORTEX包含每个移液后，磁性颗粒，以确保颗粒悬浮留在离心管中。 </li></ol></li><li>放置在板振荡器上的板15分钟孵育的样品中的颗粒。 </li><li>后该温育期间，将板在96孔平板磁铁，等待30秒的颗粒拉出的溶液。 </li><li>使用多道移液器，拉断的原始样品，丢弃废物。从磁体取出平板。 </li><li>加入200μlHBST对使用多道移液器各孔中。泵缓冲上下数次以洗涤磁性粒子。 </li><li>将板放回磁铁，等待30秒，颗粒拔出的解决方案。 </li><li>使用多道移液器，拉断的200微升缓冲和丢弃废物。从磁体取出平板。 </li><li>重复步骤5.8通过量ħ5.10两次以完成颗粒的洗涤三次。 </li><li>加入100微升HBST向每孔含有磁性粒子接着2.5微升2mM的IR1。 </li><li>孵育板振荡器上1小时与IR1的颗粒。 </li><li> 1小时后，读出的发光强度在使用96孔板读数器510纳米（激发在365nm处）。 <ol><li>将板件仪器，打开平板阅读器软件来建立一个新的实验。 </li><li>设置新的实验来读取荧光端点365 nm，发射510 nm的波长的激发波长。设置狭缝宽度为两种波长至9纳米，与光学器件在顶部位置。 </li><li>阅读板块和导出数据进行分析。 </li></ol></li><li>转移样品到一个明确的96孔板和图像使用UV透照滴定。 </li></ol>

<ol>
	<li>Gubala, V., Harris, L. F., Ricco, A. J., Tan, M. X., Williams, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point+of+Care+Diagnostics:+Status+and+Future.">Point of Care Diagnostics: Status and Future.</a> Analytical Chemistry. 84, 487-515 (2011).</li><li>Kuzmenko, A., 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=Single+molecule+tracking+fluorescence+microscopy+in+mitochondria+reveals+highly+dynamic+but+confined+movement+of+Tom40.">Single molecule tracking fluorescence microscopy in mitochondria reveals highly dynamic but confined movement of Tom40.</a> Scientific Reports. 1, (2011).</li><li>Sassolas, A., Leca-Bouvier, B. D., Blum, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Biosensors+and+Microarrays.">DNA Biosensors and Microarrays.</a> Chemical Reviews. 108, 109-139 (2007).</li><li>Miyawaki, A., Sawano, A., Kogure, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Lighting+up+cells:+labelling+proteins+with+fluorophores.">Review: Lighting up cells: labelling proteins with fluorophores.</a> Nature Cell Biology. 5, S1-S7 (2003).</li><li>Liu, 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=Highly+sensitive+protein+detection+using+enzyme-labeled+gold+nanoparticle+probes.">Highly sensitive protein detection using enzyme-labeled gold nanoparticle probes.</a> Analyst. 135, 327-331 (2010).</li><li>Almeida, M., Carabineiro, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+nanogold+in+human+tropical+diseases:+research,+detection+and+therapy.">The role of nanogold in human tropical diseases: research, detection and therapy.</a> Gold Bulletin. 46, 65-79 (2013).</li><li>Holder, E., Langeveld, B. M. W., Schubert, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Trends+in+the+Use+of+Transition+Metal&#8211;Ligand+Complexes+for+Applications+in+Electroluminescent+Devices.">New Trends in the Use of Transition Metal&#8211;Ligand Complexes for Applications in Electroluminescent Devices.</a> Advanced Materials. 17, 1109-1121 (2005).</li><li>Wagenknecht, P. S., Ford, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+centered+ligand+field+excited+states:+Their+roles+in+the+design+and+performance+of+transition+metal+based+photochemical+molecular+devices.">Metal centered ligand field excited states: Their roles in the design and performance of transition metal based photochemical molecular devices.</a> Coordination Chemistry Reviews. 255, 591-616 (2011).</li><li>Lamansky, 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=Highly+Phosphorescent+Bis-Cyclometalated+Iridium+Complexes:+Synthesis,+Photophysical+Characterization,+and+Use+in+Organic+Light+Emitting+Diodes.">Highly Phosphorescent Bis-Cyclometalated Iridium Complexes: Synthesis, Photophysical Characterization, and Use in Organic Light Emitting Diodes.</a> Journal of the American Chemical Society. 123, 4304-4312 (2001).</li><li>Ho, M. -. L., 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=Synthesis,+structure+and+oxygen-sensing+properties+of+Iridium(iii)-containing+coordination+polymers+with+different+cations.">Synthesis, structure and oxygen-sensing properties of Iridium(iii)-containing coordination polymers with different cations.</a> Dalton Transactions. 41, 2592-2600 (2012).</li><li>McDaniel, N. D., Coughlin, F. J., Tinker, L. L., Bernhard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclometalated+Iridium(III)+Aquo+Complexes:+Efficient+and+Tunable+Catalysts+for+the+Homogeneous+Oxidation+of+Water.">Cyclometalated Iridium(III) Aquo Complexes: Efficient and Tunable Catalysts for the Homogeneous Oxidation of Water.</a> Journal of the American Chemical Society. 130, 210-217 (2007).</li><li>Ma, D. -. L., 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=A+Highly+Selective+Luminescent+Switch-On+Probe+for+Histidine/Histidine-Rich+Proteins+and+Its+Application+in+Protein+Staining.">A Highly Selective Luminescent Switch-On Probe for Histidine/Histidine-Rich Proteins and Its Application in Protein Staining.</a> Angewandte Chemie International Edition. 47, 3735-3739 (2008).</li><li>Ma, D. -. L., He, H. -. Z., Leung, K. -. H., Chan, D. S. -. H., Leung, C. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioactive+Luminescent+Transition-Metal+Complexes+for+Biomedical+Applications.">Bioactive Luminescent Transition-Metal Complexes for Biomedical Applications.</a> Angewandte Chemie International Edition. 52, 7666-7682 (2013).</li><li>Leung, C. -. H., 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=A+Metal-Based+Inhibitor+of+Tumor+Necrosis+Factor-&#945;.">A Metal-Based Inhibitor of Tumor Necrosis Factor-&#945;.</a> Angewandte Chemie International Edition. 51, 9010-9014 (2012).</li><li>You, Y., Nam, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photofunctional+triplet+excited+states+of+cyclometalated+Ir(iii)+complexes:+beyond+electroluminescence.">Photofunctional triplet excited states of cyclometalated Ir(iii) complexes: beyond electroluminescence.</a> Chemical Society Reviews. 41 (21), 7061-7084 (2012).</li><li>Zhao, Q., 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=Cationic+Iridium(III)+Complexes+with+Tunable+Emission+Color+as+Phosphorescent+Dyes+for+Live+Cell+Imaging.">Cationic Iridium(III) Complexes with Tunable Emission Color as Phosphorescent Dyes for Live Cell Imaging.</a> Organometallics. 29, 1085-1091 (2010).</li><li>Li, 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=A+Nonemissive+Iridium(III)+Complex+That+Specifically+Lights-Up+the+Nuclei+of+Living+Cells.">A Nonemissive Iridium(III) Complex That Specifically Lights-Up the Nuclei of Living Cells.</a> Journal of the American Chemical Society. 133, 11231-11239 (2011).</li><li>Jones, A. L., Hulett, M. D., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histidine-rich+glycoprotein:+A+novel+adaptor+protein+in+plasma+that+modulates+the+immune,+vascular+and+coagulation+systems.">Histidine-rich glycoprotein: A novel adaptor protein in plasma that modulates the immune, vascular and coagulation systems.</a> Immunology, &amp; Cell Biology. 83, 106-118 (2005).</li><li>Panton, L. 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=Purification+and+partial+characterization+of+an+unusual+protein+of+Plasmodium+falciparum:+histidine-rich+protein+II.">Purification and partial characterization of an unusual protein of Plasmodium falciparum: histidine-rich protein II.</a> Molecular and Biochemical Parasitology. 35, 149-160 (1989).</li><li>Schneider, E. L., Marletta, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heme+Binding+to+the+Histidine-Rich+Protein+II+from+Plasmodium+falciparum.">Heme Binding to the Histidine-Rich Protein II from Plasmodium falciparum.</a> Biochemistry. 44, 979-986 (2004).</li><li>Mabey, D., Peeling, R. W., Ustianowski, A., Perkins, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tropical+infectious+diseases:+Diagnostics+for+the+Developing+World.">Tropical infectious diseases: Diagnostics for the Developing World.</a> Nature Reviews Microbiology. 2, 231-240 (2004).</li><li>Ziegler, J., Chang, R. T., Wright, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple-Antigenic+Peptides+of+Histidine-Rich+Protein+II+of+Plasmodium+falciparum:+Dendrimeric+Biomineralization+Templates.">Multiple-Antigenic Peptides of Histidine-Rich Protein II of Plasmodium falciparum: Dendrimeric Biomineralization Templates.</a> Journal of the American Chemical Society. 121, 2395-2400 (1999).</li><li>Schmid, B., Garces, F. O., Watts, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+characterizations+of+cyclometalated+iridium(III)+solvento+complexes.">Synthesis and characterizations of cyclometalated iridium(III) solvento complexes.</a> Inorganic Chemistry. 33, 9-14 (1994).</li><li>Davis, K. M., Swartz, J. D., Haselton, F. R., Wright, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-Resource+Method+for+Extracting+the+Malarial+Biomarker+Histidine-Rich+Protein+II+To+Enhance+Diagnostic+Test+Performance.">Low-Resource Method for Extracting the Malarial Biomarker Histidine-Rich Protein II To Enhance Diagnostic Test Performance.</a> Analytical Chemistry. 84, 6136-6142 (2012).</li><li>Giljohann, D. A., Mirkin, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drivers+of+biodiagnostic+development.">Drivers of biodiagnostic development.</a> Nature. 462, 461-464 (2009).</li><li>Li, M. -. 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=High+electrochemiluminescence+of+a+new+water-soluble+iridium(iii)+complex+for+determination+of+antibiotics.">High electrochemiluminescence of a new water-soluble iridium(iii) complex for determination of antibiotics.</a> Analyst. 136, 205-210 (2011).</li></ol>

The authors have nothing to disclose.

作为微粒为基础的诊断工具来现代诊断技术的前沿，直接作用于颗粒的表面的检测对象的生物分子的是一大优势。传统的分子的检测方法，如ELISA和PCR，是有利的，因为它们能够实现检测25的非常低的限制。但是，这些测定法需要大量的试剂和冗长的协议。该测定被设计为模仿ELISA类型夹心相互作用不受时间和试剂的要求（ 图5）。另外，每个样品的IR1探针的成本估计是围绕一个便士，而抗体的一个典型的ELISA的成本为$ 0.20-0.30每个样品。 因为上述方法依赖于一个环金属铱探针用于检测疟疾生物标志物的，该探针将不容易受到共同的其他检测方法的故障模式（ 即荧光团淬火时NG和抗体/酶降解）。组氨酸是在其金属结合特性是唯一的。概述的工作利用了这一现象通过替换抗体中的典型ELISA夹心与含有金属配合物的优势。镍（Ⅱ）NTA捕获rcHRP-Ⅱ的颗粒的表面上和IR1信号的蛋白质的存在。在测定中最关键的步骤是允许磁性颗粒混合的样品和探针。在一个96孔板的ELISA，生物分子和试剂达到与井的底部的二维表面平衡。磁性颗粒具有沉降出来的溶液中的倾向，由于其致密氧化铁核心，这将减少了可用结合位点。因此，颗粒必须在测定时间进行混合，以确保最大结合。 当设计一个试剂为疾病的诊断，必须牢记患者样品的形式，以及所述生物标志物的生理浓度。为疟疾， 粉煤的HRP-Ⅱ的在病人的血液中的浓度可以从低皮摩尔到高纳摩尔变化。而该测定是临床相关更高水平的感染，需要检测极限，以检测无症状患者低皮摩尔循环粉煤的HRP-Ⅱ得到改善。在以上概述的方法中，"接通"，在组氨酸存在下通过IR1产生的信号出现。而疟疾生物标志物是富含组氨酸，其它血清蛋白，如人血清白蛋白和组氨酸丰富的糖蛋白，会引起与探针的信号的响应。这反过来又导致假阳性诊断。这使得粒子的有益步骤，在该感兴趣的蛋白质，可以迅速地从患者样品中提取的表面上的蛋白质的捕获。此外，设计一个双功能探头，即夫妻富含组氨酸肽与强大的分子识别元件（ 即适体），可以添加特异性另一层到测定。肽将与铱装载联接到所述适体之前。在这样的设计中，稳定IR1探针仍然可以同时实现靶特异性与适体使用。这将允许应用的探针来检测天然的HRP-II在一个复杂的基质（ 即血浆或全血），并减少非特异性结合效果。为了进一步提高快速诊断测试信号，该测定可纳入电化学发光（ECL）系统，其中， 粉煤的HRP-II可以在快速诊断被检测为电化学读数26。这种新一代铱探测器将大大提高我们的小球上的ELISA检测的疾病生物标志的检测分析。

比色和荧光标记是用于生化分子的检测和跟踪的重要方法和处理1,2-。最常见的荧光标记物是低分子量的有机染料3,4-，但这些分子并不总是理想的光学性质。荧光染料容易发生光漂白，常常有小的斯托克斯位移，并且可具有重叠的激发和发射光谱。比色标记通常通过使用酶标记，其具有一个放大的信号在免疫测定定量5,6-有用实现。这些酶也有其缺点，包括光敏性，反应条件，和短基片的保质期。这些性质往往需要免疫测定和蛋白标记方法使用昂贵的试剂良好控制的条件下进行。 发射过渡金属配合物已探索作为替代标记方法FO- [R生化检测。特别是，环金属化铱（Ⅲ）已经研究了在有机发光二极管（OLED）7-9氧传感10的上下文中，催化11，和蛋白/细胞染色12-14。高光稳定性和量子效率使这一类探头的一个很好的候选人生物分子检测15,16。这是以前发现，环金属化的形式的铱（Ⅲ）配合物，[铱（C ^ N）2（SOLV）2] +，不可逆地结合组氨酸和引发蓝绿信号响应12,17。这些配合物是不发光的solvento状态，但是当组氨酸置换的溶剂分子与结合至金属中心，它们释放长波紫外线照射后的强烈的磷光信号。这个信号只发生配位体置换后，并且是一个三线态的电子通过的金属配位体的电荷转移活化放松到基态的结果（3毫升CT）和配体为中心的转移（3 LC）途径8,15。这些配合物可以潜在地用作检测探针对富含组氨酸的蛋白质。 富含组氨酸的蛋白质和它们的调节的水平是很重要的，在许多疾病，包括肝硬化，癌症，和凝血酶病症。18恶性疟原虫组氨酸丰富蛋白II（pfHRP-Ⅱ），特别是良好的验证生物标记疟原虫感染。这种蛋白质是67 kDa的并含有34％组氨酸，多内特性AHHAHHAAD重复图案19。这些重复的组氨酸可结合不含金属离子19和血红素络合物20主机的血液。 pfHRP-II是在低资源设置常用检测使用免疫层析快速诊断测试（主任小组），但这些测试往往是不准确的，由于样本的条件下，低浓度的生物标记物，可怜的制造标准，以及抗体降解21。 [Ir（PPY）2（H 2 O）2] +（IR1）来检测BNT-II，pfHRP-II的分支肽模拟进行了探讨22。这种相互作用的动力学实时使用生物层干涉测量技术监测。该测定也适合于在小球上的ELISA类型的格式，在那里分别实现检测纳摩尔限制。该测定中保持优于传统的ELISA，因为它可以在2小时来进行的，而不是在4-5小时，并用典型的ELISA需要生物试剂与抗体的游离的试剂。

<table><tbody><tr><td>Dicholortetrakis(2-(2-pyridinyl)phenyl)diiridium(III)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>658383</td><td></td></tr><tr><td>silver trifluoromethanesulfonate</td><td>Sigma-Aldrich</td><td>176435</td><td></td></tr><tr><td>Silica gel</td><td>Sigma-Aldrich</td><td>6858</td><td></td></tr><tr><td>L-Amino acids</td><td>Fisher Scientific</td><td>varies</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>BNT-II peptide</td><td>Synthesized in house</td><td>N/A</td><td></td></tr><tr><td>recombinant HRPII</td><td>Immunology Consultants Laboratory inc.</td><td>AGPF-55</td><td></td></tr><tr><td>Costar black polypropylene 96-well plate</td><td>Fisher Scientific</td><td>07-200-567</td><td></td></tr><tr><td>Costar white polypropylene 96-well plate</td><td>Fisher Scientific</td><td>07-200-589</td><td></td></tr><tr><td>Grenier black polypropylene 96-well plate</td><td>VWR</td><td>89089-582</td><td></td></tr><tr><td>Ni(II)NTA magnetic agarose particles</td><td>Qiagen</td><td>36111</td><td></td></tr><tr><td>Ni(II)NTA biolayer interferometry sensors</td><td>ForteBio</td><td>18-5101</td><td></td></tr><tr><td>Octet Red96 Biolayer Interferometry Instrumet</td><td>ForteBio</td><td></td><td></td></tr><tr><td>Biotek Synergy H4 Microplate Reader</td><td>Biotek</td><td></td><td></td></tr><tr><td>Fisher Biotech Transilluminator</td><td>Fisher Scientific</td><td>FBDLT-88</td><td></td></tr></tbody></table>

iridium(iii) luminescent probe for detection of the malarial protein biomarker histidine rich protein-ii

这项工作概述了非发射的合成，环金属化铱（III）络合物，铱（PPY）2（H 2 O）2 +（IR1），当配位到一个组氨酸可引起一个快速，长寿命的磷光信号含蛋白质固定化磁性粒子的表面上。 IR1的合成，产量高，是完整的O / N和涉及母公司环金属铱（III）氯桥联二聚体成两个​​当量的溶解复杂的分裂。 以确认特异性，数个氨基酸进行探测的协调活动时加入到合成的探针，只有组氨酸引起的信号响应。使用BNT-II中，疟疾生物标记组氨酸丰富蛋白II（pfHRP-II）的支链的肽模拟物中，铱探针验证作为HRP-Ⅱ检测的工具。淬火效果注意到在BNT-II / IR1滴定相比L-组氨酸/ IR1，但这些都归因于空间位阻和三诗人盖伊状态淬火。生物层干涉测量被用来确定IR1与BNT-Ⅱ相互作用的实时动力学。一旦系统进行了优化，检测用探针rcHRP-Ⅱ的限制被认为是12.8纳米在溶液中。当这种蛋白质被固定在50微米磁性琼脂糖颗粒的表面上，检测限为14.5纳米。此无机探针的健壮信号的响应，以及​​在溶液中利用其柔性或固定在表面上，可借本身朝向各种应用，从诊断用成像。

如图1，合成在父二聚体氯化物桥的IR1 solvento络合物涉及分裂通过的氯化物沉淀中不溶性氯化银的形式，并且水分子的关联与金属中心。 IR1的形成通过H 1 NMR和ESI确认。此外，紫外可见波段的金属配位体的电荷转移特性和π-π*跃迁被分配到的光谱，进一步验证IR1的形成。这种溶剂化的离子配合物没有发光的字符时在365nm处激发。 <img alt="图1" src="/files/ftp_upload/52856/52856fig1.jpg" /> 图1:合成IR1的 Dichlorotetrakis（2-（2-吡啶基）苯基）氯化桥分解反应和表征二铱（III）来创建复杂的水合IR（PPY）2（H 2 O）2 </子&gt; +（IR1）。在加入的左旋组氨酸，以在水性缓冲液中水合配合物，发光信号被接通。 一旦合成的复杂性和特征，所述氨基酸选择性进行了分析，如用类似的铱类似物17先前所描述。 图2A示出，只有组氨酸引起在510nm处的信号响应中，当在365nm激发。 <img alt="图2" src="/files/ftp_upload/52856/52856fig2.jpg" /> 图2:氨基酸选择性和IR1与组氨酸肽丰富的滴定（A）为200微米的各种氨基酸（半胱氨酸，丝氨酸，天门冬氨酸，谷氨酸，苯丙氨酸，赖氨酸，精氨酸，酪氨酸，色氨酸，他的）用50μM的相互作用。在IR1哈佛商学院。光谱扫描取自400-700纳米处的365纳米的激发波长在黑色96孔板中。信号以相对荧光单位（RFU）从所有氨基酸besides组氨酸（虚线黑色线）是可以忽略不计。 BNT-II（▪）和rcHRP-II（♦）（二）纳摩尔浓度进行滴定IR1在哈佛商学院。读取的发射在510nm激发与365纳米的光前后的组氨酸丰富的肽温育用探针在溶液中15分钟。检测限计算为x的当y =3σ 空白值。 这种"开关"的铱探针的磷光的发生是因为铱的单重激发态（1 MLCT / 1 LC）（Ⅲ）的生物共轭经历系间窜越到三重激发态（3 MLCT）中，当组氨酸配位与金属中心。该探头应用于BNT-II肽模仿的生物标志物的疟疾疟原虫富组氨酸蛋白II（PF HRP-Ⅱ）。在BNT-Ⅱ与IR1的滴定，以浓度依赖性的信号反应，观察（ <strong&gt;的图2B）。重组HRP-II（rcHRP-II）也展出的解决方案类似的反应。在溶液中检测BNT-II和rcHRP-II的限分别为54.8 nm和12.8纳米，分别。使用的生物层干涉测量技术，IR1所述K D结合到BNT-Ⅱ固定在镍（Ⅱ）NTA表面被发现是2.05微米（ 图3）。 <img alt="图4" src="/files/ftp_upload/52856/52856fig4.jpg" /> 图3:IR1与BNT-Ⅱ的生物层干涉测量为不同浓度IR1的镍（Ⅱ）NTA玻璃传感器的表面上的结合BNT-Ⅱ的动力学分析实时动力学分析 。平衡传感器中的KB（区域A）后，将传感器装入0.5μMBNT-II（区域B）。一旦肽上的传感器装，基线确立（C区）测量IR1到BNT-II（D区）协会前。后一段关联的，所述传感器被放回KB到测量解离（区域E）。整个动力学分析过程用时不到30分钟。 当从基于溶液的测定法，以磁性粒子平台过渡，所添加的系统中的颗粒的复杂性必须加以考虑。它是从以 ​​前的工作已知的，这些颗粒有效结合的疟疾生物标志pf的 HRP-II 24黑色荧光板的溶液测定行之有效以上描述，但白色板是更适合于对粒子检测平台，如在可见图4A。在白色板时，光被反射回进入样品，因而允许由IR1更好吸光度结合到粒子的表面上。在对粒子检测，检测rcHRP-II的极限被确定为14.5米（ 图4B）。检测的rcHRP-II在溶液和小球上的限制是统计的的角度讲同一基础上的不成对t检验（p值= 0.731）。 <img alt="图5" src="/files/ftp_upload/52856/52856fig5.jpg" style="font-size: 14px; line-height: 28px;" /> <img alt="图5" src="/files/ftp_upload/52856/52856fig5.jpg" style="font-size: 14px; line-height: 28px;" /> 图4:从IR1结合的BNT-Ⅱ为50μm的Ni的表面上检测到的信号之间在珠检测BNT-Ⅱ和HRP-Ⅱ（A）的差（Ⅱ）NTA磁性琼脂糖颗粒在黑色96-孔板（实线）与一白色96孔板（虚线）。这些颗粒激发与365纳米的光，并发射测量在510纳米。 rcHRP-II（B）的滴定固定在磁性颗粒和使用IR1检测。检测限计算为VAx的略时，Y =3σ 空白 。 <img alt="图5" src="/files/ftp_upload/52856/52856fig5.jpg" /> 图5:在珠检测rcHRP-Ⅱ与IR1的示意图 HRP-II结合的Ni（II）的NTA粒子和标记IR1的表面一般方案。将颗粒孵育15分钟时，组氨酸丰富肽。在此之后的潜伏期，该颗粒物用使用HBST磁体，以拉颗粒从溶液中析出。最后，肽结合的粒子正在阅读在激发与365纳米的光后510nm处的发射之前孵育IR1 1小时。

Watch this Scientific Journal Video about IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II at JoVE.com

IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

铱（III）荧光探针检测疟疾蛋白质生物标志物组氨酸丰富的蛋白质-Ⅱ

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived ...

iridium-iii-luminescent-probe-for-detection-malarial-protein

Research

JoVE Journal

Chemistry

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

9.1K 次观看.  Vanderbilt University. Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

视频: IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

生物分子检测的干涉反射成像传感器（IRIS）

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

科研

生物工程

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

We demonstrate a method of quantification and detection of parasites in aqueous red blood cells (RBCs) by using a simple benchtop Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectrometer in conjunction with Multivariate Data Analysis (MVDA). 3D7 P. falciparum were cultured to 10% parasitemia ring stage parasites and used to spike fresh donor isolated RBCs to create a dilution series between 0&#8211;1%. 10 &#181;L of each sample were placed onto the center of the ATR diamond window to acquire the spectrum. The sample data was treated to improve the signal to noise ratio and to remove the contribution of water, and then the second derivative was applied to resolve spectral features. The data were then analyzed using two types of MVDA: first Principal Component Analysis (PCA) to determine any outliers and then Partial Least Squares Regression (PLS-R) to build the quantification model.

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

Here, we present a protocol for the detection and quantification of Plasmodium falciparum in infected aqueous red blood cells using an attenuated total reflection infrared spectrometer and multivariate data analysis.

衰减全反射红外光谱和多变量数据分析检测和定量水红血球中恶性疟原虫

We demonstrate a method of quantification and detection of parasites in aqueous red blood cells (RBCs) by using a simple benchtop Attenuated Total ...

化学

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.
One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.
SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.
While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.
In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.
This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

方法来提高高分辨率的敏感性疟疾熔单核苷酸多态性基因分型

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

免疫与感染

High Spatial Resolution Chemical Imaging of Implant-Associated Infections with X-ray Excited Luminescence Chemical Imaging Through Tissue

Microbial infections associated with implantable medical devices are a major concern in fracture fixation failure. Early diagnosis of such infection will allow successful eradication with antibiotics without an extra cost for a second surgery. Herein, we describe XELCI as a technique with high X-ray resolution, implant specificity, and chemical sensitivity to noninvasively image chemical concentrations near the surface of implanted medical devices. The devices are coated with chemically reporting surfaces. This chemically responsive surface consists of two layers coated on an implantable medical device; a pH-sensitive layer (bromothymol blue or bromocresol green incorporated hydrogel) which is coated over a red-light emitting scintillator (Gd2O2S: Eu) layer for monitoring. A focused X-ray beam irradiates a spot on the implant, and the red light generated by the scintillator (with 620 nm and 700 nm peaks) is transmitted through the sensing layer which alters the spectral ratio depending on the pH. An image is generated by scanning the X-ray beam across the implant and measuring the spectral ratio of light passing through the tissue point-by-point. We used this imaging technique for monitoring implant-associated infections previously on the bone surface of the femur with a modified implantable plate sensor. Now we are studying pH changes that occur from tibial intramedullary rod infections. Two different types of intramedullary rod designs are used in pre-pilot rabbit studies, and we learned that the XELCI technique could be used to monitor any chemical changes that occur not only on the bone surface but also inside the bone. Thus, this enables noninvasive, high spatial resolution, low background local pH imaging to study implant-associated infection biochemistry.

Here, we present a protocol for high-resolution optical detection of chemical information around implanted medical devices with X-ray excited luminescence chemical imaging (XELCI). This novel imaging technique is developed in our lab which enables studying implant-associated infection biochemistry.

通过组织的X射线激发发光化学成像对植入物相关感染进行高空间分辨率化学成像

Microbial infections associated with implantable medical devices are a major concern in fracture fixation failure. Early diagnosis of such infection will ...

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

超超磁氧化铁纳米探针在肺外结核检测的合成、表征及应用

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

医学

Optimized Griess Reaction for UV-Vis and Naked-eye Determination of Anti-malarial Primaquine

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening infections caused by P. vivax and ovale. However, PMQ has unwanted adverse effects that lead&#160;to acute hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. There is a need to develop simple and reliable methods for PMQ determination with the purpose of dosage monitoring. In early 2019, we have reported an UV-Vis and naked-eye based approach for PMQ colorimetric quantification. The detection was based on a Griess-like reaction between PMQ and anilines, which can generate colored azo products. The detection limit for direct measurement of PMQ in synthetic urine is in the nanomolar range. Moreover, this method has shown great potential for PMQ quantification from human serum samples at clinically relevant concentrations. In this protocol, we will describe the technical details regarding the syntheses and characterization of colored azo products, the reagent preparation, and the procedures for PMQ determination.

This protocol describes a novel colorimetric method for antimalarial primaquine (PMQ) detection in synthetic urines and human serums.

抗疟药的紫外线-Vis和裸眼测定优化的格里斯反应

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening ...

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron regulatory proteins, IRP1 and IRP2. They bind to iron responsive elements (IREs) within the untranslated regions (UTRs) of several target mRNAs, thereby controlling the mRNAs translation or stability. IRE/IRP interactions have been widely studied by EMSA. Here, we describe the EMSA protocol for analyzing the IRE-binding activity of IRP1 and IRP2, which can be generalized to assess the activity of other RNA-binding proteins as well. A crude protein lysate containing an RNA-binding protein, or a purified preparation of this protein, is incubated with an excess of32 P-labeled RNA probe, allowing for complex formation. Heparin is added to preclude non-specific protein to probe binding. Subsequently, the mixture is analyzed by non-denaturing electrophoresis on a polyacrylamide gel. The free probe migrates fast, while the RNA/protein complex exhibits retarded mobility; hence, the procedure is also called &#8220;gel retardation&#8221; or &#8220;bandshift&#8221; assay. After completion of the electrophoresis, the gel is dried and RNA/protein complexes, as well as free probe, are detected by autoradiography. The overall goal of the protocol is to detect and quantify IRE/IRP and other RNA/protein interactions. Moreover, EMSA can also be used to determine specificity, binding affinity, and stoichiometry of the RNA/protein interaction under investigation.

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Here we present a protocol to analyze RNA/protein interactions. The electrophoretic mobility shift assay (EMSA) is based on the differential migration of RNA/protein complexes and free RNA during native gel electrophoresis. By using a radiolabeled RNA probe, RNA/protein complexes can be visualized by autoradiography.

凝胶电泳迁移分析（EMSA）为RNA，蛋白质相互作用的研究:IRE / IRP示例

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...

生物学

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

基于海拉细胞自由表达系统中的表达<em&gt;疟原虫</em&gt;棒状体蛋白

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

A Bright NIR-II Fluorescence Probe for Vascular and Tumor Imaging

As an emerging imaging technology, near-infrared II (NIR-II, 1000-1700 nm) fluorescence imaging has significant potential in the biomedical field, owing to its high sensitivity, deep tissue penetration, and superior imaging with spatial and temporal resolution. However, the method to facilitate the implementation of NIR-II fluorescence imaging for some urgently needed fields, such as medical science and pharmacy, has puzzled relevant researchers. This protocol describes in detail the construction and bioimaging applications of a NIR-II fluorescence molecular probe, HLY1, with a D-A-D (donor-acceptor-donor) skeleton. HLY1 showed good optical properties and biocompatibility. Furthermore, NIR-II vascular and tumor imaging in mice was performed using a NIR-II optics imaging device. Real-time high-resolution NIR-II fluorescence images were acquired to guide the detection of tumors and vascular diseases. From probe preparation to data acquisition, the imaging quality is greatly improved, and the authenticity of the NIR-II molecular probes for data recording in intravital imaging is ensured.

The present protocol describes a detailed, real-time NIR-II fluorescence imaging operation of a mouse using a NIR-II optics imaging device.

用于血管和肿瘤成像的明亮NIR-II荧光探针

As an emerging imaging technology, near-infrared II (NIR-II, 1000-1700 nm) fluorescence imaging has significant potential in the biomedical field, owing ...

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

单细胞多重荧光成像，可可视化病毒核酸和蛋白质，并监测 HIV、HTLV、HBV、HCV、寨卡病毒和流感感染

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...