Name: iridium(iii) luminescent probe for detection of the malarial protein biomarker histidine rich protein-ii
Uploaded: 2015-07-07T15:00:00.000+00:00
Duration: 12 min 52 s
Description: Watch this Scientific Journal Video about IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II at JoVE.com

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>

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	<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

Nanyang Technological University

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

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

科研

生物化学

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, ...

生物工程

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their involvement in two-component signal transduction, a ubiquitous system through which bacteria sense and respond to environmental stimuli. Two-component signaling features autophosphorylation of a histidine kinase, followed by phosphotransfer to the receiver domain of a response regulator protein, which ultimately leads to an output response. Autophosphorylation of the histidine kinase is responsive to the presence of a cognate environmental stimulus, thereby giving bacteria a means to detect and respond to changes in the environment. Despite their importance in bacterial biology, histidine kinases remain poorly understood due to the inherent lability of phosphohistidine. Conventional methods for studying these proteins, such as SDS-PAGE autoradiography, have significant shortcomings. We have developed a nitrocellulose binding assay that can be used to characterize histidine kinases. The protocol for this assay is simple and easy to execute. Our method is higher throughput, less time-consuming, and offers a greater dynamic range than SDS-PAGE autoradiography.

We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.

使用硝酸纤维素结合分析细菌组氨酸激酶自身磷酸化的定量分析

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

体内在肿瘤微环境中 pH 值、 pO2、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

癌症研究

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards understanding its mechanism. Protonation and conformational changes affect the vibration pattern of amino acid side chains and of the peptide bond, respectively, both of which can be probed by infrared (IR) difference spectroscopy. For proteins whose function can be repetitively and reproducibly triggered by light, it is possible to obtain infrared difference spectra with (sub)microsecond resolution over a broad spectral range using the step-scan Fourier transform infrared technique. With ~102-103 repetitions of the photoreaction, the minimum number to complete a scan at reasonable spectral resolution and bandwidth, the noise level in the absorption difference spectra can be as low as ~10-4, sufficient to follow the kinetics of protonation changes from a single amino acid. Lower noise levels can be accomplished by more data averaging and/or mathematical processing. The amount of protein required for optimal results is between 5-100 &#181;g, depending on the sampling technique used. Regarding additional requirements, the protein needs to be first concentrated in a low ionic strength buffer and then dried to form a film. The protein film is hydrated prior to the experiment, either with little droplets of water or under controlled atmospheric humidity. The attained hydration level (g of water / g of protein) is gauged from an IR absorption spectrum. To showcase the technique, we studied the photocycle of the light-driven proton-pump bacteriorhodopsin in its native purple membrane environment, and of the light-gated ion channel channelrhodopsin-2 solubilized in detergent.

Key steps of protein function, in particular backbone conformational changes and proton transfer reactions, often take place in the microsecond to millisecond time scale. These dynamical processes can be studied by time-resolved step-scan Fourier-transform infrared spectroscopy, in particular for proteins whose function is triggered by light.

质子转移和蛋白质构象动力学的光敏蛋白的时间分辨步进扫描傅立叶变换红外光谱

Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards ...

工程学

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein film electrochemistry, in which a protein is immobilized on an electrode surface such that the electrode replaces physiological electron donors or acceptors, has provided functional insight into the redox reactions of a range of different proteins. Full chemical understanding requires electrochemical control to be combined with other techniques that can add additional structural and mechanistic insight. Here we demonstrate a technique, protein film infrared electrochemistry, which combines protein film electrochemistry with infrared spectroscopic sampling of redox proteins. The technique uses a multiple-reflection attenuated total reflectance geometry to probe a redox protein immobilized on a high surface area carbon black electrode. Incorporation of this electrode into a flow cell allows solution pH or solute concentrations to be changed during measurements. This is particularly powerful in addressing redox enzymes, where rapid catalytic turnover can be sustained and controlled at the electrode allowing spectroscopic observation of long-lived intermediate species in the catalytic mechanism. We demonstrate the technique with experiments on E. coli hydrogenase 1 under turnover (H2 oxidation) and non-turnover conditions.

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

Here, we describe a technique, protein film infrared electrochemistry, which allows immobilized redox proteins to be studied spectroscopically under direct electrochemical control at a carbon electrode. Infrared spectra of a single protein sample can be recorded at a range of applied potentials and under a variety of solution conditions.

用 [NiFe] 氢对 H2氧化的研究表明, 蛋白质膜红外电化学

Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

鉴定蛋白质相互作用的网站使用多肽阵列

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

生物学

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 ...

医学

Amplified Luminescent Proximity Homogeneous Assay: A Bead-Based Proximity Assay to Screen Small Molecules Inhibiting Protein-Protein Interactions

Source: Wang, L. et al., <a href="https://app.jove.com/v/62762">Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay.</a> J. Vis. Exp. (2021)
In this video, we demonstrate the amplified luminescent proximity homogeneous assay&#8212;a bead-based proximity assay. It utilizes homogeneously-sized acceptor and donor beads, immobilized with two proteins that tend to interact to screen potential small molecules inhibiting protein interactions.

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

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