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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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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dicholortetrakis(2-(2-pyridinyl)phenyl)diiridium(III)&#160;</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 ...

iridiumiii-luminescent-probe-for-detection-malarial-protein-biomarker

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

9.0K 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

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish1, plants2, 3, arthropods4, 5, fungi6, and bacteria7. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested8-10, see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears11, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 &deg;C), and temperature recordings as a function of time were not an available option for these instruments.
We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously9, 10, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details.
To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)12. This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies' group (Queens University)10. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs5, 13. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations14, 15. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber13. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere16.
The Cold Stage System
The cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 &mu;m in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).

Ice binding proteins (IBPs), also known as antifreeze proteins, inhibit ice growth and are a promising additive for use in the cryopreservation of tissues. The main tool used to investigate IBPs is the nanoliter osmometer. We developed a home-designed cooling stage mounted on an optical microscope and controlled using a custom-built LabVIEW routine. The nanoliter osmometer described here manipulated the sample temperature in an ultra-sensitive manner.

LabVIEW操作的新型纳升渗透压仪冰结合蛋白的调查

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition ...

科研

化学

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

粒子粒子相互作用采用胶体探针纳米显微定量和定性检查

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo- and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge. AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single &#945;-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein. While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured.&#160;&#160;

Sedimentation equilibrium (SE) can be used to study protein-protein interactions in a physiological environment. This manuscript describes the use of this technique to determine the effect of pH on the stability of a homo-pentamer formed by the small hydrophobic (SH) protein encoded by the human syncytial respiratory virus (hRSV).

小型低聚物形成膜蛋白的沉降平衡：组氨酸质子对五聚体稳定性

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and ...

Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are rapidly oxidized, for example by atmospheric oxygen, to indigo type dyes. This reaction enables fast and easy screening in vivo without isolation or purification of enzymes, as well as rapid tests on agar plates or in solution (e.g., blue-white screening, micro-wells) and is used in biochemistry, histochemistry, bacteriology and molecular biology. Unfortunately the synthesis of such substrates proved to be difficult, due to various side reactions and the low reactivity of the indoxyl hydroxyl function. Especially for glucose type structures low yields were observed. Our novel approach employs indoxylic acid ester as key intermediates. Indoxylic acid esters with varied substitution patterns were prepared on scalable pathways. Phase transfer glycosylations with those acceptors and peracetylated glycosyl halides can be performed under common conditions in high yields. Ester cleavage and subsequent mild silver mediated glycosylation yields the peracetylated indoxyl glycosides in high yields. Finally deprotection is performed according to Zempl&#233;n.

Indoxyl glycosides are well-established and widely used tools for enzyme screening and enzyme activity monitoring. Especially for glucose type structures previous syntheses proved to be challenging and low yielding. Our novel approach employs indoxylic acid esters as precious intermediates to yield a considerable number of indoxyl glycosides in good yields.

的吲哚酚苷的检测糖苷酶活动的合成

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

蛋白质生物缀合物的合成<em&gt;通过</em&gt;半胱氨酸马来酰亚胺化学

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Color Spot Test As a Presumptive Tool for the Rapid Detection of Synthetic Cathinones

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and other border protection agencies globally. Color testing is a presumptive identification technique indicating the presence or absence of a particular drug class using rapid and uncomplicated chemical methods. Owing to their relatively recent emergence, a color test for the specific identification of synthetic cathinones is not currently available. In this study, we introduce a protocol for the presumptive identification of synthetic cathinones, employing three aqueous reagent solutions: copper(II) nitrate, 2,9-dimethyl-1,10-phenanthroline (neocuproine) and sodium acetate. Small pin-head sized amounts (approximately 0.1-0.2 mg) of the suspected drugs are added to the wells of a porcelain spot plate, and each reagent is then added dropwise sequentially before heating on a hotplate. A color change from very light blue to yellow-orange after 10 min indicates the likely presence of synthetic cathinones. The highly stable and specific test reagent has the potential for use in the presumptive screening of unknown samples for synthetic cathinones in a forensic laboratory. However, the nuisance of an added heating step for the color change result limits the test to laboratory application and decreases the likelihood of an easy translation to field testing.

Here we present a simple, inexpensive, and selective chemical spot test protocol for the detection of synthetic cathinones, a class of new psychoactive substances. The protocol is suitable for use in various areas of law enforcement that encounter illicit material.

色斑试验作为合成 Cathinones 快速检测的推定工具

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and ...

Breath Collection from Children for Disease Biomarker Discovery

Breath collection and analysis can be used to discover volatile biomarkers in a number of infectious and non-infectious diseases, such as malaria, tuberculosis, lung cancer, and liver disease. This protocol describes a reproducible method for sampling breath in children and then stabilizing breath samples for further analysis with gas chromatography-mass spectrometry (GC-MS). The goal of this method is to establish a standardized protocol for the acquisition of breath samples for further chemical analysis, from children aged 4-15 years. First, breath is sampled using a cardboard mouthpiece attached to a 2-way valve, which is connected to a 3 L bag. Breath analytes are then transferred to a thermal desorption tube and stored at 4-5 &#176;C until analysis. This technique has been previously used to capture breath of children with malaria for successful breath biomarker identification. Subsequently, we have successfully applied this technique to additional pediatric cohorts. The advantage of this method is that it requires minimal cooperation on part of the patient (of particular value in pediatric populations), has a short collection period, does not require trained staff, and can be performed with portable equipment in resource-limited field settings.

This protocol describes a simple method for the acquisition of breath samples from children. Briefly, samples of mixed air are pre-concentrated in sorbent tubes prior to gas chromatography-mass spectrometry analysis. Breath biomarkers of infectious and non-infectious diseases can be identified using this breath collection method.

儿童疾病生物标志物发现的呼吸收集

Breath collection and analysis can be used to discover volatile biomarkers in a number of infectious and non-infectious diseases, such as malaria, ...

Preparation and Application of a New Bacterial Biosensor for the Presumptive Detection of Gunshot Residue

MicRoboCop is a biosensor that has been designed for a unique application in forensic chemistry. MicRoboCop is a system made up of three devices that, when used together, can indicate the presence of gunshot residue (GSR) by producing a fluorescence signal in the presence of three key analytes (antimony, lead, and organic components of GSR). The protocol describes the synthesis of the biosensors using Escherichia coli (E. coli), and the analytical chemistry methods used to evaluate the selectivity and sensitivity of the sensors. The functioning of the system is demonstrated by using GSR collected from the inside of a spent cartridge casing. Once prepared, the biosensors can be stored until needed and can be used as a test for these key analytes. A positive response from all three analytes provides a presumptive positive test for GSR, while each individual device has applications for detecting the analytes in other samples (e.g., a detector for lead contamination in drinking water). The main limitation of the system is the time required for a positive signal; future work may involve studying different organisms to optimize the response time.

A protocol is presented using synthetic biology techniques to synthesize a set of bacterial biosensors for the analysis of gunshot residue, and to test the functioning of the devices for their intended use using fluorescence spectroscopy.

一种用于射击残留量推定检测的新型细菌生物传感器的研制与应用

MicRoboCop is a biosensor that has been designed for a unique application in forensic chemistry. MicRoboCop is a system made up of three devices that, ...

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands for in vivo investigations. Within this protocol, a robust and reliable remote-controlled radiosynthesis of [11C]SNAP-7941, an antagonist to the melanin-concentrating hormone receptor 1, is described. The radiosynthesis starts with cyclotron produced [11C]CO2 that is subsequently further reacted via a gas-phase transition to [11C]CH3OTf. Then, this reactive intermediate is introduced to the precursor solution and forms the respective radiotracer. Chemical as well as the radiochemical purity are determined by means of RP-HPLC, routinely implemented in the radiopharmaceutical quality control process. Additionally, the molar activity is calculated as it is a necessity for the following real-time kinetic investigations. Furthermore, [11C]SNAP-7941 is applied to MDCKII-WT and MDCKII-hMDR1 cells for evaluating the impact of P-glycoprotein (P-gp) expression on cell accumulation. For this reason, the P-gp expressing cell line (MDCKII-hMDR1) is either used without or with blocking prior to experiments by means of the P-gp substrate (&#177;)-verapamil and the results are compared to the ones observed for the wildtype cells. The overall experimental approach demonstrates the importance of a precise time-management that is essential for every preclinical and clinical study using PET tracers radiolabelled with short-lived nuclides, such as carbon-11 (half-life: 20 min).

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Here, we represent a protocol for the fully automated radiolabelling of [11C]SNAP-7941 and the analysis of the real-time kinetics of this PET-tracer on P-gp expressing and non-expressing cells.

[11C_SNAP-7941] 自动合成和实时动力学评估的技术方面

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands ...

Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. The single-probe, a microscale sampling and ionization device, can be coupled with a mass spectrometer for on-line, rapid SCMS analysis of cellular constituents under ambient conditions. Previously, the single-probe SCMS technique was primarily used to measure cells immobilized onto a substrate, limiting the types of cells for studies. In the current study, the single-probe SCMS technology has been integrated with a cell manipulation system, typically used for in vitro fertilization. This integrated cell manipulation and analysis platform uses a cell-selection probe to capture identified individual floating cells and transfer the cells to the single-probe tip for microscale lysis, followed by immediate mass spectrometry analysis. This capture and transfer process removes the cells from the surrounding solution prior to analysis, minimizing the introduction of matrix molecules in the mass spectrometry analysis. This integrated setup is capable of SCMS analysis of targeted patient-isolated cells present in body fluids samples (e.g., urine, blood, saliva, etc.), allowing for potential applications of SCMS analysis to human medicine and disease biology.

An integrated cell manipulation platform is developed for use in conjunction with a single-probe mass spectrometry setup for the on-line analysis of individual suspension cells under ambient conditions.

集成细胞操作平台与单悬架细胞中药物和代谢物质谱分析的单探针相结合

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. ...