这项研究部分得到了美国国家癌症研究所（斯旺森生物技术中心）的科赫研究所支持资助 P30-CA14051、美国国家环境健康科学研究所的核心中心资助 P30-ES002109、科赫研究所的大理石癌症纳米医学中心、科赫研究所前沿研究计划通过 Kathy 和 Curt 大理石癌症研究基金， 以及弗吉尼亚和D.K.路德维希癌症研究基金。A.E.V.H. 由 NIH 资助的博士前培训奖学金 （T32GM130546） 提供支持。S.N.B. 是霍华德休斯医学研究所的研究员。L.H. 得到了美国国家癌症研究所的 K99/R00 独立之路奖和波士顿大学的启动资金的支持。

所有动物研究均由作者所在机构的机构动物护理和使用委员会（IACUC）批准。需要标准的动物护理设施，包括饲养室、无菌罩、麻醉和伦理终点安乐死，才能正确进行这些实验。所有实验均按照机构和国家指南进行，并由该机构的兽医人员监督。用于实验的雌性BALB / c小鼠是从商业来源获得的（参见 材料表），在研究开始时的年龄范围为6至8周。 补充表1中提供了定制合成的DNA、crRNA、基于FRET的肽底物探针和传感器肽的序列。
1. 蛋白酶激活肽底物选择
<ol><li>根据先前发表的报告13，从健康肺或具有肿瘤结节的肺组织中收集和制备组织样本。<ol><li>使用组织解离管在预冷的磷酸盐缓冲盐水（PBS，pH 7.4）（200mg组织/ mL PBS）中匀浆组织，以将组织自动解离成单细胞悬浮液。</li>
		<li>离心组织在4°C下以6,000× g 匀浆5分钟。 将上清液保留在新管中。</li>
		<li>在4°C下以14,000× g 离心上清液25分钟。</li>
		<li>使用二辛可宁酸（BCA）蛋白检测试剂盒测量蛋白质浓度（参见 材料表）。</li>
		<li>向样品中加入 PBS 以制备 0.33 mg/mL 溶液。</li>
	</ol></li>
	<li>按照以下步骤评估蛋白水解活性。<ol><li>在 384 孔板中，向每个孔中加入 5 μL、6 μM 基于 FRET 的肽底物探针。对于每个探针，一式三份地进行反应。 注意：基于FRET的肽底物探针包含一个短肽序列（6-8个氨基酸，设计为对靶蛋白酶或一组蛋白酶具有特异性），以荧光团和淬灭剂对终止。 补充表1 中描述了两个FRET探头作为示例。完整的探针库可以在Hao等人中找到。13.</li>
		<li>在室温下以180× g 离心孔板10秒，以确保探针位于板的底部。</li>
		<li>向每个孔中加入 25 μL 0.33 mg/mL 组织样品或 40 μM 重组蛋白酶 13 。 注意：组织样品或重组蛋白酶的最终浓度可能需要根据其内在的蛋白水解活性进行优化。例如，高度蛋白水解的组织（如肠道）可能需要更高的稀释因子，以便监测初始酶裂解。</li>
		<li>以180× g （室温）短暂离心孔板，以混合探针和组织裂解物。</li>
		<li>通过在37°C下每2分钟测量荧光1小时（λex：485nm;λem：535nm）来监测解裂，立即开始检测探针切割。 注意：对特定的荧光团和淬灭剂对使用适当的激发和发射波长。在本研究中，为FAM荧光团设置了参数（λex：485 nm和λem：535 nm）。</li>
		<li>要分析荧光测量数据，请使用 https://github.com/nharzallah/NNanotech-Kinetic 提供的 Python（参见 材料表）软件包进行酶动力学分析。此脚本使用前 8-10 个初始时间点的线性拟合斜率计算初始反应速度 （V0）。</li>
	</ol></li>
</ol>2. 传感器配方和表征
<ol><li>合成DNA和蛋白酶活化肽（PAP）的偶联物。<ol><li>在室温下，将20-mer硫代磷酸化DNA报告基因与3'-DBCO基团（1.1当量，参见 材料表）与C末端半胱氨酸末端的叠氮化物末端PAP一起在室温下孵育>4小时。如果放置过夜，则在4°C下孵育。</li>
		<li>在配备生物分子理想色谱柱的高效液相色谱（HPLC）系统上纯化产品（参见材料表）。将HPLC梯度设置为从5%A缓冲液（0.05%TFA在H2O中）开始，保持等度20分钟，并在65分钟达到80%B缓冲液（0.05%TFA，99.95%乙腈），流速为0.3mLmin-1，并收集保留时间为~31分钟的偶联产物。</li>
		<li>使用α-氰基-4-羟基肉桂酸作为基质14，通过基质辅助激光解吸/电离飞行时间 （MALDI-TOF） 质谱法验证纯化的偶联物（参见 材料表）。</li>
	</ol></li>
	<li>合成DNA编码的基于活性的纳米传感器。<ol><li>将 2 mg 多价 PEG（40 kDa，8 臂，参见 材料表）与马来酰亚胺反应性基团溶解在 1 mL 100 mM 磷酸盐缓冲液 （pH 7.0） 和过滤器（截止值：0.2 μm）中。</li>
		<li>将半胱氨酸终止的DNA-肽偶联物（2当量，参见 材料表）加入PEG中，并在室温下反应>4小时。如果放置过夜，则在4°C下孵育。</li>
		<li>在快速蛋白质液相色谱（FPLC）上使用市售的葡聚糖-琼脂糖复合基质柱使用尺寸排阻色谱除去未偶联的材料（参见 材料表）。在PBS中运行样品，并在260nm处监测DNA的吸光度，在280nm处监测肽的吸光度。</li>
		<li>根据制造商推荐的速度，用离心过滤管（MWCO = 10 kDa）浓缩合成的纳米传感器（参见 材料表）。</li>
		<li>使用ssDNA检测试剂盒（参见 材料表）和λex：485nm和λem：535nm的酶标仪定量DNA的浓度。将纳米传感器储存在4°C。</li>
	</ol></li>
	<li>表征基于DNA编码活性的纳米传感器。<ol><li>通过动态光散射 （DLS） 测量 DNA 编码纳米传感器的流体动力学粒径。 注：纳米传感器的预期尺寸范围为15-50纳米，平均尺寸为20-30纳米。如果需要有限的尺寸范围，FPLC（在步骤2.3中）可用于分离具有不同分子量的较窄级分。</li>
		<li>用离心过滤管（MWCO = 10 kDa）将纳米传感器浓缩至0.5mg / mL（通过DNA浓度），并将样品加载到安装在低温支架上的碳膜涂层铜网格上。使用低温透射电子显微镜（200 kV，放大倍率10,000-60,000）观察形态14。</li>
	</ol></li>
</ol>3. 传感器注射和尿液收集
<ol><li>收集尿液进行基线测量。<ol><li>将鼠标放入以96孔板为底的定制壳室（参见 补充图1）中。</li>
		<li>束缚鼠标并在膀胱上轻轻按压，以将任何剩余的尿液排到板上。</li>
		<li>将小鼠更换到正常外壳后，将收集的尿液（~100-200μL）从96孔板移液到1.5mL管中。</li>
	</ol></li>
	<li>建立临床前小鼠肿瘤模型。<ol><li>通过静脉注射表达荧光素酶的MC26-Fluc细胞系（100k细胞/小鼠）接种6至8周龄的BALB / c雌性小鼠（参见 材料表）。每周使用 体内 荧光成像系统监测肿瘤进展。 注意：发光信号指示的可见肿瘤负荷大约发生在注射该特定细胞系的第 2 周。在肿瘤进展过程中定期仔细检查荷瘤动物。</li>
	</ol></li>
	<li>在肿瘤植入后的不同时间点注射纳米传感器。<ol><li>在无菌PBS中通过DNA条形码制备含有浓度为1nmol的纳米传感器的注射溶液（最大体积为200μL）。</li>
		<li>将PBS中的200μL传感器溶液静脉注射到每只实验小鼠中。</li>
	</ol></li>
	<li>如步骤1.1-1.3所述，在传感器注射后1小时从健康对照和荷瘤小鼠收集尿液样本。 注意：新鲜尿液样本可以直接进行DNA条形码分析，也可以立即在冰上冷冻。</li>
</ol>4. DNA条形码的CRISPR检测：基于荧光
<ol><li>使用新鲜的尿液样本或在冰上解冻冷冻样本。在室温下以800× g 离心尿液样品5分钟。</li>
	<li>将 补充表2中的试剂混合，最后加入Cas12a酶（见 材料表），并通过上下移液轻轻混合反应。将反应在37°C孵育30分钟。</li>
	<li>如 补充表3所示，使用步骤2的产物一式三份运行报告反应。最后加入步骤2的反应，并快速送至酶标仪。</li>
	<li>通过在37°C下每2分钟测量荧光3小时（λex：485nm和λem：535nm）来检测LbaCas12a活化，以监测DNA报告基因的切割动力学。</li>
	<li>要分析荧光测量数据，请利用 https://github.com/nharzallah/NNanotech-Kinetic 提供的 Python 包进行酶动力学分析。此脚本使用前 8-10 个初始时间点的线性拟合斜率计算初始反应速度 （V0）。</li>
</ol>5. DNA条形码的CRISPR检测：纸质
<ol><li>在室温下以800× g 离心尿液样品5分钟。 注意：同时运行尿液样本进行基于荧光和纸张的CRISPR检测。</li>
	<li>合并 附表2中的试剂。在37°C孵育30分钟。 注意：该孵育步骤与基于荧光的CRISPR检测相同。</li>
	<li>使用FAM-生物素标记的DNA报告基因在纸条上进行侧向层析测定（参见 材料表）。使用步骤2的产物将 补充表4 中的试剂合并在96孔板中。用铝箔覆盖并在37°C下孵育1小时。 注意：根据上述基于荧光的CRISPR检测测定中的实时动力学监测选择最佳孵育时间。</li>
	<li>向 96 孔板的新鲜孔中加入 80 μL PBS。将步骤 3 中的 20 μL 样品加入该孔中。</li>
	<li>将一个侧流纸条放在每个孔中，等待液体到达条带顶部（<5分钟）。在纸条上查找对照和/或样品条的外观。</li>
	<li>拍摄侧流带的照片并使用 ImageJ 量化条带强度。</li>
</ol>

用于癌症诊断的 CRISPR-Cas 合成尿液生物标志物测试

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S.N.B.、L.H. 和 R.T.Z. 在与本作品内容相关的专利申请中被列为发明人。S.N.B. 持有 Glympse Bio、Satellite Bio、Lisata Therapeutics、Port Therapeutics、Intergalactic Therapeutics、Matrisome Bio 的股权，并且是 Vertex 的董事;为Moderna提供咨询，并获得强生公司（Johnson & Johnson）、Revitope和Owlstone公司赞助的研究资金。

这里介绍的是一个高度可定制的平台，用于多路复用癌症检测，使用便携式尿液检测，使用微创注射传感器评估与疾病相关的蛋白水解活性。当被肿瘤蛋白酶激活时，肽底物裂解 通过 释放到尿液中的 DNA 条形码进行扩增。尿液样本中的合成DNA报告基因可以通过使用荧光检测或简单的纸质检测的二次CRISPR-Cas介导的酶扩增来读出。DNA条形码是一种有吸引力的标记合成生物标志物的方法，可提?...

尽管在鉴定脱落蛋白和 DNA 等肿瘤生物标志物方面付出了巨大努力，但癌症诊断领域仍因其在循环中的低丰度或快速降解而备受关注1。作为一种补充策略，选择性地响应疾病特征以产生放大信号的生物工程合成生物标志物代表了实现准确和可及诊断的新途径2,3。为了帮助检测，这些合成生物标志物利用肿瘤依赖性激活机制（如酶扩增）来生产具有改进信噪比的分析物4。本文利用一类癌症相关酶蛋白酶来激活可注射的纳米级传感器，以释放可从生物体液（如血液或尿液）中检测到的疾病报告基因5,6。鉴于肿瘤的异质性，开发一组蛋白酶激活的传感器可以进行多分析物测试，将不同的蛋白酶切割事件组合成一个“疾病特征”，以更特异性、多重的方式评估癌症的发病率和进展。
已经开发了蛋白酶激活的合成生物标志物，其包含偶联到惰性载体表面的肽底物7。当在体内注射时，这些肽被带到肿瘤中，然后肿瘤蛋白酶的酶裂解将报告基因释放到血液或尿液中进行检测。使用蛋白酶激活的合成生物标志物进行多重检测时，混合物中的每种合成生物标志物都必须使用唯一的分子条形码进行标记。为此，已经开发了各种方法，包括质量条形码和配体编码报告基因8,9,10。与这些可能仅限于几种不同信号可能性的多重方法相反，DNA条形码根据人类疾病状态的高度复杂性和异质性提供了更多的组合。为了扩大合成生物标志物的多重性，传感器通过用独特的DNA序列标记每个报告基因来对传感器进行条形码编码，以便通过CRISPR-Cas核酸酶进行检测，以放大离体生物流体信号。这些单链 DNA （ssDNA） 条形码旨在与互补的 CRISPR 向导 RNA （crRNA） 结合，激活 CRISPR-Cas12a11 的靶向触发的侧支核酸酶活性。这种核酸酶活性可用于切割通过荧光动力学或使用纸条检测到的报告基因 DNA 链。
除了通过蛋白酶（体内）和 CRISPR-Cas（离体）进行分子扩增外，蛋白酶激活的合成生物标志物的另一个关键设计特征涉及利用纳米材料药代动力学来增加生物流体中的诊断信号浓度10。一种方法是使用纳米颗粒载体来增加表面共轭肽底物的循环时间。选择聚乙二醇（PEG）树枝状聚合物作为具有相对较小的流体动力学直径和多价的纳米载体，以增加向肿瘤的递送。虽然 PEG 载体足够小以促进肿瘤递送，但其尺寸大于肾小球滤过屏障的 ~5 nm 尺寸截止值，因此只有裂解的肽底物才能清除到尿液中，利用肾脏的大小过滤12。在该协议中，概述了在临床前小鼠模型中合成和应用基于 DNA 条形码活性的纳米传感器的多步骤工作流程，重点介绍了 CRISPR-Cas 介导的多分析物合成尿液生物标志物测试的设置，该小组已采用该测试对多种癌症类型小鼠模型中的疾病状态进行分类 13.由于多功能设计原理，纳米传感器的所有三个功能组件——纳米载体（PEG聚合物）、刺激响应模块（蛋白酶激活底物）和生物流体报告基因（DNA条形码）——都可以根据特定应用需求精确互换，通过定制靶标和释放特异性来实现模块化。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x NEB Buffer 2.1</td>
			<td>New England Biolabs</td>
			<td>B6002SVIAL</td>
			<td></td>
		</tr>
		<tr>
			<td>20-mer phosphorothioated DNA reporters with 3&#8217;-DBCO group</td>
			<td>IDT</td>
			<td></td>
			<td>Custom DNA</td>
		</tr>
		<tr>
			<td>Agilent 1100 High Performance Liquid Chromatography system with Vydac 214TP510 C4 column</td>
			<td>&#160;Agilent</td>
			<td></td>
			<td>HPLC</td>
		</tr>
		<tr>
			<td>&#196;KTA fast protein liquid chromatography (FPLC)</td>
			<td>GE Healthcare</td>
			<td></td>
			<td>FPLC</td>
		</tr>
		<tr>
			<td>Amicon ultracentrifuge tubes (MWCO = 10 kDa)</td>
			<td>EMD millipore</td>
			<td></td>
			<td>Various volumes available</td>
		</tr>
		<tr>
			<td>Azide-terminated PAPs with C-terminus cysteine</td>
			<td>CPC Scientific</td>
			<td></td>
			<td>Custom peptide</td>
		</tr>
		<tr>
			<td>crRNAs&#160;</td>
			<td>IDT</td>
			<td></td>
			<td>See Supplementary Table 1</td>
		</tr>
		<tr>
			<td>Cryogenic transmission electron microscopy</td>
			<td>JEM-2100F</td>
			<td>JEOL</td>
			<td>cyroTEM</td>
		</tr>
		<tr>
			<td>Cysteine terminated DNA-peptide conjugates</td>
			<td>CPC Scientific</td>
			<td></td>
			<td>Custom peptide</td>
		</tr>
		<tr>
			<td>Dynamic light scattering (DLS)</td>
			<td></td>
			<td></td>
			<td>DLS</td>
		</tr>
		<tr>
			<td>EnGen LbaCas12a (Cpf1), 100 &#181;M</td>
			<td>New England Biolabs</td>
			<td>M0653T</td>
			<td></td>
		</tr>
		<tr>
			<td>Experimental animals</td>
			<td>Taconic Biosciences</td>
			<td>BALB/cAnNTac</td>
			<td>6&#8211;8&#8201;weeks of age</td>
		</tr>
		<tr>
			<td>gentleMACS C tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-237</td>
			<td>tissue homogenization</td>
		</tr>
		<tr>
			<td>HybriDetect Universal Lateral Flow Assay Kit</td>
			<td>Miltenyi Biotec</td>
			<td>MGHD 1</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrix-assisted laser desorption/ionization&#8211;time of flight (MALDI&#8211;TOF) mass spectrometry&#160;</td>
			<td>Bruker</td>
			<td></td>
			<td>Microflex MALDI&#8211;TOF</td>
		</tr>
		<tr>
			<td>MC26-Fluc cell line</td>
			<td></td>
			<td></td>
			<td>Kenneth K. Tanabe Laboratory, Massachusetts General Hospital</td>
		</tr>
		<tr>
			<td>multivalent PEG (40 kDA, 8-arm) with maleimide-reactive group</td>
			<td>JenKem</td>
			<td>A10020-1 / 8ARM(TP)-MAL-40K,1 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Python, Version 3.9</td>
			<td></td>
			<td></td>
			<td>https://www.python.org/</td>
		</tr>
		<tr>
			<td>Quant-iT OliGreen ssDNA Assay Kit and Quant-iT OliGreen ssDNA Reagent</td>
			<td>Invitrogen</td>
			<td>O11492</td>
			<td>ssDNA assay kit</td>
		</tr>
		<tr>
			<td>ssDNA FAM-T10-Quencher and&#160; FAM-T10-Biotin reporter substrates</td>
			<td>IDT</td>
			<td></td>
			<td>Custom DNA</td>
		</tr>
		<tr>
			<td>Superdex 200 Increase 10/300 GL column</td>
			<td>GE Healthcare</td>
			<td>GE28-9909-44</td>
			<td>For FPLC</td>
		</tr>
		<tr>
			<td>Tecan Infinite Pro M200 plate reader</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ThermoFisher Pierce BCA Protein Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
	</tbody>
</table>

crispr-cas-mediated multianalyte synthetic urine biomarker test for portable diagnostics

为开发精确诊断创建合成生物标志物，可以通过超越传统生物流体测量的途径检测疾病。合成生物标志物通常利用在生物流体中提供可读信号的报告基因来反映疾病发病和进展过程中局部疾病微环境中的生化变化。报告基因的药代动力学浓度和疾病信号的生化扩增对于在诊断测试中实现高灵敏度和特异性至关重要。在这里，使用一种形式的合成生物标志物构建了一个癌症诊断平台：基于活性的纳米传感器携带化学稳定的DNA报告基因，可以通过肿瘤微环境中异常的蛋白水解特征释放出来。合成DNA作为疾病报告基因，通过将其用作条形码提供多重检测能力，允许一次读取多个蛋白水解特征。通过与CRISPR RNA杂交，使用CRISPR核酸酶检测释放到尿液中的DNA报告基因，CRISPR RNA在酶激活时产生荧光或比色信号。在该协议中，构建了DNA条形码，基于活性的纳米传感器，并在转移性结直肠癌的临床前小鼠模型中举例说明了它们的应用。该系统可根据疾病生物学进行高度修改，并同时产生多种疾病信号，通过微创过程提供对疾病特征的全面了解，只需纳米传感器给药、尿液收集和纸质测试即可实现即时诊断。

提名蛋白酶激活肽底物 为了设计能够反映组织蛋白水解活性变化的传感器，首先使用肽探针库13 表征组织中的蛋白酶活性（图1）。通过将组织样本与设计用于检测底物切割的FRET探针相结合，新鲜和冷冻的组织样品可以提供有关肿瘤微环境蛋白水解活性的大量信息。将具有 FAM 荧光基团和 CPQ-2 淬灭剂的 FRET探针库与组织样品一起孵育。通过荧...

Watch this Scientific Journal Video about CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics at JoVE.com

作者聚焦：用于疾病检测和成像的工程分子工具

该协议描述了一种CRISPR-Cas介导的多分析物合成尿液生物标志物测试，该测试通过对肿瘤相关蛋白酶活性的离体分析来实现即时癌症诊断。

CRISPR-Cas介导的多分析物合成尿液生物标志物检测用于便携式诊断

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for ...

心脏实验室正在设计分子和细胞工具，以精确跟踪和控制疾病生物学。我们使用有关病变组织的特殊信息来为异质性疾病的响应性检测、成像和治疗工具的设计提供信息。生物流体为疾病检测和评估提供了便利的机会，但这些机会往往受到相关分析物数量少的限制。因此，该领域的大部分都在寻求放大疾病相关信号的策略，以提高诊断测试的灵敏度，如BCR和表面等离子体共振。我们的方法不是依赖循环中的内源性疾病生物标志物，而是使用合成生物标志物来报告从注射的纳米传感器释放时的疾病活动。我们的合成生物标志物是化学稳定的DNA条形码，可以同时检测多种疾病微环境信号，并使用相应的条形码进行识别。

crispr-cas-mediated-multianalyte-synthetic-urine-biomarker-test-for

Research

JoVE Journal

Bioengineering

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics

712 次观看.  Boston University. 心脏实验室正在设计分子和细胞工具，以精确跟踪和控制疾病生物学。我们使用有关病变组织的特殊信息来为异质性疾病的响应性检测、成像和治疗工具的设计提供信息。生物流体为疾病检测和评估提供了便利的机会，但这些机会往往受到相关分析物数量少的限制。因此，该领域的大部分都在寻求放大疾病相关信号的策略，以提高诊断测试的灵敏度，如BCR和表面等离子?...

视频: 作者聚焦：用于疾病检测和成像的工程分子工具

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.

Development of custom assays on the ViBE platform for more sensitive, reproducible, automated results in complex matrices is described. The universal cartridge allows assays to be easily adapted for use with custom assays. This versatility enables rapid development and validation of novel assays or automated versions of existing manual assays, exemplified in this video.

开发自定义的中国仓鼠卵巢宿主细胞蛋白检测使用声膜微粒子技术

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

科研

生物工程

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

血浆或尿液的水凝胶纳米粒子收获检测低丰度蛋白

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for specialized equipment and trained personnel require that many diagnostic tests be performed at remote, centralized clinical laboratories. Magnetic levitation is a simple yet powerful technique and can be applied to levitate cells, which are suspended in a paramagnetic solution and placed in a magnetic field, at a position determined by equilibrium between a magnetic force and a buoyancy force. Here, we present a versatile platform technology designed for point-of-care diagnostics which uses magnetic levitation coupled to microscopic imaging and automated analysis to determine the density distribution of a patient's cells as a useful diagnostic indicator. We present two platforms operating on this principle: (i) a smartphone-compatible version of the technology, where the built-in smartphone camera is used to image cells in the magnetic field and a smartphone application processes the images and to measures the density distribution of the cells and (ii) a self-contained version where a camera board is used to capture images and an embedded processing unit with attached thin-film-transistor (TFT) screen measures and displays the results. Demonstrated applications include: (i) measuring the altered distribution of a cell population with a disease phenotype compared to a healthy phenotype, which is applied to sickle cell disease diagnosis, and (ii) separation of different cell types based on their characteristic densities, which is applied to separate white blood cells from red blood cells for white blood cell cytometry. These applications, as well as future extensions of the essential density-based measurements enabled by this portable, user-friendly platform technology, will significantly enhance disease diagnostic capabilities at the point of care.

We present a magnetic levitation technique coupled with automated imaging and analysis in both a smartphone-compatible device and a device with embedded imaging and processing. This is applied to measure the density distribution of cells with two demonstrated biomedical applications: sickle cell disease diagnosis and separating white and red blood cells.

磁悬浮加上便携式成像与分析诊断疾病

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

纳米传感器检测体内蛋白酶活性的无创诊断

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Methylation Specific Multiplex Droplet PCR using Polymer Droplet Generator Device for Hematological Diagnostics

A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, along with a thermoplastic elastomer (TPE) microfluidic droplet generation device. Epigenetic markers are used for WBC&#160;subtyping which is of important prognostic value in different diseases. This is achieved through the quantification of DNA methylation patterns of specific CG-rich regions in the genome (CpG loci). In this paper, bisulfite-treated DNA from peripheral blood mononuclear cells (PBMCs) is encapsulated in droplets with mdPCR reagents including primers and hydrolysis fluorescent probes specific for CpG loci that correlate with WBC sub-populations. The multiplex approach allows for the interrogation of many CpG loci without the need for separate mdPCR reactions, enabling more accurate parametric determination of WBC sub-populations using epigenetic analysis of methylation sites. This precise quantification can be extended to different applications and highlights the benefits for clinical diagnosis and subsequent prognosis.

Epigenetic markers are used for white blood cell (WBC) subtyping through the quantification of DNA methylation patterns. This protocol presents a multiplex droplet polymerase chain reaction (mdPCR) method using a thermoplastic elastomer (TPE)-based microfluidic device for droplet generation allowing for precise and multiplex methylation-specific target quantification of WBC differential counts.

甲基化特异性多路复用液滴PCR，使用聚合物液滴发生器设备进行血液诊断

A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, ...

Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression of a wide range of diseases including breast, ovarian, kidney, and pancreatic cancers. Freely available fiber quantification software tools are mainly focused on the calculation of fiber alignment or orientation, and they are subject to limitations such as the requirement of manual steps, inaccuracy in detection of the fiber edge in noisy background, or lack of localized feature characterization. The collagen fiber quantitation tool described in this protocol is characterized by using an optimal multiscale image representation enabled by curvelet transform (CT). This algorithmic approach allows for the removal of noise from fibrillar collagen images and the enhancement of fiber edges to provide location and orientation information directly from a fiber, rather than using the indirect pixel-wise or window-wise information obtained from other tools. This CT-based framework contains two separate, but linked, packages named &#8220;CT-FIRE&#8221; and &#8220;CurveAlign&#8221; that can quantify fiber organization on a global, region of interest (ROI), or individual fiber basis. This quantification framework has been developed for more than ten years and has now evolved into a comprehensive and user-driven collagen quantification platform. Using this platform, one can measure up to about thirty fiber features including individual fiber properties such as length, angle, width, and straightness, as well as bulk measurements such as density and alignment. Additionally, the user can measure fiber angle relative to manually or automatically segmented boundaries. This platform also provides several additional modules including ones for ROI analysis, automatic boundary creation, and post-processing. Using this platform does not require prior experience of programming or image processing, and it can handle large datasets including hundreds or thousands of images, enabling efficient quantification of collagen fiber organization for biological or biomedical applications.

Here, we present a protocol to use a curvelet transform-based, open-source MATLAB software tool for quantifying fibrillar collagen organization in the extracellular matrix of both normal and diseased tissues. This tool can be applied to images with collagen fibers or other types of line-like structures.

使用基于曲线的转换工具量化纤维胶原蛋白组织

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

在微流体平台内开发 3D 组织人类心脏组织

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications

Streptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a natural producer of chloramphenicol, jadomycin, and pikromycin, which makes it an attractive candidate&#160;as a next-generation synthetic biology chassis. Therefore, genetic tools that accelerate the development of S. venezuelae ATCC 10712, as well as other Streptomyces spp. models, are highly desirable for natural product engineering and discovery. To this end, a dedicated S. venezuelae ATCC 10712 cell-free system is provided in this protocol to enable high-yield heterologous expression of high G+C (%) genes. This protocol is suitable for small-scale (10-100 &#956;L) batch reactions in either 96-well or 384-well plate format, while reactions are potentially scalable. The cell-free system is robust and can achieve high yields (~5-10 &#956;M) for a range of recombinant proteins in a minimal setup. This work also incorporates a broad plasmid toolset for real-time measurement of mRNA and protein synthesis, as well as in-gel fluorescence staining of tagged proteins. This protocol can also be integrated with high-throughput&#160;gene expression characterization workflows or the study of enzyme pathways from high G+C (%) genes present in Actinomycetes genomes.

A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications

This protocol details an enhanced method for synthesizing high yields of recombinant proteins from a Streptomyces venezuelae cell-free transcription-translation (TX-TL) system.

用于合成生物学和天然产物应用的高产 链霉菌 转录翻译工具包

Streptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a ...

膜蛋白在巨型单层囊泡中的无细胞表达

Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.

作者聚焦：合成细胞中的膜蛋白重构

This protocol describes the inverted emulsion method used to encapsulate a cell-free expression (CFE) system within a giant unilamellar vesicle (GUV) for the investigation of the synthesis and incorporation of a model membrane protein into the lipid bilayer.