这项工作由 NIH 主任的新创新奖 (奖号) 资助。DP2HD091793)。Q.D.M. 得到 NSF 研究生研究奖学金计划的支持 (批准号:DGE-1650044)。H 获得美国国立卫生研究院 GT BioMAT 训练补助金的支持, 5T32EB006343 和格鲁吉亚技术总裁的奖学金。G.A.K. 在 "宝来欢迎基金" 的科学界面上担任职业奖。内容完全是作者的责任, 不一定代表国家卫生研究院的官方意见。

在研究机构的机构动物护理和使用委员会 (IACUC) 的机构批准是进行以下动物实验的必要。此外, 标准的动物保育设施 (例如,住房室、无菌的动物罩、麻醉的 isofluorane 室和为道德端点 euthanization 的 CO2室) 是必要的, 以便适当地执行这些实验。对这些设施的特殊培训和协助可以由一个机构的生理学研究实验室提供。所有的动物工作都是通过 IACUC 在佐治亚理工大学 (礼宾: A14100) 批准的。
1. 氧化铁纳米颗粒 (IONP) 合成
注: 安全: 整个氧化铁纳米颗粒的合成应使用个人防护设备和在化学油烟机下面。
<ol><li>在一个15毫升圆锥管, 冷却1毫升30% 氢氧化铵在一个冰桶30分钟。</li>
	<li>用去离子水洗250毫升锥形瓶, 加入10毫升双蒸馏水 (ddH2O)。将瓶子的外面浸入一个放在搅拌/热板上的冰浴中。使用塑料吸管将 N2气体流动到瓶中的 ddH2O。气泡15分钟, N2到 deoxygenate。</li>
	<li>称224µmol (4.5 克) 葡聚糖 (分子量 (兆瓦) = 20 kDa) 为50毫升圆锥管。添加 H2O, 使混合物的最终体积, 包括葡聚糖, 是20毫升。漩涡大力溶解葡聚糖。</li>
	<li>将290µmol (78.5 克) 的氯化铁 (III) 的重量加到六水合物中, 加入葡聚糖溶液和涡旋溶解。</li>
	<li>通过0.2 µm 筛选器筛选结果解决方案。</li>
	<li>将1毫升冷却氢氧化铵转化为9毫升的脱氧水。返回到4°c。</li>
	<li>重367µmol (73.0 克) 的氯化铁 (II), 溶解在1毫升无氧 ddH2O, 并过滤通过0.2 µm 过滤器。</li>
	<li>将葡聚糖铁 (III) 溶液转化成250毫升锥形瓶和 deoxygenate, 在冰上15分钟。与 1600 rpm 旋转的磁力搅拌杆混合。</li>
	<li>要创造一个均匀的氮气气氛, 用橡胶隔膜盖上烧瓶。用18口径的针刺穿隔膜, 将2到烧瓶中流动。插入一个单独的18测量针作为流量出口。</li>
	<li>添加467µL 的铁 (II) 溶液的葡聚糖铁 (III) 溶液与1毫升注射器, 搅拌与磁搅拌杆在 1600 rpm。铁 (II) 与铁 (III) 的比值导致了平衡反应, 将产生磁铁矿, 或 Fe3O4。</li>
	<li>加入冷稀释氢氧化铵滴入铁 (II)-铁 (III)-葡聚糖溶液, 以启动成核过程21。确保每个液滴在掉入另一粒之前混合良好。在加入1-2 毫升氢氧化铵后完成反应。</li>
	<li>停止流动的 N2和消除橡胶隔膜。除去冰浴, 用温水浴代替, 同时继续搅拌溶液。确保溶液的温度达到75摄氏度, 孵育75分钟。</li>
	<li>从搅拌/热板中取出烧瓶, 然后通过0.2 µm 0.1 µm 过滤器将溶液加倍过滤, 去除粗颗粒。</li>
	<li>缓冲液将微粒转化成 ddH2O, 使用 100 kDa 分子量切断 (m.w.c.) 选矿厂, 从溶液中去除多余的葡聚糖, 这应该是非常粘性的。如果在 2-3 旋转之后, 即使流通过仍然是暗的, 则替换筛选器, 这表明筛选器可能已损坏。<ol>
<li>要使用自旋过滤器进行缓冲交换, 离心机在4°c 4800 X g 处15分钟, 丢弃流程, 并向 IONP 解决方案中添加新缓冲区。重复3-5 次。</li>
	</ol>
</li>
	<li>用分光光度计/平板阅读器测定纳米粒子的浓度。吸光度测量在400毫微米和使用 IONP 的摩尔吸收率在这个波长 (ε = 2.07 x 106 cm-1 M-1) 确定 IO 微粒的浓度。</li>
	<li>并用重悬 IONPs 到10毫克/毫升在 ddH2O (通常总体积是 ~ 3 毫升), 并确保使用聚丙烯管这一步骤的化学相容性。添加1.6 卷5米氢氧化钠, 然后添加0.65 容量的环氧氯丙烷。严格混合在室温下的平板振动筛12小时, 以交联葡聚糖。</li>
	<li>使用一个20毫升注射器与一个18口径的针将 IONP 溶液转移到 50 kDa m.w.c. 透析膜。将透析膜放入4升 ddH2o, 并更换 H2o 数次 (每 2-3 小时)。孵化过夜。</li>
	<li>测量浓度, 使 IONPs 5 毫克/毫升 (见步骤 1.15)。加入氢氧化铵达到 20% (v/v), 在室温下摇动 > 12 小时, aminate IONPs 表面。使用 30 kDa m.w.c. 过滤器进行缓冲交换 (见 1.14)。</li>
	<li>用快速蛋白液相色谱法 (FPLC) 在最终纯化前, 将音量调低至 2.5-3.5 毫升; 见凝胶过滤柱材料表)。</li>
	<li>使用动态光散射 (dl) 确定 IONPs 的水动力半径 (预期范围 10-100 nm, 平均大小 40-50 nm)。这样做, 通过 aliquoting 1 毫升100X 稀释 IONP 样品在试管, 在机器的地方, 并使用制造商的软件采取的测量。 注: IONPs 的总人口将在10和100毫微米之间由 dl 测量, 但多数人口是在平均直径附近, 范围从 40-50 毫微米。如果你想要限制大小范围, 你可以进一步使用大小排除色谱分离分数以不同的直径。IONPs 应存放在4摄氏度。</li>
</ol>2. 肽的设计, 共轭到 IONP, 并在体外验证
<ol><li>合成一种多肽基质 (例如,由核心设施或商业) 的目标蛋白酶与 n-终端荧光记者, 如荧光素异硫氰酸酯 (FITC) 和 C 末端半胱氨酸残渣, 以允许硫醇介导的耦合。 注: 在本研究的情况下 MMP9, 使用的肽是 FITC-PLGVRGK C, 与 k猫/km ~ 2.0 x 105 m-1s-1。</li>
	<li>整除0.5 毫克的 IONP (1 毫克/毫升) 和缓冲交换成耦合缓冲 (50 毫米硼酸钠与1毫米 EDTA, pH = 8.5) 使用 10 kDa m.w.c. o. 旋转过滤器 (见步骤 1.14)。</li>
	<li>溶解琥珀酰亚胺碘乙酸 (新加坡) 在甲基酰胺 (DMF) 达到浓度为30毫克/毫升, 并增加新加坡航空 IONP 摩尔比500新加坡航空: IONP。 注意: 新加坡航空是一种 heterobifunctional 交叉链接器, 它介导胺类在 IONPs 上的偶联与肽基板上的巯基团。</li>
	<li>在黑暗的室温下孵育 1-2 小时。如果一夜之间离开, 孵育4摄氏度。</li>
	<li>使用 10 kDa m.w.c. 旋转过滤器将缓冲区交换到耦合缓冲区中以删除反应 (参见步骤 1.14)。</li>
	<li>将最终产品解决方案带到1毫克/毫升 (0.5 毫升溶液)。将感兴趣的肽与 90:1 (肽: IONP) 的摩尔比与 20 kDa 硫醇终止的聚乙二醇 (peg) 混合, 摩尔比为 20:1 (peg: IONP)。将这种肽 PEG 溶液与 IONP 混合。</li>
	<li>在室温下以最高速度在平板振动筛上孵化, 以防止荧光分子漂白。</li>
	<li>添加 l-半胱氨酸的摩尔比为 500:1 (C: IONP), 以中和任何反应的新加坡航空分子。在室温下以最高速度在25摄氏度上孵育1小时。</li>
	<li>通过 FPLC 净化 (见 1.1.19)。使用分光光度计测量样品溶液的吸光度, 并计算肽: IONP 比根据以下。提纯后, 在 PBS 的4摄氏度贮存1毫克/毫升的产品。<ol>
<li>使用分光光度计测量样品溶液在 400 nm (sample,400) 的吸光度, 以及荧光所用的最大吸光度波长, 为 FITC (sample,488) 的 488 nm。测量同一两个波长 (400 nm 和 488 nm) 的股票胺 IONP 溶液的吸光度, 分别表示为NP,400和NP,488。</li>
		<li>使用等式1和2计算规范化的400和488值。 <img alt="Equation 1" src="/files/ftp_upload/57937/57937eq1.jpg">等式1 <img alt="Equation 2" src="/files/ftp_upload/57937/57937eq2.jpg">等式2</li>
		<li>400和488表示样本的归一化值。使用这些值计算肽的比率: IONP 与等式3。 <img alt="Equation 3" src="/files/ftp_upload/57937/57937eq3.jpg">等式3</li>
		<li>IONP <img alt="Equation 4" src="/files/ftp_upload/57937/57937eq4.jpg">和<img alt="Equation 5" src="/files/ftp_upload/57937/57937eq5.jpg">荧光的摩尔 absorptivities 分别在哪里和哪里。为这些微粒<img alt="Equation 4" src="/files/ftp_upload/57937/57937eq4.jpg"> = 2.06 x 106 M-1cm-1和为 FITC <img alt="Equation 5" src="/files/ftp_upload/57937/57937eq5.jpg"> = 7.2万 M-1cm-1, 因此价值<img alt="Equation 6" src="/files/ftp_upload/57937/57937eq6.jpg">应该是28.75。典型肽: IONP 比应在20:1 到50:1 的范围内。</li>
	</ol>
</li>
	<li>通过进行体外裂解试验验证探针的功能。<ol>
<li>与 PBS (1% BSA), 使一个18µL 纳米粒子溶液与 200 nM 浓度的肽。混合2µL 的蛋白酶的兴趣 (MMP9; 0.1-1 毫克/毫升)。</li>
		<li>在37摄氏度的平板阅读器中孵育1小时, 进行荧光测量 (使用适当的激发和发射波长, 分别为 FITC 485 nm 和 528 nm), 每 1-2 分钟来监测裂解。</li>
	</ol>
</li>
</ol>3. 癌症的纳米传感器和尿检管理
注: 有关示例模型的详细信息, 请参阅我们以前的报告13。
<ol><li>建立一个新陈代谢的笼子, 通过确保一个圆柱套到96井板顶部的尿收集。在收集尿液时, 将一只老鼠放在袖子内, 用培养皿盖盖住, 以防动物逃跑。</li>
	<li>在无菌盐水中, 用肽在3000毫克/千克 (~ 50 µmol) 的浓度下, 准备注射液 (200 µL 最大容积)。</li>
	<li>在雌性, 8 周大的裸鼠, 携带异种 LS174T 大肠肿瘤的负担约100-300 毫米3, 这应该发生在大约10天, 通过尾静脉注射管理 nanosensor 溶液。注射后立即将老鼠放入新陈代谢的笼子里, 注意每只老鼠注射的时间。</li>
	<li>注射后 60-90 分钟, 从96井板中取出圆柱套筒。抑制小鼠, 对膀胱施加轻微的压力, 诱导小鼠将剩余的尿液放到盘子里。收集所有尿液 (200-500 µL)。</li>
	<li>通过免疫沉淀分析尿样, 从尿液中纯化 FITC, 提高灵敏度。使用磁性珠子加上抗 FITC 抗体。<ol>
<li>首先, 用涂层缓冲器清洗25毫克的磁珠3次, 并将最终体积带到225µL。 注意: 要使涂层缓冲 (0.1 米硼酸钠) 是很重要的, ph 值 9.5), 阻断缓冲 (pbs, 0.5% BSA, 0.05% Tween-20, pH 7.4) 和洗涤缓冲 (PBS, 0.1% BSA, 0.05% Tween-20, pH 7.4) 每次新鲜, 每一个大约20毫升是必要的。</li>
		<li>添加200µL 抗 FITC (5 毫克/毫升) 和200µL 硫酸铵 (3 米)。在旋转的 37 C 上孵育16-24 小时。</li>
		<li>将解决方案添加到磁选机上, 取出上清, 替换<img alt="Equation 7" src="/files/ftp_upload/57937/57937eq7.jpg">为625Λ阻塞缓冲器, 并在一夜之间孵化 37 C。然后用洗涤缓冲器清洗3次, 然后在1.25 毫升的洗涤缓冲器中储存。</li>
		<li>孵化2µL 尿与5µL 磁性珠 (20 毫克/毫升) 和带来到总容量到50µL 与 PBS (0.01% 吐温 20)。孵育60分钟。</li>
		<li>每洗一次后, 用磁选机将50µL PBS (0.01% 吐温 20) 洗两次, 收集磁性珠子。</li>
		<li>洗脱两次, 32.5 µL 5% 冰乙酸。中和池洗脱 (70 µL) 与35µL 2 米三, 以达到最终的 pH 值为7。</li>
		<li>阅读在一个板块阅读器 (见材料表) 在适当的激发和发射波长, 以量化尿荧光。计算荧光记者对已知的自由荧光浓度阶梯的浓度。</li>
	</ol>
</li>
</ol>

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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=Public+health+impact+of+achieving+80%+colorectal+cancer+screening+rates+in+the+United+States+by+2018.">Public health impact of achieving 80% colorectal cancer screening rates in the United States by 2018.</a> Cancer. 121 (13), 2281-2285 (2015).</li><li>Mehner, 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=Tumor+cell-produced+matrix+metalloproteinase+9+(MMP-9)+drives+malignant+progression+and+metastasis+of+basal-like+triple+negative+breast+cancer.">Tumor cell-produced matrix metalloproteinase 9 (MMP-9) drives malignant progression and metastasis of basal-like triple negative breast cancer.</a> Oncotarget. 5 (9), 2736-2749 (2014).</li><li>Bremer, C., Tung, C. H., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+molecular+target+assessment+of+matrix+metalloproteinase+inhibition.">In vivo molecular target assessment of matrix metalloproteinase inhibition.</a> Nature Medicine. 7 (6), 743-748 (2001).</li><li>Warren, A. D., 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=Disease+detection+by+ultrasensitive+quantification+of+microdosed+synthetic+urinary+biomarkers.">Disease detection by ultrasensitive quantification of microdosed synthetic urinary biomarkers.</a> Journal of the American Chemical Society. 136 (39), 13709-13714 (2014).</li><li>Warren, A. D., Kwong, G. A., Wood, D. K., Lin, K. Y., Bhatia, S. N. <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+for+noncommunicable+diseases+using+synthetic+urinary+biomarkers+and+paper+microfluidics.">Point-of-care diagnostics for noncommunicable diseases using synthetic urinary biomarkers and paper microfluidics.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (10), 3671-3676 (2014).</li><li>Kwong, G. 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=Mathematical+framework+for+activity-based+cancer+biomarkers.">Mathematical framework for activity-based cancer biomarkers.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (41), 12627-12632 (2015).</li><li>Dudani, J. S., Jain, P. K., Kwong, G. A., Stevens, K. R., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivated+spatiotemporally-responsive+nanosensors+of+in+vivo+protease+activity.">Photoactivated spatiotemporally-responsive nanosensors of in vivo protease activity.</a> ACS Nano. 9 (12), 11708-11717 (2015).</li><li>Palmacci, S., Josephson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+polysaccharide+covered+superparamagnetic+oxide+colloids.">Synthesis of polysaccharide covered superparamagnetic oxide colloids.</a> US patent. , (1991).</li><li>Choi, H. 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=Renal+clearance+of+nanoparticles.">Renal clearance of nanoparticles.</a> Nature Biotechnology. 25 (10), 1165-1170 (2007).</li><li>Harris, T. J., von Maltzahn, G., Derfus, A. M., Ruoslahti, E., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+actuation+of+nanoparticle+self-assembly.">Proteolytic actuation of nanoparticle self-assembly.</a> Angewandte Chemie (International edition in English). 45 (19), 3161-3165 (2006).</li><li>Kwon, E. J., Dudani, J. S., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasensitive+tumour-penetrating+nanosensors+of+protease+activity.">Ultrasensitive tumour-penetrating nanosensors of protease activity.</a> Nature Biomedical Engineering. 1, (2017).</li><li>Villanueva, J., Nazarian, A., Lawlor, K., Tempst, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+Peptidase+activities+in+complex+proteomes+by+MALDI-TOF+mass+spectrometry.">Monitoring Peptidase activities in complex proteomes by MALDI-TOF mass spectrometry.</a> Nature Protocols. 4 (8), 1167-1183 (2009).</li><li>Villanueva, 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=Differential+exoprotease+activities+confer+tumor-specific+serum+peptidome+patterns.">Differential exoprotease activities confer tumor-specific serum peptidome patterns.</a> The Journal of Clinical Investigation. 116 (1), 271-284 (2006).</li></ol>

邝其志是联合创始人, 并担任 Glympse 的顾问, 正在开发与本文所述研究相关的产品。这项研究可能影响他的个人财务状况。格鲁吉亚技术委员会根据其利益冲突政策审查并批准了这一安排的条款。

该方法描述了由共轭纳米粒核的蛋白酶基质组成的活性纳米传感器的发展。蛋白水解的事件被称为 "药代动力学开关", 因为裂解肽产品小于肾脏大小的过滤限制5毫微米23和过滤成尿产生无创信号。因此, 重要的是使用纳米粒子或载体的水动力半径大于 5 nm, 因为任何更小的将被肾脏迅速清除和混淆尿信号。我们使用 pegylated IONPs, 因为他们是 FDA 批准, 耐受性好, 并有循环半衰期 3-5 小...

蛋白酶是多功能酶, 专门研究肽键的水解, 对许多生物过程有显著的控制作用, 包括稳态、allostasis 和疾病1。蛋白酶活性的改变状态与各种疾病相关, 包括癌症和心血管疾病, 使蛋白酶有吸引力的候选者发展成为临床生物标志物2,3。此外, 蛋白酶活性在功能上与不同的梗死再发, 患者的结局和疾病的预后4。广泛地, 生物传感器被开发用于检测各种生物学现象和疾病, 如癌症, 神经退行性疾病, 电子转移过程5,6,7,8,9. 更具体地说, 以基板为基础的蛋白酶传感器用于检测蛋白酶活性, 包括荧光探针, 用于诊断成像10和 isotopically 标记的肽基片体外质谱检测11。此外, 还开发了基于活动的探针, 其中含有与基片类似的区域, 可以约束或修改目标蛋白酶12。采用这种方法, 当活性部位改变时, 目标蛋白酶不可逆转地受到抑制, 分析需要组织的收获, 这限制了体内的应用。然而,在体内对蛋白酶活性的感知是很重要的, 因为对蛋白酶活性的调节严重依赖于其他生物活动的上下文, 如内源性抑制剂的存在。
这项工作的目的是描述以活动为基础的纳米传感器的配方, 通过产生一个可测量的尿液来检测体内蛋白酶的活性。这个平台被用来作为一种无创诊断, 以鉴别复杂的疾病, 如癌症, 使用失调蛋白酶活动作为功能生物标志物。我们的 nanosensor 平台由氧化铁纳米粒子 (IONP) 与蛋白酶基体结合而成。这些基底被一个荧光记者终止, 当蛋白酶切割基体时释放。这些 IONPs在体内循环, 定位到疾病部位, 并暴露基质到活性疾病相关的蛋白酶。乳沟后, 荧光记者被释放, 由于其体积小, 被过滤成尿液, 而 uncleaved 基质上的 IONP 留在体内。因此,体内蛋白酶活性的增加会导致尿液中的记者浓度增高 (图 1)。由于我们的平台是尿液测试, 不需要成像平台和诊断信号丰富的尿液。
这个平台可以被设计来检测各种疾病, 包括癌症, 纤维化, 血栓形成13,14。在这里, 我们描述了纳米传感器的设计, 以检测基质 metallopeptidase 9 (MMP9) 活性作为结直肠癌的生物标志物的海拔。大肠癌是美国癌症死亡的第二大原因, 估计仅在2014年就有136800例新病例和50300人死亡.结直肠肿瘤细胞产生 MMP9, 这已经显示出推动恶性进展, 基质退化, 以及转移16。此外, 我们确定了一个合适的肽基质 (PLGVRGK) 为 MMP9 从文献17。该平台可用于早期癌症检测和低成本的护理点诊断13,14,18,19,20,21。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57937/57937fig1.jpg"> 图 1:体内Nanosensor 活动示意图.纳米传感器循环通过身体和本地化到疾病的地点。然后, IONPs 提出了与疾病相关的蛋白酶劈裂肽基。切割碎片的大小允许肾脏清除, 导致他们在尿中定位。在动物小便后, 这些肽片段可以通过他们的记者分子来分析。<a href="https://www.jove.com/files/ftp_upload/57937/57937fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table><tbody><tr><td>0.2 &amp;micro;m syringe filters</td><td>VWR</td><td>4652</td><td></td></tr><tr><td>18G needle</td><td>VWR</td><td>89134-024</td><td></td></tr><tr><td>15 mL conicals</td><td>VWR</td><td>89039-670</td><td></td></tr><tr><td>250 mL Erlenmeyer flask</td><td>VWR</td><td>89000-362</td><td></td></tr><tr><td>Stir bar</td><td>VWR</td><td>58949-006</td><td></td></tr><tr><td>Hot Plate/Magnetic Stirrer</td><td>VWR</td><td>97042-634</td><td></td></tr><tr><td>Glacial acetic acid</td><td>VWR</td><td>97064-482</td><td></td></tr><tr><td>Albumin from Bovine Serum (BSA)</td><td>Thermo Fisher</td><td>A13100</td><td></td></tr><tr><td>Iron (III) chloride hexahydrate</td><td>Sigma</td><td>236489</td><td></td></tr><tr><td>Iron (II) chloride tetrahydrate</td><td>Sigma</td><td>44939</td><td></td></tr><tr><td>Epichlorohydrin</td><td>Sigma</td><td>45340-500ML-F</td><td></td></tr><tr><td>DMF</td><td>Sigma</td><td>D4551</td><td></td></tr><tr><td>Ammonium Hydroxide</td><td>Sigma</td><td>320145-500ML</td><td></td></tr><tr><td>Sodium Hydroxide pellets</td><td>Sigma</td><td>221465-500G</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E9884</td><td></td></tr><tr><td>Sodium Borate</td><td>Sigma</td><td>B9876</td><td></td></tr><tr><td>L-Cysteine</td><td>Sigma</td><td>168149-100G</td><td></td></tr><tr><td>Tris-HCl</td><td>Sigma</td><td>T5941</td><td></td></tr><tr><td>Tris base</td><td>Sigma</td><td>T6066</td><td></td></tr><tr><td>PBS tablets</td><td>Sigma</td><td>P4417</td><td></td></tr><tr><td>Dextran</td><td>Pharmacosmos</td><td>5510 0020 9006</td><td></td></tr><tr><td>Amicon 15 mL 10k filters, 24 pk</td><td>Millipore</td><td>UFC901024</td><td></td></tr><tr><td>Amicon 15 mL 30k filters, 24 pk</td><td>Millipore</td><td>UFC903024</td><td></td></tr><tr><td>Amicon 15 mL 100k filters, 24 pk</td><td>Millipore</td><td>UFC910024</td><td></td></tr><tr><td>Zetasizer Nano ZS</td><td>Malvern Panalytical</td><td>NanoZS</td><td></td></tr><tr><td>Slide-A-Lyzer Dialysis Cassette</td><td>LifeTech</td><td>66130</td><td></td></tr><tr><td>Dynabeads MyOne Tosylactivated</td><td>LifeTech</td><td>65501</td><td></td></tr><tr><td>SIA</td><td>Life Tech</td><td>22349</td><td></td></tr><tr><td>PEG 20k</td><td>Laysan Bio</td><td>MPEG-SH-20K-1g</td><td></td></tr><tr><td>Fluorescein Antibody [2A3]</td><td>GeneTex</td><td>GTX10257</td><td></td></tr><tr><td>Hiload 16/600 superdex 200</td><td>GE Healthcare</td><td>45-002-490</td><td></td></tr><tr><td>Plate Reader</td><td>Fisher</td><td>BTCYT5M</td><td></td></tr><tr><td>BD Insulin Syringes</td><td>Fisher</td><td>NC0872854</td><td></td></tr></tbody></table>

nanosensors to detect protease activity in vivo for noninvasive diagnostics

蛋白酶是多功能酶, 专门研究肽键的水解和控制广泛的生物过程, 包括稳态和 allostasis。此外, 失调蛋白酶活性驱动发病机制, 是疾病的功能性生物标志物, 如癌症;因此, 检测体内蛋白酶活性的能力可以为生物医学诊断提供临床相关信息。该协议的目的是创造纳米传感器, 通过产生一个可量化的尿液信号, 探索体内蛋白酶的活性。这些蛋白酶纳米传感器由两个成分组成: 纳米粒子和基体。纳米微粒的作用是增加循环半衰期和基质输送到目标疾病地点。基板是一个短肽序列 (6-8 AA), 这是专门针对目标蛋白酶或一组蛋白酶。基体与纳米微粒的表面共轭, 并由记者 (如荧光标记) 终止, 用于检测。当失调蛋白酶切割肽基板时, 记者被过滤成尿液, 作为蛋白酶活性的生物标志物。本文描述了基质金属蛋白酶 9 (MMP9) nanosensor 的构建, 它与肿瘤的进展和转移有关, 用于在小鼠模型中检测大肠癌。

IONPs 的人口的多数是在平均直径附近, 范围从 40-50 毫微米。在聚乙二醇化之后, 这个大小范围有循环半衰期大约6小时13 在体内(参见图 2a)。如果你想选择一个特定的大小范围, 你可以使用大小排除色谱分离 IONP 分数与不同的直径。纳米粒子的透射电镜将显示为单独的球形氧化铁纳米粒子交联在一起的外层葡聚糖。...

Watch this Scientific Journal Video about Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics at JoVE.com

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

蛋白酶是严格调控的酶参与基本的生物学过程, 失调蛋白酶活动推动复杂疾病, 如癌症的进展。该方法的目的是创造纳米传感器, 以测量体内蛋白酶的活动, 产生一个可从宿主尿和歧视性疾病检测到的裂解信号。

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 ...

nanosensors-to-detect-protease-activity-vivo-for-noninvasive

Research

JoVE Journal

Bioengineering

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

16.2K 次观看.  Georgia Tech College of Engineering and Emory School of Medicine. 蛋白酶是严格调控的酶参与基本的生物学过程, 失调蛋白酶活动推动复杂疾病, 如癌症的进展。该方法的目的是创造纳米传感器, 以测量体内蛋白酶的活动, 产生一个可从宿主尿和歧视性疾病检测到的裂解信号。

视频: Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Detection of Protease Activity by Fluorescent Peptide Zymography

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using electrophoresis through a resolving gel embedded with a degradable substrate. This method differs from traditional gel zymography in that a quenched fluorogenic peptide is covalently incorporated into the resolving gel instead of full length proteins, such as gelatin or casein. Use of the fluorogenic peptides enables direct detection of proteolytic activity without additional staining steps. Enzymes within the biological samples cleave the quenched fluorogenic peptide, resulting in an increase in fluorescence. The fluorescent signal in the gels is then imaged with a standard fluorescent gel scanner and quantified using densitometry. The use of peptides as the degradable substrate greatly expands the possible proteases detectable with zymographic techniques.

Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

荧光肽酶谱法检测蛋白酶活性

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

科研

癌症研究

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes.
In this assay system, the applied substrates contain N-terminal hexahistidine (His6) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

Here, we present the detailed procedure of a recently developed protease assay platform utilizing N-terminal hexahistidine/maltose-binding protein and fluorescent protein-fused recombinant substrates attached to the surface of nickel-nitrilotriacetic acid magnetic agarose beads. A subsequent in-gel analysis of the assay samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is also presented.

重组融合蛋白在荧光蛋白酶检测平台中的应用及其凝胶内再生

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

生物化学

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

使用核酸探针进行核酸活性的动力学筛选

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

蛋白酶荧光检测试剂盒，用它来确定蛋白酶活性

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

生物学

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

全自动离心微流体装置从全血超灵敏蛋白检测

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

生物工程

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical communities. The resolution of results obtained from current methods of measuring certain biomarkers can take several days or weeks to obtain, as they can be limited in resolution both spatially and temporally (e.g., fluid compartment microdialysis of interstitial fluid analyzed by enzyme-linked immunosorbent assay [ELISA], high-performance liquid chromatography [HPLC], or mass spectrometry); thus, their guidance of timely diagnosis and treatment is disrupted. In the present study, a unique technique for detecting and measuring peptide transmitters in vivo through the use of a capacitive immunoprobe biosensor (CI probe) is reported. The fabrication protocol and in vitro characterization of these probes are described. Measurements of sympathetic stimulation-evoked neuropeptide Y (NPY) release in vivo are provided. NPY release is correlated to the sympathetic release of norepinephrine for reference. The data demonstrate an approach for the fast and localized measurement of neuropeptides in vivo. Future applications include intraoperative real-time assessment of disease progression and minimally invasive catheter-based deployment of these probes.

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

Established immunochemical methods to measure peptide transmitters in vivo rely on microdialysis or bulk fluid draw to obtain the sample for offline analysis. However, these suffer from spatiotemporal limitations. The present protocol describes the fabrication and application of a capacitive immunoprobe biosensor that overcomes the limitations of the existing techniques.

通过电容式免疫探 针在猪 心中对神经肽动力学进行时间分辨体内测量

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical ...

Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples

Proteases are regulators of countless physiological processes and the precise investigation of their activities remains an intriguing biomedical challenge. Among the ~600 proteases encoded by the human genome, neutrophil serine proteases (NSPs) are thoroughly investigated for their involvement in the onset and progression of inflammatory conditions including respiratory diseases. Uniquely, secreted NSPs not only diffuse within extracellular fluids but also localize to plasma membranes. During neutrophil extracellular trap (NETs) formation, NSPs become an integral part of the secreted chromatin. Such complex behavior renders the understanding of NSPs pathophysiology a challenging task. Here, detailed protocols are shown to visualize, quantify and discriminate free and membrane-bound neutrophil elastase (NE) and cathepsin G (CG) activities in sputum samples. NE and CG are NSPs whose activities have pleiotropic roles in the pathogenesis of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD): they promote tissue remodeling, regulate downstream immune responses and correlate with lung disease severity. The protocols show how to separate fluid and cellular fraction, as well as the isolation of neutrophils from human sputum for enzymatic activity quantification via small-molecule F&#246;rster resonance energy transfer-based (FRET) reporters. To gather specific insights into the relative role of NE and CG activities, a FRET readout can be measured by different technologies: i) in vitro plate reader measurements allow for high-throughput and bulk detection of protease activity; ii) confocal microscopy spatiotemporally resolves membrane-bound activity at the cell surface; iii) small-molecule FRET flow cytometry enables for the rapid evaluation of anti-inflammatory treatments via single-cell protease activity quantification and phenotyping. The implementation of such methods opens the doors to explore NSPs pathobiology and their potential as biomarkers of disease severity for CF and COPD. Given their standardization potential, their robust readout and simplicity of transfer, the described techniques are immediately shareable for implementation across research and diagnostic laboratories.

The protocols herein described provide a guide to visualize and quantify the activity of neutrophil proteases in human sputum. The applications of such analysis span from the evaluation of anti-inflammatory treatments, to biomarker validation, drug screening and large cohort clinical studies.

监测人类痰样中的中性粒细胞和导管素 G 活性

Proteases are regulators of countless physiological processes and the precise investigation of their activities remains an intriguing biomedical ...

免疫与感染

用于癌症诊断的 CRISPR-Cas 合成尿液生物标志物测试

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

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for traditional biofluid measurements. Synthetic biomarkers generally make use of reporters that provide readable signals in the biofluid to reflect the biochemical alterations in the local disease microenvironment during disease incidence and progression. The pharmacokinetic concentration of the reporters and biochemical amplification of the disease signal are paramount to achieving high sensitivity and specificity in a diagnostic test. Here, a cancer diagnostic platform is built using one format of synthetic biomarkers: activity-based nanosensors carrying chemically stabilized DNA reporters that can be liberated by aberrant proteolytic signatures in the tumor microenvironment. Synthetic DNA as a disease reporter affords multiplexing capability through its use as a barcode, allowing for the readout of multiple proteolytic signatures at once. DNA reporters released into the urine are detected using CRISPR nucleases via hybridization with CRISPR RNAs, which in turn produce a fluorescent or colorimetric signal upon enzyme activation. In this protocol, DNA-barcoded, activity-based nanosensors are constructed and their application is exemplified in a preclinical mouse model of metastatic colorectal cancer. This system is highly modifiable according to disease biology and generates multiple disease signals simultaneously, affording a comprehensive understanding of the disease characteristics through a minimally invasive process requiring only nanosensor administration, urine collection, and a paper test which enables point-of-care diagnostics.

作者聚焦:用于疾病检测和成像的工程分子工具

This protocol describes a CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test that enables point-of-care cancer diagnostics through the ex vivo analysis of tumor-associated protease activities.

纳米传感器检测体内蛋白酶活性的无创诊断

摘要

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荧光肽酶谱法检测蛋白酶活性

重组融合蛋白在荧光蛋白酶检测平台中的应用及其凝胶内再生

使用核酸探针进行核酸活性的动力学筛选

蛋白酶荧光检测试剂盒，用它来确定蛋白酶活性

全自动离心微流体装置从全血超灵敏蛋白检测

通过电容式免疫探针在猪心中对神经肽动力学进行时间分辨体内测量

监测人类痰样中的中性粒细胞和导管素 G 活性

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

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

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

纳米传感器检测体内蛋白酶活性的无创诊断

摘要

探索更多视频

荧光肽酶谱法检测蛋白酶活性

重组融合蛋白在荧光蛋白酶检测平台中的应用及其凝胶内再生

使用核酸探针进行核酸活性的动力学筛选

蛋白酶荧光检测试剂盒，用它来确定蛋白酶活性

全自动离心微流体装置从全血超灵敏蛋白检测

通过电容式免疫探 针在猪 心中对神经肽动力学进行时间分辨体内测量

监测人类痰样中的中性粒细胞和导管素 G 活性

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

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

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

通过电容式免疫探针在猪心中对神经肽动力学进行时间分辨体内测量