Micropatterned sensing hydrogels integrated with reconfigurable microfluidics for detecting protease release from cells.
Micropatterned sensing hydrogels integrated with reconfigurable microfluidics for detecting protease release from cells.
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DOI：
10.1021/ac402660z
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发表时间：
2013-12-17
影响因子：
7.4
通讯作者：
Revzin, Alexander
中科院分区：
文献类型：
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作者：
Son, Kyung Jin;Shin, Dong-Sik;Kwa, Timothy;Gao, Yandong;Revzin, Alexander
基质金属蛋白酶（MMP）在细胞外基质的分解中起核心作用，并且通常在癌细胞中上调。本研究的目的是开发适合于捕获细胞和检测细胞分泌的蛋白酶的微孔。将由聚（乙二醇）（PEG）组成的水凝胶微孔在玻璃上进行微图案化，并用配体修饰以促进细胞粘附。为了检测蛋白酶的释放，将MMP 9可裂解的肽设计为含有供体/受体FRET对（FITC和DABCYL）。将这些传感分子结合到水凝胶威尔斯孔壁中以实现检测方案，其中在威尔斯孔内捕获的细胞分泌蛋白酶分子，所述蛋白酶分子扩散到凝胶中，切割肽，并引起荧光信号出现。检测限测定为0.625 nM，线性范围延伸至40 nM。为了提高灵敏度并限制相邻传感位点之间的串扰，将含有淋巴瘤细胞的小组（~20个细胞/孔）的微孔阵列整合到可重构PDMS微流体装置中。使用感测水凝胶微孔和可重构微流体的这种组合，证明了从少至11个细胞的MMP 9释放的检测。智能水凝胶微结构能够隔离小细胞群和传感细胞功能，具有从诊断到细胞/组织工程的多种应用。该技术的进一步发展将包括单细胞分析和基于功能的细胞分选能力。
Matrix metalloproteinases (MMPs) play a central role in the breakdown of the extracellular matrix and are typically upregulated in cancer cells. The goal of the present study is to develop microwells suitable for capture of cells and detection of cell-secreted proteases. Hydrogel microwells comprised of poly(ethylene glycol) (PEG) were photopatterned on glass and modified with ligands to promote cell adhesion. To sense protease release, peptides cleavable by MMP9 were designed to contain a donor/acceptor FRET pair (FITC and DABCYL). These sensing molecules were incorporated into the walls of the hydrogel wells to enable a detection scheme where cells captured within the wells secreted protease molecules which diffused into the gel, cleaved the peptide, and caused a fluorescence signal to come on. By challenging sensing hydrogel microstructures to known concentrations of recombinant MMP9, the limit of detection was determined to be 0.625 nM with a linear range extending to 40 nM. To enhance sensitivity and to limit cross-talk between adjacent sensing sites, microwell arrays containing small groups (~20 cells/well) of lymphoma cells were integrated into reconfigurable PDMS microfluidic devices. Using this combination of sensing hydrogel microwells and reconfigurable microfluidics, detection of MMP9 release from as few as 11 cells was demonstrated. Smart hydrogel microstructures capable of sequestering small groups of cells and sensing cell function have multiple applications ranging from diagnostics to cell/tissue engineering. Further development of this technology will include single-cell analysis and function-based cell sorting capabilities.
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通过表面等离子体共振对蛋白质毒素的高度敏感检测，其基于生物素化的内联原子转移自由基聚合扩增。
影响因子：
7.4
作者：
Liu Y;Dong Y;Jauw J;Linman MJ;Cheng Q
通讯作者：
Cheng Q
影响因子：
3.2
作者：
Revzin, Alexander;Maverakis, Emanual;Chang, H. -C.
通讯作者：
Chang, H. -C.
影响因子：
6.4
作者：
Ranuncolo, SM;Armanasco, E;Puricelli, L
通讯作者：
Puricelli, L
影响因子：
2.9
作者：
Shoji, Atsushi;Kabeya, Mitsutaka;Sugawara, Masao
通讯作者：
Sugawara, Masao
影响因子：
19.6
作者：
通讯作者：
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