Glucose oxidase-mediated polymerization as a platform for dual-mode signal amplification and biodetection.
Glucose oxidase-mediated polymerization as a platform for dual-mode signal amplification and biodetection.
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DOI：
10.1002/bit.23101
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发表时间：
2011-07
影响因子：
3.8
通讯作者：
Bowman, Christopher N.
中科院分区：
文献类型：
--
作者：
Berron, Brad J.;Johnson, Leah M.;Ba, Xiao;McCall, Joshua D.;Alvey, Nicholas J.;Anseth, Kristi S.;Bowman, Christopher N.
我们报告了首次使用基于聚合的 ELISA 底物溶液，采用酶介导的自由基聚合作为双模式扩增策略。酶选择性地耦合到表面以产生自由基，随后导致基于聚合的扩增（PBA）和生物检测。使用带有葡萄糖氧化酶 (GOx)-亲和素缀合物的生物素化微孔表面，以微孔条形式优化基于聚合的检测系统的灵敏度和扩增。固定化的 GOx 用于引发聚合，从而能够通过视觉或通过使用读板器来检测生物识别事件。在使用生物素化孔的简化测定中，将测定响应与利用硝基蓝四唑的酶底物的响应进行比较。该聚合底物表现出与传统酶底物相当的灵敏度（2 µg/mL GOx-亲和素）和三倍以上的信号放大能力，因为酶促生成的每个自由基都会导致大量聚合事件。酶介导的聚合反应在环境气氛中进行，无需外部能源，这是对之前 PBA 平台的改进。底物配方在最低酶浓度下对葡萄糖和铁浓度高度敏感。扩增时间的增加对应于更高的检测灵敏度，但非特异性信号没有增加。最后，在转化生长因子-β 测定中，聚合底物在检测限 (156 ng/mL) 下产生了 14 的信噪比。生物技术。生物工程。 2011； 108：1521–1528。 © 2011 Wiley 期刊公司。
We report the first use of a polymerization-based ELISA substrate solution employing enzymatically mediated radical polymerization as a dual-mode amplification strategy. Enzymes are selectively coupled to surfaces to generate radicals that subsequently lead to polymerization-based amplification (PBA) and biodetection. Sensitivity and amplification of the polymerization-based detection system were optimized in a microwell strip format using a biotinylated microwell surface with a glucose oxidase (GOx)–avidin conjugate. The immobilized GOx is used to initiate polymerization, enabling the detection of the biorecognition event visually or through the use of a plate reader. Assay response is compared to that of an enzymatic substrate utilizing nitroblue tetrazolium in a simplified assay using biotinylated wells. The polymerization substrate exhibits equivalent sensitivity (2 µg/mL of GOx-avidin) and over three times greater signal amplification than this traditional enzymatic substrate since each radical that is enzymatically generated leads to a large number of polymerization events. Enzyme-mediated polymerization proceeds in an ambient atmosphere without the need for external energy sources, which is an improvement upon previous PBA platforms. Substrate formulations are highly sensitive to both glucose and iron concentrations at the lowest enzyme concentrations. Increases in amplification time correspond to higher assay sensitivities with no increase in non-specific signal. Finally, the polymerization substrate generated a signal to noise ratio of 14 at the detection limit (156 ng/mL) in an assay of transforming growth factor-beta. Biotechnol. Bioeng. 2011; 108:1521–1528. © 2011 Wiley Periodicals, Inc.
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影响因子：
6.2
作者：
Hansen, Ryan R.;Sikes, Hadley D.;Bowman, Christopher N.
通讯作者：
Bowman, Christopher N.
影响因子：
41.2
作者：
Sikes, Hadley D.;Hansen, Ryan R.;Bowman, Christopher N.
通讯作者：
Bowman, Christopher N.
影响因子：
2.1
作者：
HOLLAND, VR;SAUNDERS, BC;WALPOLE, AL
通讯作者：
WALPOLE, AL
影响因子：
7.4
作者：
AUSES, JP;COOK, SL;MALOY, JT
通讯作者：
MALOY, JT
影响因子：
6.2
作者：
Johnson, Leah M.;Fairbanks, Benjamin D.;Anseth, Kristi S.;Bowman, Christopher N.
通讯作者：
Bowman, Christopher N.