Hybrid Nucleic Acid-Quantum Dot Assemblies as Multiplexed Reporter Platforms for Cell-Free Transcription Translation-Based Biosensors.
Hybrid Nucleic Acid-Quantum Dot Assemblies as Multiplexed Reporter Platforms for Cell-Free Transcription Translation-Based Biosensors.
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DOI：
10.1021/acssynbio.2c00394
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发表时间：
2022-11
影响因子：
4.7
通讯作者：
Divita Mathur;Meghna Thakur;S. A. Díaz;K. Susumu;M. Stewart;E. Oh;Scott A. Walper;Igor L. Medintz
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文献类型：
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作者：
Divita Mathur;Meghna Thakur;S. A. Díaz;K. Susumu;M. Stewart;E. Oh;Scott A. Walper;Igor L. Medintz
无细胞合成生物学已经成为开发快速，便携式生物传感器的宝贵工具，可以很容易地以冻干形式运输到需要的地方，从而消除冷链要求。与无细胞传感器相关的挑战之一是，由于一组有限的荧光和比色报告器的可用性，无法在单个反应中同时检测多种分析物。为了潜在地为无细胞生物传感器提供多路复用能力，我们设计了一个基于模块化半导体量子点（QD）的报告平台，该平台插入无细胞反应中转录-翻译功能的下游，并将反应中的酶活性转化为不同的光信号。我们通过将限制性内切酶活性（用作我们的原型传感输出）转换为跨几个不同光谱输出通道的光学变化来证明概念的证明，这些通道都使用共同的激发波长。这些杂交Förster共振能量转移（FRET）为基础的QD肽rna -DNA-染料报告（QD- pdd）是完全自组装的，由差异发射的QD供体与染料受体配对组成，显示在编码给定酶裂解位点的独特DNA上。三种基于qd的pdd，分别由BamHI、EcoRI和NcoI酶独立激活，在混合酶实验中进行了原型化，这三种酶都证明了将酶活性转化为荧光输出的能力。在无细胞生物传感反应中，同时监测三个配对的量子点供体染料受体光谱通道，并添加编码每种酶的线性基因，证实了至少两种酶在共表达时的强大复用能力。模块化的qd - pdd很容易适应其他限制性内切酶甚至蛋白酶，如果需要。
Cell-free synthetic biology has emerged as a valuable tool for the development of rapid, portable biosensors that can be readily transported in the freeze-dried form to the point of need eliminating cold chain requirements. One of the challenges associated with cell-free sensors is the ability to simultaneously detect multiple analytes within a single reaction due to the availability of a limited set of fluorescent and colorimetric reporters. To potentially provide multiplexing capabilities to cell-free biosensors, we designed a modular semiconductor quantum dot (QD)-based reporter platform that is plugged in downstream of the transcription-translation functionality in the cell-free reaction and which converts enzymatic activity in the reaction into distinct optical signals. We demonstrate proof of concept by converting restriction enzyme activity, utilized as our prototypical sensing output, into optical changes across several distinct spectral output channels that all use a common excitation wavelength. These hybrid Förster resonance energy transfer (FRET)-based QD peptide PNA-DNA-Dye reporters (QD-PDDs) are completely self-assembled and consist of differentially emissive QD donors paired to a dye-acceptor displayed on a unique DNA encoding a given enzyme's cleavage site. Three QD-based PDDs, independently activated by the enzymes BamHI, EcoRI, and NcoI, were prototyped in mixed enzyme assays where all three demonstrated the ability to convert enzymatic activity into fluorescent output. Simultaneous monitoring of each of the three paired QD-donor dye-acceptor spectral channels in cell-free biosensing reactions supplemented with added linear genes encoding each enzyme confirmed robust multiplexing capabilities for at least two enzymes when co-expressed. The modular QD-PDDs are easily adapted to respond to other restriction enzymes or even proteases if desired.