Sensitive Methods to Detect Single-Stranded Nucleic Acids of Food Pathogens Based on Cell-Free Protein Synthesis and Retroreflection Signal Detection,Journal of Agricultural and Food Chemistry

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Sensitive Methods to Detect Single-Stranded Nucleic Acids of Food Pathogens Based on Cell-Free Protein Synthesis and Retroreflection Signal Detection
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2024-02-12 , DOI: 10.1021/acs.jafc.3c07785
Sunjoo Choi ₁ , Ye Seop Park ₁ , Kyung Won Lee ₁ , Yu Jin Park ₂ , Hee Ju Jang ₁ , Dong-Myung Kim ₂ , Tae Hyeon Yoo _{1,

3}

Affiliation

Cell-free protein synthesis (CFPS) has recently gained considerable attention as a new platform for developing methods to detect various molecules, ranging from small chemicals to biological macromolecules. Retroreflection has been used as an alternative signal to develop analytical methods because it can be detected by using a simple instrument comprising a white light source and a camera. Here, we report a novel reporter protein that couples the capability of CFPS and the simplicity of retroreflection signal detection. The design of the reporter was based on two pairs of protein–peptide interactions, SpyCatcher003-SpyTag003 and MDM2-PMI(N8A). MDM2-MDM2-SpyCatcher003 was decided as the reporter protein, and the two peptides, SpyTag003 and PMI(N8A), were immobilized on the surfaces of retroreflective Janus particles and microfluidic chips, respectively. The developed retroreflection signal detection system was combined with a previously reported CFPS reaction that can transduce the presence of a single-stranded nucleic acid into protein synthesis. The resulting methods were applied to detect 16S rRNAs of several foodborne pathogens. Concentration-dependent relationships were observed over a range of 10° fM to 10² pM, with the limits of detection being single-digit femtomolar concentrations. Considering the designability of the CFPS system for other targets, the retroreflection signal detection method will enable the development of novel methods to detect various molecules.

中文翻译：

基于无细胞蛋白质合成和回射信号检测的食品病原体单链核酸的灵敏检测方法

无细胞蛋白质合成（CFPS）作为开发检测各种分子（从小化学品到生物大分子）的方法的新平台，最近引起了相当大的关注。回射已被用作开发分析方法的替代信号，因为它可以通过使用包含白光源和相机的简单仪器来检测。在这里，我们报告了一种新型报告蛋白，它将 CFPS 的功能和逆反射信号检测的简单性结合起来。报告基因的设计基于两对蛋白质-肽相互作用：SpyCatcher003-SpyTag003 和 MDM2-PMI(N8A)。确定MDM2-MDM2-SpyCatcher003为报告蛋白，并将SpyTag003和PMI(N8A)两种肽分别固定在逆反射Janus颗粒和微流控芯片的表面。开发的逆反射信号检测系统与先前报道的 CFPS 反应相结合，可以将单链核酸的存在转导为蛋白质合成。由此产生的方法用于检测几种食源性病原体的 16S rRNA。在 10° fM 至 10 ² pM 范围内观察到浓度依赖性关系，检测限为个位数飞摩尔浓度。考虑到 CFPS 系统针对其他目标的可设计性，逆反射信号检测方法将有助于开发检测各种分子的新方法。

更新日期：2024-02-12

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