Toehold链置换辅助智能手机比色法特异性检测废水中新冠病毒靶标RNA

基于Toehold介导的链置换反应（TSDR）与DNA酶,构建了一个强特异性及低成本检测废水中SARS-CoV-2靶标RNA单碱基突变的比色生物传感器。SARS-CoV-2靶标RNA与双链DNA(dsDNA)引发TSDR,释放出完整的G-四链体序列并与氯化血红素（Hemin）结合,形成G-四链体/Hemin DNA酶,进而催化双氧水氧化3,3’,5,5’-四甲基联苯胺,使溶液呈蓝色。当SARS-CoV-2靶标RNA存在单碱基突变时,TSDR被抑制,导致裸眼观察到的溶液颜色与未突变异的靶标相比更浅。同时采用智能手机摄像头捕捉溶液的RGB值变化（ΔRGB）,对裸眼观察结果的准确性进行验证。在最优条件下,构建的比色生物传感器能够特异性识别单个碱基突变的SARS-CoV-2靶标RNA,具有用于废水中SARS-CoV-2变异毒株检测的潜力。     

CRISPR/Cas技术在核酸检测中的应用进展

CRISPR/Cas是大多数细菌及古细菌基于RNA的后天免疫系统。由CRISPR/Cas系统改造而成的CRISPR/Cas技术已成为一种强大的基因编辑工具,广泛应用于基因功能研究和基因修饰与治疗。除了作为基因编辑工具,Ⅱ类Cas蛋白具有的"附属切割"特性,已被开发成一种快速、低成本且高灵敏的核酸检测工具,在核酸分子诊断领域具有重要的应用潜力。该文总结了3种Ⅱ类Cas蛋白在核酸检测领域的代表性研究进展,并对CRISPR/Cas系统在该领域的应用前景进行了展望。     

CRISPR-Cas12a信号放大策略与便携式血糖仪耦合定量检测黄曲霉毒素B1

建立了CRISPR-Cas12a与便携式血糖仪耦合定量检测黄曲霉毒素B1(AFB1)的方法。体系中AFB1能够激活Cas12a的反式切割活性,激活后的Cas12a切割电极上蔗糖酶修饰的发夹探针,使得蔗糖酶游离到电极表面的溶液中。蔗糖酶催化蔗糖产生可以被血糖仪监测的响应信号,进而实现对AFB1的检测。在浓度0.001～0.1 ng/mL范围内,AFB1浓度与血糖信号呈良好的线性关系,线性方程为S=3.5+229.1c,检出限为0.3 pg/mL。该方法特异性强,适用于实际样品中AFB1的检测。     

基于CRISPR-Cas12a与G-四链体构建免标记电化学生物传感器检测microRNA

CRISPR-Cas12a是一种功能强大且可编程的分子诊断技术。本文基于CRISPR-Cas12a的附属切割活性与G-四链体/氯化血红素（Hemin）复合物,设计了一个免标记电化学生物传感器,实现对miRNA的强特异性检测。靶标miRNA-21与双链DNA探针上的Toehold区域结合并发生链置换反应,置换出双链DNA探针中较短的DNA。置换下来的DNA可以有效地激活CRISPR-Cas12a的附属切割活性。随后,具有附属切割活性的Cas12a切割电极表面上形成G-四链体/Hemin的DNA序列,导致电流信号减弱。在最优条件下,电流信号强度变化与10～100 pmol/L范围内的miRNA-21浓度呈良好的线性关系,检出限为4.2 pmol/L。该电化学生物传感器能够实现对单个碱基突变的miRNA-21或其它miRNA序列特异性识别,并可用于人血清样本（10%）中miRNA-21的检测。     

Cascaded strand displacement for non-enzymatic target recycling amplification electrochemical detection of SARS-CoV-2EI北大核心CSCD

An electrochemical biosensor for low cost and highly sensitive and selective detection of SARS-CoV-2 target nucleic acid was developed based on two cascaded toehold-mediated strand displacement reactions (TSDRs). Driven by thermodynamic entropy, the target nucleic acid bound to the first toehold region of the probes, leading to the first TSDR and the second toehold region exposed. Subsequently, the methylene blue (MB)-modified signal probe triggered the second TSDR and led to cyclic reuse of the target nucleic acid. Based on cascaded TSDRs, a large number of signal probes were combined on the sensor surface to produce significantly enhanced square wave voltammetry (SWV)electrochemical signals. The results showed that the SWV signal intensity was proportional to the logarithm of the target nucleic acid concentration, and had a good linear relationship in the range of 5×10-14-5×10-10 mol/L with a detection limit of 1.8×10-14 mol/L. Moreover, the sensor could be employed to monitor SARS-CoV-2 nucleic acid in 10% serum samples. © 2023, Youke Publishing Co.,Ltd. All rights reserved.     

Portable biosensor for quantitative detection of melamine based on CRISPRCas12a and a personal glucose meterEI北大核心CSCD

A CRISPR-Cas system holds great promise as a next-generation biosensing technology for molecular diagnostics. In this paper, a portable biosensor based on the trans-cleavage activity of CRISPR-Cas12a and a personal glucose meter has been developed for quantitative, sensitive and specific detection of melamine. The presence of the target melamine binds to the aptamer, leading to the release of locker DNA. And then, the leasing locker DNA activates the trans-cleavage activity of CRISPR-Cas12a to cleave the single-strand DNA (ssDNA) linker on sucrase-ssDNA modified electrode, releasing a short DNA fragment labeled with sucrase in the resulting solution. The sucrase could further catalyze sucrose to glucose, which could be detected by the PGM. Under the optimized conditions, the increase of PGM signal was relative with the concentration of melamine ranging from 0.1 to 2.5 μmol/L and the limit of detection (LOD) was 37 nmol/L. Moreover, the portable biosensor has strong specificity and can be used for the quantitative detection of melamine in milk samples. © 2023, Youke Publishing Co.,Ltd. All rights reserved.