Exploring a CRISPR/Cas12a-powered impedimetric biosensor for amplification-free detection of a pathogenic bacterial DNA

IF 10.7 1区生物学 Q1 BIOPHYSICS

Biosensors and Bioelectronics Pub Date : 2025-05-26 DOI:10.1016/j.bios.2025.117607

Dagwin Wachholz Junior , Rafael Gonçalves Pontes , Bruna M. Hryniewicz , Lauro Tatsuo Kubota

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引用次数: 0

Abstract

Timely and precise detection of bacterial infections is essential for improving patient outcomes and reducing healthcare costs, especially for sepsis, where delayed diagnosis increases mortality. Traditional culture- and PCR-based methods are time consuming and require complex sample processing, making them unsuitable for rapid diagnostics in resource-limited settings. CRISPR/Cas-based methods, particularly when combined with electrochemical sensing, offer a promising alternative for rapid point-of-care (POC) diagnostics of bacterial infections due to their simplicity and specificity. This study proposes a label-free impedimetric biosensor using the CRISPR/Cas12a system for rapid and amplification-free detection of Staphylococcus aureus DNA, a primary pathogen responsible for sepsis. By leveraging CRISPR/Cas12a′s target-activated collateral cleavage on non-specific DNA reporters we investigated the impact of using a protospacer adjacent motif (PAM) sequence on detection sensitivity and specificity. Our biosensor demonstrated ultra-sensitive detection, with limit of detection as low as 20 aM for dsDNA targets in buffer and without any pre-amplification steps. The study also confirmed CRISPR specificity's dependence on the PAM sequence, showing that mismatches on targeting sequences reduces cleavage efficiency, with a drastic reduction in trans-cleavage activity for single mismatch in PAM-containing sequences. Additionally, we examined how the DNA reporter affects performance, noting reduced cleavage efficiency when a ssDNA target was paired with a dsDNA reporter. Furthermore, validation experiments using human serum samples confirmed the biosensor's accuracy for bacterial DNA detection in clinical settings. This work advances CRISPR-powered electrochemical biosensors, providing a detailed discussion on developing a highly sensitive, fast and amplification-free tool for early detection of sepsis-causing bacteria.

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探索一种CRISPR/ cas12a驱动的无扩增检测病原菌DNA的阻抗生物传感器。

及时准确地检测细菌感染对于改善患者预后和降低医疗保健成本至关重要，特别是对于败血症，延迟诊断会增加死亡率。传统的基于培养和pcr的方法耗时且需要复杂的样品处理，这使得它们不适合在资源有限的环境中进行快速诊断。基于CRISPR/ cas的方法，特别是当与电化学传感相结合时，由于其简单性和特异性，为细菌感染的快速即时诊断（POC）提供了一种有希望的替代方法。本研究提出了一种使用CRISPR/Cas12a系统的无标记阻抗生物传感器，用于快速和无扩增检测金黄色葡萄球菌DNA，这是导致败血症的主要病原体。通过利用CRISPR/Cas12a在非特异性DNA报告基因上的靶激活侧切，我们研究了使用原间隔器相邻基序（PAM）序列对检测灵敏度和特异性的影响。我们的生物传感器显示出超灵敏的检测，对缓冲液中的dsDNA目标的检测限低至20 aM，无需任何预扩增步骤。该研究还证实了CRISPR特异性对PAM序列的依赖性，表明靶向序列的错配降低了切割效率，在含有PAM的序列中，单次错配会显著降低反式切割活性。此外，我们研究了DNA报告基因如何影响性能，注意到当ssDNA靶标与dsDNA报告基因配对时，切割效率会降低。此外，使用人类血清样本的验证实验证实了生物传感器在临床环境中用于细菌DNA检测的准确性。这项工作推进了crispr驱动的电化学生物传感器，为开发一种高灵敏度、快速和无扩增的工具来早期检测败血症引起的细菌提供了详细的讨论。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Biosensors and Bioelectronics 工程技术-电化学

CiteScore

20.80

自引率

7.10%

发文量

1006

审稿时长

29 days

期刊介绍： Biosensors & Bioelectronics, along with its open access companion journal Biosensors & Bioelectronics: X, is the leading international publication in the field of biosensors and bioelectronics. It covers research, design, development, and application of biosensors, which are analytical devices incorporating biological materials with physicochemical transducers. These devices, including sensors, DNA chips, electronic noses, and lab-on-a-chip, produce digital signals proportional to specific analytes. Examples include immunosensors and enzyme-based biosensors, applied in various fields such as medicine, environmental monitoring, and food industry. The journal also focuses on molecular and supramolecular structures for enhancing device performance.