基于微流控的无需预扩增CRISPR-Cas生物传感快速检测技术及其应用
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北京工业大学材料循环低碳再生全国重点实验室 心肺脑复苏创新转化北京市重点实验室 环境安全与生物效应卓越中心 北京工业大学化学与生命科学学院 化学系 北京 100124

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065;Q7

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国家自然科学基金(22174007),北京工业大学杰出学子(雏鹰)计划和北京工业大学本科生星火基金资助项目。


Microfluidic-based Pre-amplification-free CRISPR-Cas Biosensing for Rapid Detection: Technologies and Applications
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State Key Laboratory of Materials Low-Carbon Recycling,Beijing Key Laboratory of in Cardiopulmonary-Cerebral Resuscitation Innovation and Translation,Center of Excellence for Environmental Safety and Biological Effects,Department of Chemistry,College of Chemistry and Life Science,Beijing University of Technology

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This work was supported by grants from The National Natural Science Foundation of China (22174007), Outstanding Students Project for undergraduates BJUT, and the Spark Fund for undergraduates, Beijing University of Technology.

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    摘要:

    CRISPR-Cas生物传感系统凭借高特异性和可编程性,已成为核酸快速检测的重要技术平台。传统CRISPR诊断高度依赖PCR或等温扩增等预扩增步骤,虽可提高灵敏度,却增加操作流程、温控要求和污染控制负担,限制其在现场快速检测(POCT)中的现场化应用。无需预扩增CRISPR检测因此受到关注,而微流控器件凭借微型化、高通量和精确流体操控能力,可实现反应限域、数字化分区、信号读出和流程集成,是推动该类方法实用化的重要载体。本文综述CRISPR-Cas系统与微流控技术融合的最新进展,重点讨论CRISPR RNA(crRNA)工程化、液滴与微腔阵列数字化检测、物理信号转导界面及片上级联信号放大等策略,比较不同技术路线在灵敏度提升、操作复杂度、成本和POCT适配性方面的特点。本文强调,微流控并非单一信号增强手段,而是连接分子识别、反应控制和现场读出的工程平台。最后,总结其在食品真实性鉴别和病原体快速筛查中的应用,并从低丰度靶标检测、样本基质干扰、器件集成化和自动化“样本入-结果出”系统等方面展望未来发展。

    Abstract:

    CRISPR-Cas-based biosensing systems have become important platforms for rapid nucleic acid detection because of their programmable sequence recognition and high specificity. In many diagnostic workflows, the CRISPR reaction is coupled with polymerase chain reaction or isothermal pre-amplification to enrich low-abundance targets before readout. This design improves sensitivity, but it also introduces additional primers and enzymes, requirements for temperature control and reaction compatibility, and a higher demand for contamination management. These factors make the whole assay less convenient for integrated and field-deployable point-of-care testing. Developing pre-amplification-free CRISPR assays is therefore not simply a pursuit of shorter protocols, but a way to simplify molecular diagnosis at the system level, especially in settings where rapid decisions, closed workflows, and minimal manual operation are required. Microfluidic devices provide an important engineering route for this purpose. Their small reaction volumes, precise fluid manipulation, high-throughput partitioning, and compatibility with portable readout make it possible to integrate target confinement, reaction control, signal acquisition, and quantitative analysis on a chip. Droplet microfluidics and microwell arrays can divide a sample into large numbers of independent microreactors, enabling digital counting of rare recognition events. Continuous-flow, centrifugal, and paper-based microfluidic formats further provide options for automated operation, low-cost fabrication, and on-site use. In this context, microfluidics is not only a signal-enhancement method, but also a platform that connects CRISPR molecular recognition with practical assay implementation. This review summarizes recent progress in microfluidic-based pre-amplification-free CRISPR-Cas biosensing for rapid detection. We focus on four related technical directions. crRNA engineering, including spacer-length tuning, multi-crRNA design, chemical modification, and allosteric regulation, can improve recognition kinetics, nuclease stability, and mismatch discrimination. Digital microfluidic detection based on droplets or microwell arrays converts single-molecule recognition events into countable positive partitions, thereby improving quantitative capability without target pre-amplification. Physical signal transduction interfaces couple CRISPR activity to electrochemical, electrochemiluminescent, fluorescent, or surface-enhanced Raman scattering readouts, making weak molecular signals easier to detect with portable instruments. On-chip cascade signal amplification strategies further enhance output intensity through enzymatic reactions, DNA circuits, nanomaterials, or cross-domain amplification modules while avoiding direct amplification of the target nucleic acid. Together, these strategies show that the performance of a pre-amplification-free assay depends not only on biochemical recognition, but also on how the reaction is confined, amplified, transduced, and operated. In addition to describing these mechanisms, this review compares different strategies in terms of sensitivity improvement, operational complexity, cost, and suitability for point-of-care testing. Representative applications in food authenticity identification and rapid pathogen screening are also discussed to illustrate how these platforms perform in practical scenarios. Finally, we analyze the remaining challenges, including the detection of low-abundance targets in complex matrices, on-chip sample preparation, device-to-device reproducibility, long-term reagent storage, and standardization. Future development will likely depend on tighter integration of sample processing, CRISPR reaction, signal readout, and data interpretation, ultimately moving pre-amplification-free CRISPR diagnostics toward automated and user-friendly sample-to-answer systems.

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陆安妍,赵亮,郭广生,汪夏燕.基于微流控的无需预扩增CRISPR-Cas生物传感快速检测技术及其应用[J].生物化学与生物物理进展,,():

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  • 收稿日期:2026-04-08
  • 最后修改日期:2026-07-16
  • 录用日期:2026-07-17
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