Microfluidic-based Pre-amplification-free CRISPR-Cas Biosensing for Rapid Detection: Technologies and Applications
10.3724/j.pibb.2026.0180CSTR：32369.14.pibb.20260180
- VernacularTitle: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
- Author:
An-Yan LU
1
;
Liang ZHAO
1
;
Guang-Sheng GUO
1
;
Xia-Yan WANG
1
Author Information
1. State Key Laboratory of Materials Low-Carbon Recycling, Beijing Key Laboratory of 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, Beijing 100124, China
- Publication Type:Journal Article
- Keywords:
CRISPR-Cas system;
microfluidic device;
pre-amplification-free detection;
crRNA engineering;
digital detection;
signal transduction;
cascade signal amplification;
point-of-care testing
- From:
Progress in Biochemistry and Biophysics
2026;53(9):2430-2448
- CountryChina
- Language:Chinese
-
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.
