A 3D printed microfluidic device for scalable multiplexed CRISPR-cas12a biosensing

IF 3 4区医学 Q3 ENGINEERING, BIOMEDICAL

Biomedical Microdevices Pub Date : 2023-08-29 DOI:10.1007/s10544-023-00675-x

Kathrine Curtin, Jing Wang, Bethany J. Fike, Brandi Binkley, Peng Li

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引用次数: 0

Abstract

Accurate, rapid, and multiplexed nucleic acid detection is critical for environmental and biomedical monitoring. In recent years, CRISPR-Cas12a has shown great potential in improving the performance of DNA biosensing. However, the nonspecific trans-cleavage activity of Cas12a complicates the multiplexing capability of Cas12a biosensing. We report a 3D-printed composable microfluidic plate (cPlate) device that utilizes miniaturized wells and microfluidic loading for a multiplexed CRISPR-Cas12a assay. The device easily combines loop-mediated isothermal amplification (LAMP) and CRISPR-Cas12a readout in a simple and high-throughput workflow with low reagent consumption. To ensure the maximum performance of the device, the concentration of Cas12a and detection probe was optimized, which yielded a four-fold sensitivity improvement. Our device demonstrates sensitive detection to the fg mL^− 1 level for four waterborne pathogens including shigella, campylobacter, cholera, and legionella within 1 h, making it suitable for low-resource settings.

Graphical Abstract

Abstract Image

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一种用于可扩展多路复用CRISPR-cas12a生物传感的3D打印微流体设备。

准确、快速和多重核酸检测对于环境和生物医学监测至关重要。近年来，CRISPR-Cas12a在提高DNA生物传感性能方面显示出巨大的潜力。然而，Cas12a的非特异性反式切割活性使Cas12a生物传感的多路复用能力复杂化。我们报道了一种3D打印的可组合微流体板（cPlate）装置，该装置利用小型化孔和微流体负载进行多重CRISPR-Cas12a测定。该设备以简单、高通量的工作流程轻松地将环介导的等温扩增（LAMP）和CRISPR-Cas12a读数结合在一起，试剂消耗低。为了确保设备的最大性能，对Cas12a和检测探针的浓度进行了优化，使灵敏度提高了四倍。我们的设备显示出对fg mL的灵敏检测- 在1小时内对四种水传播病原体（包括志贺菌、弯曲杆菌、霍乱和军团菌）进行1级检测，使其适用于低资源环境。

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

Biomedical Microdevices 工程技术-工程：生物医学

CiteScore

6.90

自引率

3.60%

发文量

审稿时长

6 months

期刊介绍： Biomedical Microdevices: BioMEMS and Biomedical Nanotechnology is an interdisciplinary periodical devoted to all aspects of research in the medical diagnostic and therapeutic applications of Micro-Electro-Mechanical Systems (BioMEMS) and nanotechnology for medicine and biology. General subjects of interest include the design, characterization, testing, modeling and clinical validation of microfabricated systems, and their integration on-chip and in larger functional units. The specific interests of the Journal include systems for neural stimulation and recording, bioseparation technologies such as nanofilters and electrophoretic equipment, miniaturized analytic and DNA identification systems, biosensors, and micro/nanotechnologies for cell and tissue research, tissue engineering, cell transplantation, and the controlled release of drugs and biological molecules. Contributions reporting on fundamental and applied investigations of the material science, biochemistry, and physics of biomedical microdevices and nanotechnology are encouraged. A non-exhaustive list of fields of interest includes: nanoparticle synthesis, characterization, and validation of therapeutic or imaging efficacy in animal models; biocompatibility; biochemical modification of microfabricated devices, with reference to non-specific protein adsorption, and the active immobilization and patterning of proteins on micro/nanofabricated surfaces; the dynamics of fluids in micro-and-nano-fabricated channels; the electromechanical and structural response of micro/nanofabricated systems; the interactions of microdevices with cells and tissues, including biocompatibility and biodegradation studies; variations in the characteristics of the systems as a function of the micro/nanofabrication parameters.