An amplification-free ultra-sensitive electrochemical CRISPR/Cas biosensor for drug-resistant bacteria detection.

Continued development of high-performance and cost-effective diagnostic tools is vital for improving infectious disease treatment and transmission control. For nucleic acid diagnostics, moving beyond enzyme-mediated amplification assays will be critical in reducing the time and complexity of diagnostic technologies. Further, an emerging area of threat, in which diagnostics will play an increasingly important role, is antimicrobial resistance (AMR) in bacterial infections. Herein, we present an amplification-free electrochemical CRISPR/Cas biosensor utilizing silver metallization (termed E-Si-CRISPR) to detect methicillin-resistant (MRSA). Using a custom-designed guide RNA (gRNA) targeting the gene of MRSA, the Cas12a enzyme allows highly sensitive and specific detection when employed with silver metallization and square wave voltammetry (SWV). Our biosensor exhibits excellent analytical performance, with detection and quantitation limits of 3.5 and 10 fM, respectively, and linearity over five orders of magnitude (from 10 fM to 0.1 nM). Importantly, we observe no degradation in performance when moving from buffer to human serum samples, and achieve excellent selectivity for MRSA in human serum in the presence of other common bacteria. The E-Si-CRISPR method shows significant promise as an ultrasensitive field-deployable device for nucleic acid-based diagnostics, without requiring nucleic acid amplification. Finally, adjustment to a different disease target can be achieved by simple modification of the gRNA protospacer. [Abstract copyright: This journal is © The Royal Society of Chemistry.

Paper-based laser-pyrolyzed electrofluidics: an electrochemical platform for capillary-driven diagnostic bioassays

Microfluidic paper-based analytical devices (uPADs) are indispensable tools for disease diagnostics. The integration of electronic components into uPADs enables new device functionalities and facilitates the development of complex quantitative assays. Unfortunately, current electrode fabrication methods often hinder capillary flow, considerably restricting uPAD design architectures. Here, we present laser-induced graphenization as an approach to fabricate porous electrodes embedded into cellulose paper. The resulting electrodes not only have high conductivity and electrochemical activity, but also retain wetting properties for capillary transport. We demonstrate paper-based electrofluidics, including (i) a lateral flow device for injection analysis of alkaline phosphatase in serum and (ii) a vertical flow device for quantitative detection of HPV16 with a CRISPR-based assay. We expect that this platform will streamline the development of diagnostic devices that combine the operational simplicity of colorimetric lateral flow tests with the added benefits and possibilities offered by electronic signaling

Paper-Based Laser-Pyrolyzed Electrofluidics: An Electrochemical Platform for Capillary-Driven Diagnostic Bioassays

Microfluidic paper-based analytical devices (µPADs) are indispensable tools for disease diagnostics. The integration of electronic components into µPADs enables new device functionalities and facilitates the development of complex quantitative assays. Unfortunately, current electrode fabrication methods often hinder capillary flow, considerably restricting µPAD design architectures. Here, laser-induced graphenization is presented as an approach to fabricate porous electrodes embedded into cellulose paper. The resulting electrodes not only have high conductivity and electrochemical activity, but also retain wetting properties for capillary transport. Paper-based electrofluidics, including a lateral flow device for injection analysis of alkaline phosphatase in serum and a vertical flow device for quantitative detection of HPV16 with a CRISPR-based assay are demonstrated. It is expected that this platform will streamline the development of diagnostic devices that combine the operational simplicity of colorimetric lateral flow tests with the added benefits and possibilities offered by electronic signaling.ISSN:0935-9648ISSN:1521-409

Paper-based laser-pyrolyzed electrofluidics: an electrochemical platform for capillary-driven diagnostic bioassays

Microfluidic paper-based analytical devices (uPADs) are indispensable tools for disease diagnostics. The integration of electronic components into uPADs enables new device functionalities and facilitates the development of complex quantitative assays. Unfortunately, current electrode fabrication methods often hinder capillary flow, considerably restricting uPAD design architectures. Here, we present laser-induced graphenization as an approach to fabricate porous electrodes embedded into cellulose paper. The resulting electrodes not only have high conductivity and electrochemical activity, but also retain wetting properties for capillary transport. We demonstrate paper-based electrofluidics, including (i) a lateral flow device for injection analysis of alkaline phosphatase in serum and (ii) a vertical flow device for quantitative detection of HPV16 with a CRISPR-based assay. We expect that this platform will streamline the development of diagnostic devices that combine the operational simplicity of colorimetric lateral flow tests with the added benefits and possibilities offered by electronic signaling.ISSN:2573-229