Perspective and prospects of 2D MXenes for smart biosensing

Biomedical Engineering of Two-Dimensional MXenes.

Foodborne diseases, caused by pathogenic bacteria, severely threaten global human health and cause a financial burden. Rapid, sensitive and on-site detection of pathogenic bacteria is significant. The existing methods have different defects, such as time-consuming and inconvenient. In this study, we developed a G-quadruplex-based CRISPR-Cas12a bioassay for pathogenic bacteria detection with high sensitivity and visualization capability. Salmonella was used as the detection model. Simply, the amplicons of Salmonella specific invA gene activated the trans-cleavage activity of Cas12a and triggered CRISPR-Cas12a based indiscriminate degradation of single-stranded DNAs (ssDNAs). The ssDNAs were designed with the guanine-rich sequence and formed a stable G-quadruplex DNAzyme by adding K+. This DNAzyme could catalyze the TMB-H2O2 reaction in the presence of hemin, leading to an increase in absorbance at 454 nm and a color change. This change can be readily differentiated by the naked eyes as well as a smartphone with a Color Picker App. With this strategy, the limit of detection (LOD) for Salmonella was 1 CFU/mL with no cross-reactivity. A linear relationship (R2 = 0.993) between the absorbance and the concentration of Salmonella was obtained. Furthermore, G-quadruplex-based CRISPR-Cas12a bioassay was successfully applied for sensing Salmonella in real food samples. This work not only expands the reach of CRISPR-Cas based biosensing but also provides a novel pathogenic bacteria detection method with high sensitivity, specificity and on-site capability.

Ultrasensitive pathogenic bacteria detection by a smartphone-read G-quadruplex-based CRISPR-Cas12a bioassay

An innovative and generic CRISPR/Cas12a-driven photoelectrochemical (PEC) biosensing platform was developed for screening of microRNA-21 (miR-21) by coupling with target-triggered catalytic hairpin assembly (CHA) and reduced graphene oxide-anchored Bi 2 WO 6 (rGO-BWO) as the photoactive material. CHA isothermal amplification involved two programmable hairpin DNA modules and miR-21 as an activator. In the presence of miR-21, the products of target-triggered CHA circuit were inserted into the Cas12a-crRNA duplex to initial trans-cleavage capacity of CRISPR/Cas12a nuclease, accompanying the digestion on alkaline phosphatase (ALP)-labeled single-stranded DNA (ssDNA)-encoded magnetic bead (MB) through the activated CRISPR system. The ALPs were detached from magnetic beads and promoted the generation of ascorbic acid (AA), which increased the photocurrent of rGO-BWO-modified electrode. The value of photocurrent was positively proportional to the level of AA, which was also linearly correlated with target concentrations. Under optimum conditions, the CRISPR-based PEC sensing system displayed satisfying photocurrent responses toward miR-21 within the range from 1.0 fM to 1.0 nM with a limit of detection of 0.47 fM. In addition, the biosensor exhibited acceptable stability and excellent selectivity. Impressively, CHA-mediated CRISPR-based PEC biosensing platform provides a universal and sensitive method for clinical cancer diagnostics and biomolecular research. • An enhanced photoelectrochemical biosensor was designed for detection of microRNA. • Reduced graphene oxide-anchored Bi2WO6 used as the photoactive materials. • Bi2WO6-based carbon materials to enhance photoelectric conversion efficiency. • Catalytic hairpin assembly utilized for target recycling. • CRSIPR/Cas12 reaction system employed for in-situ amplified photocurrent. 

CRISPR/Cas12a-based photoelectrochemical sensing of microRNA on reduced graphene oxide-anchored Bi2WO6 coupling with catalytic hairpin assembly

CRISPR-Cas12a-mediated luminescence resonance energy transfer aptasensing platform for deoxynivalenol using gold nanoparticle-decorated Ti3C2Tx MXene as the enhanced quencher.

With hydrophilic surface and high density of functional groups, MXene can efficiently adsorb single-stranded DNA to enhance target-induced strand release and quench the fluorescence. Herein, MXene is coupled with CRISPR-Cas12a to sensitively detect LPS and bacteria. Specifically, the aptamer is well designed to initiate the trans-cleavage activity of CRISPR-Cas12a to indiscriminately cleave single-stranded DNA, resulting it to be far away from MXene and the recovery of fluorescence. The target can effectually induce the release of the aptamer strand from the hybrid duplex with the assistance of MXene. The formed aptamer/target complex will inhibit the activation of CRISPR-Cas12a and its trans-cleavage on single-stranded DNA. The established method can selectively and sensitively quantify LPS and Gram-negative bacteria in different samples with detection limits of 11 pg/mL and 23 CFU/mL, respectively. Our study provides a new insight for exploration of universal analytical methods based on MXene coupled with CRISPR-Cas12a.

Jingyi Yang

Papers

MXene Coupled with CRISPR-Cas12a for Analysis of Endotoxin and Bacteria.