On-Site Fluorescent Detection of Sepsis-Inducing Bacteria using a Graphene-Oxide CRISPR-Cas12a (GO-CRISPR) System

Abstract

Sepsis is an extremely dangerous medical condition that emanates from the body's response to a pre-existing infection. Early detection of sepsis-inducing bacterial infections can greatly enhance the treatment process and potentially prevent the onset of sepsis. However, current point-of-care (POC) sensors are often complex and costly or lack the ideal sensitivity for effective bacterial detection. Therefore, it is crucial to develop rapid and sensitive biosensors for the on-site detection of sepsis-inducing bacteria. Herein, we developed a graphene oxide CRISPR-Cas12a (GO-CRISPR) biosensor for the detection of sepsis-inducing bacteria in human serum. In this strategy, single-stranded (ssDNA) FAM probes were quenched with single-layer graphene oxide (GO). Target-activated Cas12a trans-cleavage was utilized for the degradation of the ssDNA probes, detaching the short ssDNA probes from GO and recovering the fluorescent signals. Under optimal conditions, we employed our GO-CRISPR system for the detection of Salmonella Typhimurium (S. Typhimurium) with a detection sensitivity of as low as 3 × 10³ CFU/mL in human serum, as well as a good detection specificity toward other competing bacteria. In addition, the GO-CRISPR biosensor exhibited excellent sensitivity to the detection of S. Typhimurium in spiked human serum. The GO-CRISPR system offers superior rapidity for the detection of sepsis-inducing bacteria and has the potential to enhance the early detection of bacterial infections in resource-limited settings, expediting the response for patients at risk of sepsis.

Figure 1

Schematic illustration of Salmonella detection using the GO-CRISPR system. The invA gene from Salmonella is amplified using isothermal recombinase polymerase amplification (RPA). The amplified Salmonella target DNA is then reacted with specifically designed CRISPR systems and ssDNA-FAM probes. In the presence of the target DNA, the CRISPR system is activated, initiating robust degradation of the probes. The degraded probes cannot bind to the surface of the GO, resulting in a fluorescent signal for visual detection of Salmonella.

Figure 2

Characterization of the GO-CRISPR system. (a) Schematic illustration of distance-dependent GO fluorescent quenching. The ssDNA on the fluorescent probes attaches to the GO surface through π–π stacking interactions. The fluorescence is quenched in close proximity to the GO. (b) Fluorescent spectra of varying concentrations of GO from 510 to 600 nm. (c) Fluorescent intensity at 520 nm of varying concentrations of GO. (d) Fluorescent image and intensity for the GO-CRISPR system feasibility analysis.

Figure 3

Optimization of the GO-CRISPR system. (a,b) Fluorescent spectra and intensity at 520 nm of varying concentrations of Cas12a. (c,d) Fluorescent spectra and intensity at 520 nm of varying concentrations of crRNA. (e,f) Fluorescent intensities at 520 nm and image at 30 min of the GO-CRISPR system at 37 °C and room temperature.

Figure 4

Detection of Salmonella DNA. (a) Fluorescence spectra of varying concentrations of DNA. (b) Ratio of fluorescent intensity values to the background at 520 nm for various concentrations of DNA, with linear regression for the dynamic range. (c). Fluorescent image of the GO-CRISPR system for the detection of Salmonella DNA at varying concentrations.

Figure 5

Detection sensitivity and specificity of the GO-CRISPR system for Salmonella. (a) Schematic illustration of RPA amplification and the GO-CRISPR system for Salmonella-specific detection. (b) Gel image of RPA products and fluorescent intensity of S. Typhimurium at various concentrations (NC = negative control). (c) Gel image of RPA products and fluorescent intensity of Salmonella serovars. (d) Gel image of RPA products and fluorescent intensity of competing bacteria strains (B. subtilis, E. coli O157:H7, L. monocytogenes, S. aureus, V. cholerae, and a “mix” of all five cultures) with and without S. Typhimurium. ^**p, 0.05, Student’s unpaired t test.

Figure 6

Salmonella detection in human serum. (a) Schematic illustration of the entire GO-CRISPR process for the detection of Salmonella in human serum. (b) Fluorescent spectra of varying concentrations of S. Typhimurium spiked in human serum. (c) Gel image of RPA products and the fluorescence intensity of varying concentrations of S. Typhimurium spiked in human serum. (d) Fluorescent image of varying concentrations of S. Typhimurium spiked in human serum ^**p, 0.05, Student’s unpaired t test.