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

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.

CRISPR-based Biosensors for Human Health: A Novel Strategy to Detect Emerging Infectious Diseases.

Infectious diseases (such as sepsis, influenza, and malaria), caused by various pathogenic bacteria and viruses, are widespread across the world. Early and rapid detection of disease-related pathogens is necessary to reduce their spread in the world and prevent their potential global pandemics. The clustered regularly interspaced short palindromic repeats (CRISPR) technology, as the next-generation molecular diagnosis technique, holds immense promise in the detection of infectious diseases because of its remarkable advantages, including supreme flexibility, sensitivity, and specificity. While numerous CRISPR-based biosensors have been developed for application in environmental monitoring, food safety, and point-of-care diagnosis, there remains a critical need to summarize and explore their potential in human health. This review aims to address this gap by focusing on the latest advancements in CRISPR-based biosensors for infectious disease detection. We provide an overview of the current status, pre-amplification methods, the unique feature of each CRISPR system, and the design of CRISPR-based biosensing strategies to detect disease-associated nucleic acids. Last but not least, the review analyzes the current challenges and provides future perspectives, which will contribute to developing more effective CRISPR-based biosensors for human health.

Development of Nanobody-Displayed Whole-Cell Biosensors for the Colorimetric Detection of SARS-CoV-2.

The accurate and effective detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to preventing the spread of infectious diseases and ensuring human health. Herein, a nanobody-displayed whole-cell biosensor was developed for colorimetric detection of SARS-CoV-2 spike proteins. Serving as bioreceptors, yeast surfaces were genetically engineered to display SARS-CoV-2 binding of llama-derived single-domain antibodies (nanobodies) with high capture efficiency, facilitating the concentration and purification of SARS-CoV-2. Gold nanoparticles (AuNPs) employed as signal transductions were functionalized with horseradish peroxidase (HRP) and anti-SARS monoclonal antibodies to enhance the detection sensitivity. In the presence of SARS-CoV-2 spike proteins, the sandwiched binding will be formed by linking engineered yeast, SARS-CoV-2 spike proteins, and reporter AuNPs. The colorimetric signal was generated by the enzymatic reaction of HRP and its corresponding colorimetric substrate/chromogen system. At the optimal conditions, the developed whole-cell biosensor enables the sensitive detection of SARS-CoV-2 spike proteins in a linear range from 0.01 to 1 µg/mL with a limit of detection (LOD) of 0.037 µg/mL (about 4 × 108 virion particles/mL). Furthermore, the whole-cell biosensor was demonstrated to detect the spike protein of different SARS-CoV-2 variants in human serum, providing new possibilities for the detection of future SARS-CoV-2 variants.

Engineered Yeast Displaying Specific Norovirus-Binding Nanobodies for the Concentration and Detection of Human Norovirus in Food Matrix.

Human noroviruses pose grave threats to public health and economy. In this study, we genetically engineered yeast (Saccharomyces cerevisiae EBY100) to display specific norovirus-binding nanobodies (Nano-26 and Nano-85) on cell surface to facilitate the concentration of noroviruses for improved detection. Binding of norovirus virus-like particles (VLPs) to these nanobody-displaying yeasts was confirmed and characterized using confocal microscopy and flow cytometry. The ability of our engineered yeasts to capture norovirus VLPs can reach up to 91.3%. Furthermore, this approach was applied to concentrate and detect norovirus VLPs in a real food matrix. A wide linear detection range (1-104 pg/g) was observed, and the detection limit on spiked spinach was calculated as low as 0.071 pg/g. Overall, our engineered yeasts could be a promising approach to concentrate and purify noroviruses in food samples for easy detection, which allows us to prevent the spread of food-borne virus in the food supply chain.