Nucleic acid analysis plays an important role in disease diagnosis and treatment. The discovery of CRISPR technology has provided novel and versatile approaches to the detection of nucleic acids. However, the most widely used CRISPR-Cas12a detection platforms lack the capability to distinguish single-stranded DNA (ssDNA) from double-stranded DNA (dsDNA). To overcome this limitation, we first employed an anti-CRISPR protein (AcrVA1) to develop a novel CRISPR biosensor to detect ssDNA exclusively. In this sensing strategy, AcrVA1 cut CRISPR guide RNA (crRNA) to inhibit the cleavage activity of the CRISPR-Cas12a system. Only ssDNA has the ability to recruit the cleaved crRNA fragment to recover the detection ability of the CRISPR-Cas12 biosensor, but dsDNA cannot accomplish this. By measuring the recovered cleavage activity of the CRISPR-Cas12a biosensor, our developed AcrVA1-assisted CRISPR biosensor is capable of distinguishing ssDNA from dsDNA, providing a simple and reliable method for the detection of ssDNA. Furthermore, we demonstrated our developed AcrVA1-assisted CRISPR biosensor to monitor the enzymatic activity of helicase and screen its inhibitors.

Accurate and sensitive detection of single nucleotide polymorphism (SNP) holds significant clinical implications, especially in the field of cancer diagnosis. Leveraging its high accuracy and programmability, the CRISPR system emerges as a promising platform for advancing the identification of SNPs. In this study, we compared two type V CRISPR/Cas systems (Cas12a and Cas14a) for the identification of cancer-related SNP. Their identification performances were evaluated by characterizing their mismatch tolerance to the BRAF gene. We found that the CRISPR/Cas14a system exhibited superior accuracy and robustness over the CRISPR/Cas12a system for SNP detection. Furthermore, blocker displacement amplification (BDA) was combined with the CRISPR/Cas14a system to eliminate the interference of the wild type (WT) and increase the detection accuracy. In this strategy, we were able to detect BRAF V600E as low as 103 copies with a sensitivity of 0.1% variant allele frequency. Moreover, the BDA-assisted CRISPR/Cas14a system has been applied to identify the BRAF mutation from human colorectal carcinoma cells, achieving a high sensitivity of 0.5% variant allele frequency, which is comparable to or even superior to those of most commercially available products. This work has broadened the scope of the CRISPR system and provided a promising method for precision medicine.

Sepsis is an extremely dangerous medical condition that emanates from the bodys 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.

Pathogenic bacterial contamination is a major threat to human health and safety. In this review, we summarize recent strategies for the integration of recognition elements with nanomaterials for the detection and sensing of pathogenic bacteria. Nanoprobes can provide sensitive and specific detection of bacterial cells, which can be applied across multiple applications and industries.

A novel enzyme-induced metallization colorimetric assay is developed to monitor and measure beta-galactosidase (β-gal) activity, and is further employed for colorimetric bacteriophage (phage)-enabled detection of Escherichia coli (E. coli). This assay relies on enzymatic reaction-induced silver deposition on the surface of gold nanorods (AuNRs). In the presence of β-gal, the substrate p-aminophenyl β-d-galactopyranoside is hydrolyzed to produce p-aminophenol (PAP). Reduction of silver ions by PAP generates a silver shell on the surface of AuNRs, resulting in the blue shift of the longitudinal localized surface plasmon resonance peak and multicolor changes of the detection solution from light green to orange-red. Under optimized conditions, the detection limit for β-gal is 128 pM, which is lower than the conventional colorimetric assay. Additionally, the assay has a broader dynamic range for β-gal detection. The specificity of this assay for the detection of β-gal is demonstrated against several protein competitors. Additionally, this technique is successfully applied to detect E. coli bacteria cells in combination with bacteriophage infection. Due to the simplicity and short incubation time of this enzyme-induced metallization colorimetric method, the assay is well suited for the detection of bacteria in low-resource settings.

Traditional plating and culturing methods used to quantify bacteria commonly require hours to days from sampling to results. We present here a simple, sensitive and rapid electrochemical method for bacterial detection in drinking water based on gold nanoparticle-enzyme complexes. The gold nanoparticles were functionalized with positively charged quaternary amine headgroups that could bind to enzymes through electrostatic interactions, resulting in inhibition of enzymatic activity. In the presence of bacteria, the nanoparticles were released from the enzymes and preferentially bound to the bacteria, resulting in an increase in enzyme activity, releasing a redox-active phenol from the substrate. We employed this strategy for the electrochemical sensing of Escherichia coli and Staphylococcus aureus, resulting in a rapid detection (<1 h) with high sensitivity (10(2) CFU mL(-1)).

Isolation of circulating tumor cells (CTCs) from blood samples has important prognostic and therapeutic implications for cancer treatments, but the process is very challenging due to the low concentration of CTCs. In this study, we report a novel 3D printed microfluidic device functionalized with anti-EpCAM (epithelial cell adhesion molecule) antibodies to isolate CTCs from human blood samples. A 3D printing technology was utilized with specially designed interior structures to fabricate a microfluidic device with high surface area and fluid flow manipulation, increasing capture efficiency of tumor cells. These devices with the optimal flow rate (1 mL/h) and channel length (2 cm) were demonstrated to test three kinds of EpCAM positive cancer cell lines (MCF-7 breast cancer, SW480 colon cancer, and PC3 prostate cancer), and one kind of EpCAM negative cancer cell line (293T kidney cancer). Experimentally, the capture efficiency higher than 90% has been achieved, and the isolation of MCF-7 tumor cells from spiked human blood samples has also been demonstrated. Combined with DNA-based detection (e.g. polymerase chain reaction or DNA sequencing), the detection and analysis of released DNAs from captured tumor cells could be another future direction for clinical diagnosis and cancer treatment.

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.

The highly contagious nature and 100% fatality rate contribute to the ongoing and expanding impact of the African swine fever virus (ASFV), causing significant economic losses worldwide. Herein, we developed a cascaded colorimetric detection using the combination of a CRISPR/Cas14a system, G-quadruplex DNAzyme, and microfluidic paper-based analytical device. This CRISPR/Cas14a-G4 biosensor could detect ASFV as low as 5 copies/μL and differentiate the wild-type and mutated ASFV DNA with 2-nt difference. Moreover, this approach was employed to detect ASFV in porcine plasma. A broad linear detection range was observed, and the limit of detection in spiked porcine plasma was calculated to be as low as 42-85 copies/μL. Our results indicate that the developed paper platform exhibits the advantages of high sensitivity, excellent specificity, and low cost, making it promising for clinical applications in the field of DNA disease detection and suitable for popularization in low-resourced areas.

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.