Chao Zhao

A sandwich immunoassay for brucellosis diagnosis based on immune magnetic beads and quantum dots

&NA; Brucellosis is a serious zoonosis with a rapid increase in incidence across epidemic regions. Currently, there are numerous methods for diagnosing brucellosis. However, these studies often have a few defects, such as low sensitivity, poor specificity, time consuming, laborious, and even potential biological risk. To overcome these shortcomings, we have developed a rapid, sensitive and accurate diagnosis procedure for brucellosis based on the immune magnetic beads (IMB) probe and quantum dots (QDs) – staphylococcal protein A (SPA) probe. With the presence of Brucella antibody in the tested serum, the QDs‐SPA probe links to the IMB probe and an immune‐sandwich complex is formed. As a result, the fluorescence intensity from QDs increased significantly and was correlated with the amount of Brucella antibody. Under the optimized conditions, 248 blood samples were detected and the diagnosis effect was evaluated. The area under the receiver‐operating characteristic (ROC) curve was 0.970 (95% confidence interval (CI), 0.9479–0.9920). The diagnostic sensitivity was 96.15% (95% CI, 91.82–98.58%), the diagnostic specificity was 94.12% (95% CI, 87.64–97.81%) with a fluorescence intensity cutoff value of 150.4 and the detection time was only 100 min. This diagnostic procedure can be satisfactorily applied to the diagnosis of brucellosis. Graphical abstract Figure. No caption available. HighlightsWe design a rapid, sensitive and accurate diagnosis procedure for brucellosis.IMB probe was utilized to enrich and separate the Brucella antibodies in serum.QDs‐SPA probe could be bind to antibodies strongly and be employed to generate the detectable fluorescent signal.This sandwich immunoassay was satisfactorily applied to the diagnosis of brucellosis.

Rapid and Quantitative Detection of Vibrio parahemolyticus by the Mixed-Dye-Based Loop-Mediated Isothermal Amplification Assay on a Self-Priming Compartmentalization Microfluidic Chip

Vibrio parahemolyticus (VP) mostly isolated from aquatic products is one of the major causes of bacterial food-poisoning events worldwide, which could be reduced using a promising on-site detection method. Herein, a rapid and quantitative method for VP detection was developed by applying a mixed-dye-loaded loop-mediated isothermal amplification (LAMP) assay on a self-priming compartmentalization (SPC) microfluidic chip, termed on-chip mixed-dye-based LAMP (CMD-LAMP). In comparison to conventional approaches, CMD-LAMP was advantageous on the limit of detection, which reached down to 1 × 103 CFU/mL in food-contaminated samples without the pre-enrichment of bacteria. Additionally, as a result of the use of a mixed dye and SPC chip, the quantitative result could be easily acquired, avoiding the requirement of sophisticated instruments and tedious operation. Also, CMD-LAMP was rapid and cost-effective. Conclusively, CMD-LAMP has great potential in realizing the on-site quantitative analysis of VP for food safety.

Colorimetric immunoassay for rapid detection of Vibrio parahaemolyticus based on Mn2+-mediate the assembly of gold nanoparticles

Vibrio parahemolyticus ( V. parahemolyticus) is an important food-borne pathogen that causes food poisoning and acute gastroenteritis in humans. Herein, a novel colorimetric immunoassay was presented for rapid detection of V. parahemolyticus using gold nanoparticles (18.1 nm diameter) as chromogenic substrate, whose combination of a magnetic bead-based sandwich immunoassay and an optical sensing system via Mn2+ ions mediated aggregation of gold nanoparticles. MnO2 nanoparticles coated with polyclonal IgG antibodies (7.8 nm diameter) are used to recognize the target and can be etched to generate manganese ions by ascorbic acid. A color change ranging from red to purple to blue can be easily discerned by bare eye, corresponding to V. parahemolyticus concentration in the range between 10 and 106 cfu·mL-1. The proposed method possesses high specificity with a limit of detection of 10 cfu·mL-1 and was successfully applied to determination of V. parahemolyticus in oyster samples without pre-enrichment. In our perception, it shows promise in rapid instrumental and on-site visual detection of V. parahemolyticus.

Development of a self-priming PDMS/paper hybrid microfluidic chip using mixed-dye-loaded loop-mediated isothermal amplification assay for multiplex foodborne pathogens detection

Foodborne pathogen is the primary cause of foodborne disease outbreak. Given its great damage, a sensitive, simple and rapid detection method is demanded. Herein, we described a self-priming polydimethylsiloxane (PDMS)/paper hybrid microfluidic chip, termed SPH chip, with mixed-dye-loaded loop-mediated isothermal amplification (LAMP) for multiplex foodborne pathogens detection. Staphylococcus aureus (SA) and Vibrio parahaemolyticus (VP) were chosen to verify the novel method. Compared to other similar detection devices, the SPH chip required easier fabrication process, less operation steps and lower cost. Additionally, the reaction result, especially for the weak-positive reaction, could be judged more accurately and conveniently due to the use of mixed-dye. Without pre-enrichment of bacteria in the food contaminated sample, the limit of detection (LOD) reached down to 1000 CFU mL-1 with high specific. Additionally, for fully exploiting the potential of SPH chip, a conceptual eight-channel detection chip was also developed. Overall, the reliable and excellent result demonstrated that the novel method had great potential to be applied in the wider range of pathogens detection or disease diagnose, especially in some resource-limited area.