Fast and sensitive graphene oxide-DNAzyme-based biosensor for Vibrio alginolyticus detection.

DNAzymes have been widely and effectively used for the detection of pathogenic bacteria, which pose a serious public health threat. However, the rapid and cost-effective detection of such bacteria remains a major challenge. In this study, we successfully selected Vibrio alginolyticus-specific DNAzymes. The activity of the candidates was assessed via fluorescence intensity and gel electrophoresis. The DNAzyme DT1 had a detection limit of 31 CFU/ml for V. alginolyticus and exhibited high specificity. Graphene oxide (GO) was used to develop a DNAzyme-based fluorescent sensor for the detection of V. alginolyticus, which significantly improved detection performance and shortened the reaction time as little as 10 s. The proposed method was then validated using crab, shrimp, fish, clam, and oyster samples. This study thus provides a new method for the rapid and sensitive detection of V. alginolyticus.

Anti-Helicobacter pylori Activity of a Lactobacillus sp. PW-7 Exopolysaccharide.

Helicobacter pylori is a cause of gastric cancer. We extracted the exopolysaccharide (EPS) of Lactobacillus plajomi PW-7 for antibacterial activity versus H. pylori, elucidating its biological activity and structural characteristics. The minimum inhibitory concentration (MIC) of EPS against H. pylori was 50 mg/mL. Disruption of the cell membranes of pathogenic bacteria by EPS was indicated via the antibacterial mechanism test and confirmed through electron microscopy. EPS also has antioxidant capacity. The IC50 of EPS for 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical, superoxide anions, and hydroxyl radicals were 300 µg/mL, 180 µg/mL, and 10 mg/mL, respectively. The reducing power of EPS was 2 mg/mL, equivalent to 20 µg/mL of ascorbic acid. EPS is a heteropolysaccharide comprising six monosaccharides, with an approximate molecular weight of 2.33 × 104 Da. Xylose had a significant effect on H. pylori. EPS from L. plajomi PW-7 showed potential as an antibacterial compound and antioxidant, laying a foundation for the development of EPS-based foods.

Rapid detection of Pseudomonas aeruginosa using a DNAzyme-based sensor.

In the present study, a DNAzyme was screened in vitro through the use of a DNA library and crude extracellular mixture (CEM) of Pseudomonas aeruginosa. Following eight rounds of selection, a DNAzyme termed PAE-1 was obtained, which displayed high rates of cleavage with strong specificity. A fluorescent biosensor was designed for the detection of P. aeruginosa in combination with the DNAzyme. A detection limit as low as 1.2 cfu/ml was observed. Using proteases and filtration, it was determined that the target was a protein with a molecular weight of 10 kDa-50 kDa. The DNAzyme was combined with a polystyrene board to construct a simple indicator plate sensor which produced a color that identified the target within 10 min. The results were reliable when tap water and food samples were tested. The present study provides a novel experimental strategy for the development of sensors based on a DNAzyme to rapidly detect P. aeruginosa in the field.

Detection of Vibrio vulnificus in Seafood With a DNAzyme-Based Biosensor.

Vibrio vulnificus is an important pathogenic bacterium that is often associated with seafood-borne illnesses. Therefore, to detect this pathogen in aquatic products, a DNAzyme-based fluorescent sensor was developed for the in vitro detection of V. vulnificus. After screening and mutation, a DNAzyme that we denominated "RFD-VV-M2" exhibited the highest activity, specificity, and sensitivity. The limit of detection was 2.2 × 103 CFU/ml, and results could be obtained within 5-10 min. Our findings suggested that the target of DNAzyme RFD-VV-M2 was a protein with a molecular weight between 50 and 100 kDa. The proposed biosensor exhibited an excellent capacity to detect marine products contaminated with V. vulnificus. Therefore, our study established a rapid, simple, sensitive, and highly specific detection method for V. vulnificus in aquatic products.

Rapid Detection of Helicobacter pylori by the Naked Eye Using DNA Aptamers.

Helicobacter pylori was first isolated from gastritis patients by Barry J. Marshall and J. Robin Warren in 1982, and more than 90% of duodenal ulcers and about 80% of gastric ulcers are caused by H. pylori infection. Most detection methods require sophisticated instruments and professional operators, making detection slow and expensive. Therefore, it is critical to develop a simple, fast, highly specific, and practical strategy for the detection of H. pylori. In this study, we used H. pylori as a target to select unique aptamers that can be used for the detection of H. pylori. In our study, we used random ssDNA as an initial library to screen nucleic acid aptamers for H. pylori. We used binding rate and the fluorescence intensity to identify candidate aptamers. One DNA aptamer, named HPA-2, was discovered through six rounds of positive selection and three rounds of negative selection, and it had the highest affinity constant of all aptamers tested (K d = 19.3 ± 3.2 nM). This aptamer could be used to detect H. pylori and showed no specificity for other bacteria. Moreover, we developed a new sensor to detect H. pylori with the naked eye for 5 min using illumination from a hand-held flashlight. Our study provides a framework for the development of other aptamer-based methods for the rapid detection of pathogenic bacteria.

Selection of DNAzymes for Sensing Aquatic Bacteria: Vibrio Anguillarum.

Vibrio anguillarum is a bacterial pathogen that causes serious damage to aquatic fish, and its rapid detection and prevention are critical. DNAzymes are DNA-based catalysts with excellent stability. In this study, in vitro selection of DNAzymes was performed using the crude extracellular matrix (CEM) of V. Anguillarum as the target. Different from previous selections targeting bacterial CEM, this work used an unmodified DNA library, allowing easier adoption of the technology. After seven rounds of selection, a DNAzyme named VAE-2 with high activity and specificity was obtained. It showed the highest activity toward V. Anguillarum among eight types of tested bacterial strains. Polyvalent metal ions are needed for its activity. Protease treatment of the CEM and filtration studies indicated that the target is a protein with a molecular weight between 50 k and 100 k Da. A fluorescent biosensor was designed for V. anguillarum with a detection limit down to 4000 cfu/mL, and detection was demonstrated for real fish tissue and feeding water samples. Being the first work of DNAzyme-based sensing of aquatic bacteria, this study indicates that unmodified DNA can be used for targeting bacterial CEM, and it provides a new framework for developing other RNA-cleaving DNAzymes for rapid detection of pathogenic bacteria and water pollution.