A dual-channel electrochemical biosensor enables concurrent detection of pathogens and antibiotic resistance.

Diarrheagenic E. coli infections, commonly treated with ß-lactam antibiotics, contribute to antibiotic resistance - a pressing public health concern. Rapid monitoring of pathogen antibiotic resistance is vital to combat antimicrobial spread. Current bacterial diagnosis methods identify pathogens or determine antibiotic resistance separately, necessitating multiple assays. There is an urgent need for tools that simultaneously identify infectious agents and their antibiotic resistance at the point of care (POC). We developed an integrated electrochemical chip-based biosensor for detecting enteropathogenic E. coli (EPEC), a major neonatal diarrheal pathogen, using an antibody against a virulence marker, termed EspB, and the ß-lactam resistance marker, ß-lactamase. A dual-channel microfabricated chip, bio-functionalized with a specific EspB monoclonal antibody, and nitrocefin, a ß -lactamase substrate was utilized. The chip facilitated electrochemical impedance spectroscopy (EIS)-based detection of EspB antigen and EspB-expressing bacteria. For ß-lactam resistance profiling, a second channel enabled differential-pulse voltammetric (DPV) measurement of hydrolyzed nitrocefin. EIS-based detection of EspB antigen was calibrated (LOD: 4.3 ng/mL ±1 and LOQ: 13.0 ng/mL ±3) as well as DPV-based detection of the antibiotic resistance marker, ß-lactamase (LOD: 3.6 ng/mL ±1.65 and LOQ: 10 ng/mL ±4). The integrated EIS and DPV biosensor was employed for the simultaneous detection of EspB-expressing and ß-lactamase-producing bacteria. The combined readout from both channels allowed the distinction between antibiotic-resistant and -sensitive pathogenic bacteria. The integrated electrochemical biosensor successfully achieved simultaneous, rapid detection of double positive EspB- and ß-lactamase-expressing bacteria. Such distinction enabled by a portable device within a short assay time and a simplified sample preparation, may be highly valuable in mitigating the spread of AMR. This new diagnostic tool holds promise for the development of POC devices in clinical diagnosis.

Electrochemical Detection of Waterborne Bacteria Using Bi-Functional Magnetic Nanoparticle Conjugates.

Detection of microbial contamination in water is imperative to ensure water quality. We have developed an electrochemical method for the detection of E. coli using bi-functional magnetic nanoparticle (MNP) conjugates. The bi-functional MNP conjugates were prepared by terminal-specific conjugation of anti-E. coli IgG antibody and the electroactive marker ferrocene. The bi-functional MNP conjugate possesses both E. coli-specific binding and electroactive properties, which were studied in detail. The conjugation efficiency of ferrocene and IgG antibodies with amine-functionalized MNPs was investigated. Square-wave voltammetry enabled the detection of E. coli concentrations ranging from 101-107 cells/mL in a dose-dependent manner, as ferrocene-specific current signals were inversely dependent on E. coli concentrations, completely suppressed at concentrations higher than 107 cells/mL. The developed electrochemical method is highly sensitive (10 cells/mL) and, coupled to magnetic separation, provides specific signals within 1h. Overall, the bi-functional conjugates serve as ideal candidates for electrochemical detection of waterborne bacteria. This approach can be applied for the detection of other bacteria and viruses.

Vitagenic Effect of Specific Bioactive Fractions of Rhodiola with Trachurus sp. Extract Against Oxidative Stress-Induced Aging in Human Amnion Derived Epithelial Cell Line: In View of a Novel Senolytic.

BACKGROUND: Rhodiola rosea is a herb that has been used in traditional medicine for several years, and LF is a class of lipoproteins derived from the fish Trachurus sp. (LF-T), which exhibits known anti-inflammatory activity. OBJECTIVE: Investigating the anti-aging effect of Rhodiola specific bioactive fractions cluster in combination with LF-T (R-L compound) in H2O2 mediated oxidative stress-induced human amnion derived epithelial cell line - FL cells as normal human cell line. METHODS: FL cells were treated with H2O2 to induce cellular aging, followed by treatment of the RL compound to study its anti-aging characteristics. Based on the proliferation rate, 0.05% and 0.1% concentration of R-L compound was determined using MTT assay. Anti-aging and anti-oxidant assays, ABTS, DPPH, Hyaluronidase activity Nitric Oxide, Lipid Peroxidase, and Superoxide Dismutase were performed. qPCR for anti-aging genes and matrix metalloproteinase genes were analyzed. RESULTS: FL cells treated with R-L compound exhibited increased proliferation rate and free-radical reduction. Decreased Hyaluronidase enzyme activity and regulation of genes such as SIRT1, KLOTHO, SERPINA 6, MMP 9, and MMP 2 expression depicted the anti-aging role of the R-L compound. Chemometric profiling of the R-L compound revealed that aromatic compounds and unsaturated fatty acids along with their derivatives, were present predominantly, which might have attributed to the potent oxidative stress impeded aging activity. CONCLUSION: Specific Bioactive Fractions of Rhodiola in combination with LF-T obtained from Trachurus sp. involve in the regulation of aging genes and might be a novel approach to prevent the cells from oxidative stress damage and also it might avert the aging of cells.

Developing a Programmable, Self-Assembling Squash Leaf Curl China Virus (SLCCNV) Capsid Proteins into "Nanocargo"-like Architecture.

A new era has begun in which pathogens have become useful scaffolds for nanotechnology applications. In this research/study, an attempt has been made to generate an empty cargo-like architecture from a plant pathogenic virus named Squash leaf curl China virus (SLCCNV). In this approach, SLCCNV coat protein monomers are obtained efficiently by using a yeast Pichia pastoris expression system. Further, dialysis of purified SLCCNV-CP monomers against various pH modified (5-10) disassembly and assembly buffers produced a self-assembled "Nanocargo"-like architecture, which also exhibited an ability to encapsulate magnetic nanoparticles in vitro. Bioinformatics tools were also utilized to predict the possible self-assembly kinetics and bioconjugation sites of coat protein monomers. Significantly, an in vitro biocompatibility study using SLCCNV-Nanocargo particles showed low toxicity to the cells, which eventually proved as a potential nanobiomaterial for biomedical applications.