Rapid and cost-effective detection of dengue infection, especially during the early stages of disease and in resource-limited settings, remains a significant public health concern. Recent advances in nanomaterial-based biosensors have provided promising opportunities for the development of simple, sensitive, and affordable nucleic acid detection platforms. In the present study, a Cu2+-modified reduced graphene oxide (Cu2+/rGO) nanostructure-based colorimetric biosensor integrated with hybridization chain reaction (HCR) was developed and assessed for the detection of a dengue virus target sequence. The nanozyme exhibited peroxidase-like activity and catalyzed the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2), generating a visible colorimetric signal. Following HCR-mediated double-stranded DNA formation, the catalytic activity of the nanostructure was suppressed, resulting in reduced color intensity and enabling signal-off detection. The proposed biosensor showed a linear response over 0.5-10 nM, with detection limits of 1.36 nM in buffer and 1.6 nM in human serum samples spiked with synthetic target DNA. The color variation was directly visible to the naked eye. The assay showed good linearity (R2 = 0.9868) while avoiding the need for thermocyclers and complex instrumentation. The Cu2+/rGO nanozyme preserved nearly 79% of its catalytic performance after 10 days of storage. Overall, the results suggest that the developed platform may provide a practical and cost-effective biosensing strategy for preliminary nucleic acid screening in resource-limited settings and complement conventional polymerase chain reaction (PCR)-based assays.
S. Seraj, Mahdi Rahaie· Analytical Biochemistry· 0 citations
Antimicrobial resistance has emerged as a significant threat to global public health, necessitating accurate and rapid detection methods. To address this need, a colorimetric nanobiosensor was developed that exploits antibiotic-induced metabolic changes in bacteria, detected via the redox-dependent activity of a peroxidase-mimicking nanozyme (γ-Fe₂O₃@Prussian blue). This study aimed to differentiate resistant from susceptible E. coli strains and determine the minimum inhibitory concentration for susceptible ones using this novel principle. The experiments were conducted on two susceptible and two resistant strains of E. coli along with antibiotics, including ampicillin, cefazolin, ceftriaxone, and kanamycin. The γ-Fe2O3@PB NPs were characterized using UV-vis spectroscopy, dynamic light scattering (DLS), X-ray diffraction (XRD), transmission electron microscopy, and Fourier-transform infrared spectroscopy (FTIR). The synthesis of cubic crystalline nanoparticles with average crystallite size and hydrodynamic size of 31 nm and 219 nm, respectively, was confirmed. Upon adding H₂O₂ and the chromogenic substrate TMB to the bacterial culture supernatant, the intensity of the blue color (measured at 652 nm) in sensitive strains correlated with antibiotic concentrations. Compared to sub-MIC concentrations, a significant and sharp increase in absorbance at 652 nm was observed at the MIC, and similarly high absorbance levels were maintained at higher concentrations. By comparing the absorbance levels below and at the MIC, the MIC range can be determined. In contrast, for resistant strains, the intensity of the produced color remained nearly constant across different antibiotic concentrations. This innovative, label-free approach offers a simple and reliable method for antibiotic susceptibility testing. It achieves results in approximately 4 hours, with a detection limit (initial bacterial count) of 1.67×107 CFU/mL, without requiring expensive reagents or equipment.