Development and Optimization of LSPR-based Aptasensor for Detection of Vibrio cholerae

Introduction: Vibrio cholerae is one of the primary waterborne pathogens that can enter the biofilm phase during its lifecycle, making it difficult to detect. To address this challenge, we have designed a colorimetric aptasensor based on aptamers and gold nanoparticles (GNPs) for the effective management and treatment of this illness. Materials and Methods: The aptamer sequence was selected based on previous work and amplified using PCR with specific primers. The aptamer, which has a strong binding affinity to V. cholerae, was immobilized on the surface of GNPs. Detection was achieved by observing the aggregation of GNPs induced by the target bacteria, resulting in noticeable color changes in the reaction upon the addition of NaCl. To determine the optimal conditions, a unique method based on a Taguchi orthogonal array was used to evaluate critical parameters such as conjugation time, temperature, pH, and aptamer concentration. Results: The optimal conditions for aptamer immobilization were determined to be pH ۹, ۳ hours for incubation time ۱۰ °C for incubation temperature, and ۵۵۰ nM for aptamer concentration. Analysis of signal-to-noise ratios showed that temperature and pH levels significantly affect GNP-aptamer conjugation. Under the optimal conditions, a linear calibration relationship was established between the A۶۳۰/A۵۲۴ ratio and V. cholerae concentrations ranging from ۱۰۲ to ۱۰۷ CFU/ml. The detection limit and time were found to be ۶ CFU/ml and ۵۰ minutes, respectively. Conclusions: The developed colorimetric aptasensor demonstrates high sensitivity and specificity for the detection of V. cholerae, making it suitable for the production of rapid diagnostic kits.

کلیدواژه ها:

V. cholerae ، Diagnosis ، Aptamer ، GNPs ، Surface Plasmonic Resonance