Development and optimization of LSPR-based aptasensor for detection of Vibrio cholerae

Abstract

Abstract Vibrio cholera was one of the major water-borne pathogens that can enter into the biofilm phase in a period of life; hence, it was challenging to recognize these bacteria. For this, we reported the development of a colorimetric aptasensor based on aptamer and gold nanoparticles (GNPs) for effective management and treatment of this disease. The aptamer sequence was selected according to previous work and amplified-based PCR and specific primers. Aptamer with high binding affinity to V.cholerae was firstly immobilized on the surface of GNPs, and detection was accomplished through aggregation of GNPs induced by target bacteria, which was associated with color changes of the reaction after addition of NaCl. To find an optimum condition, a specific approach based on a Taguchi orthogonal array was therefore used to evaluate critical parameters and to optimize them such as conjugation time, temperature, pH, and aptamer concentration. Analysis of signal-to-noise ratios revealed the great influence of temperature and pH on the conjugation of GNPs-aptamers. The optimum conditions for immobilization of aptamer were determined to be 9 for pH, 3 h for incubation time, 10˚C for incubation temperature, and 550nM for aptamer concentration. Under the optimum conditions, a linear calibration relationship was obtained between the ratio of A630/A524 and concentrations of V.cholerae over the range from 102 to 107CFU/mL. The detection limit and time were 6 CFU/mL and 80min, respectively. The developed colorimetric aptasensor is simple, convenient, and suitable for on-site detection of V.cholerae.