The universal dual-mode aptasensor for simultaneous determination of different bacteria based on naked eyes and microfluidic-chip together with magnetic DNA encoded probes.

ABSTRACT

It was critically important to develop some sensitive, convenient and on-site methods for simultaneous assay of different pathogenic bacteria in foods. In this work, a dual-mode aptasensor was established for fulfilling above aims combing colorimetry with microfluidic chip. This as-prepared dual-mode aptasensor not only realized rapid screening by naked eye on-site, but also the simultaneous quantification of multiple bacteria. Namely, the presence of pathogenic bacteria was firstly judged by naked eyes with Salmonella typhimurium (S.T) and Vibrio parahaemolyticus (V.P) as models. And then, S.T and V.P in positive samples were simultaneously quantified by microfluidic chip. In order to obtain the multiple signals, a series of magnetic DNA encoded-probes (MDEs) was fabricated containing rolling cycle amplified long DNA chain (RCA-DNA) rich in G-quadruplex sequences. They can combine with hemin as DNAzyme to catalyze 3,3'-5,5'-Tetramethyl benzidine (TMB)-H2O2 system for color development and be cleaved by EcoRV endonuclease to produce DNA fragments with different lengths. The microfluidic chip was employed to separate and quantify the fragments for quantifying S.T and V.P simultaneously. For this protocol, 100 CFU·mL-1 of V.P or S.T could be observed by the naked eye and as low as 32 S.T and 30 CFU·mL-1 V.P could be detected by the chip within 3 min. The dual-mode aptasensor could quickly screen positive samples, and simultaneously perform quantitative detection of the bacteria in positive samples. Our protocol demonstrated its potential in on-site qualification & simultaneous quantification of foodborne bacteria in foods.