Label-free E. coli detection based on enzyme assay and a microfluidic slipchip.

Abstract

An enzyme assay based method in a microfluidic slipchip was proposed for the rapid and label-free detection of E. coli. The specific target analyte of E. coli was β-d-glucuronidase (GUS) which could catalyze the substrate 6-chloro-4-methyl-umbelliferyl-β-d-glucuronide (6-CMUG) to release the fluorescent molecule 6-chloro-4-methyl-umbelliferyl (6-CMU). E. coli culture, lysis and enzymatic reaction steps could be conducted in a microfluidic slipchip without any pumps and valves, which was tailored for fluorescence detection using a commercial plate reader, to achieve a rapid E. coli test. A mixture of the culture broth, enzyme inducer and E. coli was injected into the chambers on the top layer. A mixture of the substrate and lysis solution was injected into the chambers on the bottom layer. Then, the slipchip was slid to make each chamber independent. E. coli was cultured in the chamber in the LB broth for 2.5 h. After that, the slipchip was slid again to introduce the lysis solution into the culture solution for GUS release and enzyme reaction, and then incubated in the plate reader at 42 °C for another 2.5 h. During incubation, the fluorescence intensity of each chamber was recorded. This proposed label-free method can directly detect E. coli with a low concentration of 8 CFU per chamber within 5 h, thus showing great potential in on-site E. coli detection.