On-chip genotoxic bioassay based on bioluminescence reporter system using three-dimensional microfluidic network

Abstract

Microchip-based genotoxic bioassay using sensing Escherichia coli strains has been performed. In this method, the assay was conducted in three-dimensional microfluidic network constructed by a silicon perforated microwell array chip and two poly (dimethylsiloxane) (PDMS) multi-microchannel chips. The sensing strains having firefly luciferase reporter gene under transcriptional control of umuD as an SOS promoter were put into the channels on one of the PDMS chips and immobilized in the silicon microwells. Samples containing genotoxic substances and substrates for luciferase were into the channels on the other PDMS chip. The optimum conditions of the assay in the on-chip format have been investigated using mitomycin C (MMC) as a genotoxic substance. As a result, the dose-dependence of bioluminescence intensity was obtained at once on the chip. Additionally, the response ratios of the bioluminescence between mutagen-and non-induced strains were successfully enhanced by improving the on-chip assay methods and conditions. Several well-known genotoxic substances were subjected to the on-chip assay, and were detected with the detection limits comparable to those in the conventional method with reduced time.-3 -

Bacterial genotoxicity bioreporters

“…The immobilized cells were stable for at least 1 week at 4°C. Following further optimization, the detection limits of model hydrophobic and hydrophilic genotoxicants were comparable to those obtained by the reference method used ( Maehana et al. , 2006 )…”

“…The immobilized cells were stable for at least 1 week at 4°C. Following further optimization, the detection limits of model hydrophobic and hydrophilic genotoxicants were comparable to those obtained by the reference method used (Maehana et al, 2006) Electrochemical mutagen screening based on the umutest was performed on a microbial chip combined with a SECM device (Matsui et al, 2006). The microbial chip was fabricated by embedding 5 nl of collagen-immobilized genetically engineered S. typhimurium strain (TA1535/ pSK1002 in a microcavity on a glass substrate.…”

Reporter Proteins in Whole-Cell Optical Bioreporter Detection Systems, Biosensor Integrations, and Biosensing Applications

“…Obviously, the hands-on manipulations do not contribute to an ideal biosensor format. Maehana et al [ 30 ] solves the extraneous luciferin addition somewhat by incorporating microfluidics into their chip-based biosensor. Various microwells patterned onto a silicon wafer hold the sample and the bioreporter bacteria (a genotoxic sensing E. coli strain), and although the luciferin substrate is later added within the same microwells, one could envision separate wells and microfluidic mixing as a means towards forming an all-inclusive biosensor.…”

Evaluation of chrono-amperometric signal detection for the analysis of genotoxicity by a whole cell biosensor

“…In order to facilitate an easy to use field-applicable test system for the detection of genotoxicity there are several on-going activities for the construction of biosensors based on the bacterial SOS-response [12][13][14][15][16][17]. These approaches are based mainly on luminescence or fluorescence; Matsui et al proposed electrochemical signal detection via para-aminophenyl ␤-d-galactopyranoside (pAPG) by scanning electrochemical microscopy.…”