Environmental pollutants pose great risks and adverse effects to humans and therefore arouse global environmental concern. Bacterial sensors capable of assessing the bioavailability and toxicity of pollutants show great advantages in environmental sensing. This project aims at developing a bioluminescent bacteria-based microfluidic sensor for online monitoring of environmental contaminants and toxicity. Microfluidic devices immobilised with Acinetobacter sp. ADP1_lux cells as a model strain have been developed for quantitative bioassays. Three microfluidic devices were developed and tested in order to trap and culture a monolayer of bacterial cells. The terrace device is capable of trapping a monolayer of cells in a chamber for tracking single-cell growth and response. This device utilises a barrier channel lower than the cell diameter. Two flow channels can be used to load bacterial cells, deliver fresh media and inducers and wash away overgrown cells. The device was used to measure the bioluminescence induction of ADP1_lux cells and its capability to track individual cell growth was demonstrated with E.coli cells. Since bioluminescence signals from a monolayer of ADP1_lux cells were too weak to be detected after 2 h induction by 200 µM salicylate, a microwell device was developed to concentrate cells in individual microwells for population-based analysis. Cell loading procedures, dimensions of wells, carbon sources and on-chip cultures that affect bioluminescence light intensities were investigated. This device succeeded in detecting 200 µM salicylate within 1 h. However, long-term cell culture revealed that ADP1_lux cells tend to form biofilms. Cell populations in individual wells varied greatly, making quantification impossible. Therefore, this device is only suitable for rapid detection of high concentrations of contaminants if biofilm forming bacteria cells are used as biosensors. In contrast, in the case of non-adherent cells such as E.coli, a uniform population distribution in each well was achieved after 2-day culture, suggesting this method is applicable to perform long term, quantitative bioassays using suitable, non-adherent cells. To be able to detect low concentrations of contaminants and overcome potential biofilm formation, a new population array device was developed as a proof of concept to control and isolate cell populations. It consists of a network of microfluidic channels and an array of microchambers. The device was characterised with fluorescent dyes and its capability to perform quantitative bioluminescence assays was evaluated by detecting a range of concentrations of salicylate solutions (from 10 µM to 50 µM salicylate) using ADP1_lux cells. A linear correlation between bioluminescence intensities and salicylate concentrations was successfully established within 90 min induction. It is worth noting that the population array device is the first demonstration of bioluminescence detection at the length scale of microns. Therefore, it has potential to perform multiplex detection within a small footprint where different types of whole cell biosensors can be employed simultaneously. To this end, a logarithmic serial dilution device was also developed to enable quantitative, multiplex bioassays to be conducted in the same device. This integrated dilution and population device provides a powerful tool for rapid quantification of multiple contaminants simultaneously in a sample.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:726734 |
| Date | January 2017 |
| Creators | Song, Yanqing |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/8577/ |
Page generated in 0.0019 seconds