Development of novel disposable metal/coliform biosensor device for simultaneous monitoring of chemical and microbial quality of drinking water and urine

Ndubuisi Christopher Nwankwo

Research output: ThesisDoctoral Thesis

Abstract / Description of output

The main sources of human exposure to heavy metals are water and food. Metals play significant roles in the growth and development of living organisms. However, they can be lethal at elevated concentrations due to their interference with normal biological processes, although some metal species are toxic even at low concentrations (e.g. Hg, Pb). The traditional laboratory-based analytical investigations for environmental contaminants are expensive and usually require trained staff and sophisticated facilities. This study sought to develop a sensitive, specific, accurate, rapid, cheap and portable novel disposable biosensor device for the detection of arsenic and other heavy metals in drinking water and investigate its biomedical potential for testing arsenic in urine samples. A pre-existing arsenic biosensor consisting of the E. coli chromosomal ars promoter, arsR repressor gene and lacZ′α gene was tested. The expression of lacZʹα allows the fermentation of lactose with production of acid, changing the coloured pH indicator bromothymol blue from blue to yellow. Our results showed that the biosensor responded reliably to arsenate concentrations in water and urine samples below the recommended World Health Organisation limit of 10 ppb arsenic. Novel zinc and copper biosensors were developed using promoter elements regulated by endogenous zinc-binding and copper-binding transcription factors, ZntR and CueR respectively, fused to lacZʹα, and a suitable test medium, ZBM3, was developed. Initial designs showed high background activity. The constructs were redesigned using either weaker ribosome binding sites (RBS), low copy number plasmids or promoters with lower activity. The redesigned Zn biosensor accurately detected zinc, cadmium, lead and mercury concentrations (3 mg/L, 0.003 mg/L, 0.01 mg/L and 0.001 mg/L respectively) below the recommended WHO limits. Similarly, copper, silver and gold levels (2 mg/L, no guidelines for silver and gold respectively) were detected by the novel copper biosensor, below the limits recommended by the WHO. The stability of the sensor cells was independently tested by; air drying, freeze drying, or immobilised on paper, each within a re-sealable system that can then be stored and distributed, hence eliminating the need for routine culture and minimizing variation between different batches of cells. Sensor bacteria were successfully revived after 120 days storage at room temperature or 370C. Data obtained showed approximately 0.2% viable cells at the aforementioned conditions after initial inoculations of 1.3 x 108 CFU / mL of cells, an indication of reduced viability as a result of rigorous cell preparations and incomplete drying particularly air dried cells. However, data also obtained showed that dried cells with such survival rate were still effective in the assay. These growth-based biosensors supported growth on lactose medium which allows use of the same format for detection of both metals and biological contaminants (coliforms) in a single unit. Coliforms were readily detected based on lactose fermentation using a variant of the same growth medium used for biosensor organisms, allowing for easy generation of a combined coliform/metal sensor device. In conclusion, metal contaminants in environmental samples can be reliably and accurately detected at the safe limits set by the regulatory authorities by the use of biological methods of testing. These testing techniques, together with the simultaneous testing of biological contaminants, have the potential of reducing the high costs of physico-chemical methods, save time as well as make sample testing available to areas lacking modern testing facilities. However, the systems need to be improved to allow for a reduction in time needed to obtain reliable results.
Original languageEnglish
  • French, Chris, Supervisor
Publication statusPublished - 2019


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