LUMINESCENCE-BASED BIOSENSORS FOR THE DETECTION OF BIOAVAILABLE HEAVY METALS

Sisko Tauriainen, Marko Virta, Juha Lappalainen and Matti Karp

Department of Biotechnology, University of Turku, Finland

INTRODUCTION: The measurement of metal bioavailability is difficult with traditional analytical methods. However, the bioavailability of metals is an important factor in the deterrnination of metal toxicity and therefore the detection of bioavailable metals is of interest. Biosensors are regarded as a promising means of assessing bioavailability of environmental substances. We have established a new concept of analyzing bioavailability of heavy metals by creating microbial strains capable of sensing the environment by emitting light. These strains express a sensitive reporter gene, firefly luciferase, connected to a promoter element responding specifically to various heavy metals.

METHODS: The sensor plasmids were all created by inserting a heavy metal responsive element into BamHI and XhoI restriction sites of vector pCSS810 in front of the firefly luciferase gene by using standard recombinant-DNA techniques. The promoter/operator elements were isolated, and generated by Polymerase Chain Reaction (PCR) using specific oligonucleotide primers. The plasmids were expressed in different hosts, most often in E. coli and S. aureus in order to obtain maximal and specific response to each of the metal tested. Conditions for optimal performance of sensor bacteria were determined. In order to test the suitability of sensors for environmental analysis, soil samples from contaminated areas and water samples from environmental sources were measured. Water samples were from relatively clean areas and were therefore spiked with different metals. The quantification of light emission was done with a microtiter plate luminometer (Labsystems Luminoscan). Sensor cells were also freeze-dried to ensure the reagent like usage.

RESULTS: The biosensors developed responded to mercury, cadmium, lead, arsenite and antimonite. The lowest detectable concentrations were 1 fM for mercury, 10 nM for cadmium, 33 nM for lead, 100 nM for arsenite and 33 nM for antimonite. The time needed for induction to occur was only minutes, but optimal induction times were 1-2 hours. The sensors were specific and other ions tested did not notably interfere with the measurement. Bacteria survived freeze-drying and their ability to induce luciferase production in the presence of a metal was stored. Analysis of water and soil samples was done with freeze-dried bacteria and showed that it is possible to use these biosensors also for bioavailability measurements of environmental samples.

CONCLUSIONS: The described sensors complement chemical analysis of metals by distinguishing bioavailable metal from total metal contents of samples. The system together with sensors detecting total toxicity can be immobilized to perform automated real-time measurements of various environmental samples.