ENHANCED RESPONSE OF AN OLIGONUCLEOTIDE-BASED BIOSENSOR TO ENVIRONMENTAL MERCURY

One environmental pollutant of particular relevance to the coal-generated power industry is mercury. Power plants in the U.S., led by Texas, Ohio, Pennsylvania, Indiana, and Alabama, collectively emitted over 90,000 pounds of mercury into the air in 2003. Calls for increased mercury monitoring activities have come from many groups concerned with environmental contamination and mercury bioconcentration in fish. Additionally, the benefits of improvements in the reduction of mercury emissions from existing power plants cannot be seriously evaluated without extensive monitoring of the environment. Low in situ mercury concentrations and the expense of traditional laboratory analyses currently limit such routine and effective monitoring.

Microbial biosensors sensitive to mercury have been developed that quantitatively produce light in response to the amount of mercury (II) entering the cells. However, these sensors are typically difficult to prepare, can have long lag times between initial exposure and subsequent light emission, and are difficult to use in the field. Whole cell biosensors using living bacteria also require attention to the growth requirements of the cells, as well as complications brought on by the presence of other toxic compounds in addition to mercury.

A “molecular beacon” sensor for mercury (II) reported by Ono and Togashi (Angew. Chem. Int. Ed. 2004, 43:4300-4302.) was modified to enhance its sensitivity and fluorescence response. The basic detection method involves the selective binding of mercury ions to thymine-thymine (T-T) base pairs in DNA duplexes. An oligonucleotide sequence in the sensor changes its conformation upon binding with mercury ions, and causes a fluorophore at one end of the oligonucleotide sequence to come in proximity with a quencher molecule attached to the other end. Enhanced fluorescence resonance energy transfer (FRET) results in a decrease in the intensity of the fluorescence spectrum. The use of fluorescein as a harvester fluorophore and alternative emitter fluorophores dramatically increased the sensitivity of the sensor. The fluorescence spectrum generated by this sensor is analyzed using a field spectrofluorometer, and the analytical approach may be useful in environmental mercury monitoring activities.