A single-use luciferase-based mercury biosensor using Escherichia coli HB101 immobilized in a latex copolymer film

Abstract

A single-use Hg(II) patch biosensor has been developed consisting of 1.25-cm diameter patches of two acrylic vinyl acetate copolymer layers coated on polyester. The top layer copolymer was 47 microm thick whereas the bottom layer of copolymer plus E. coli cells was 30 microm thick. The immobilized E. coli HB101 cells harbored a mer-lux plasmid construct and produced a detectable light signal when exposed to Hg(II). The immobilized-cell Hg(II) biosensor had a sensitivity similar to that of suspended cells but a significantly larger detection range. The levels of mercury detected by the patches ranged from 0.1 nM to 10 000 nM HgCl2 in pyruvate buffer, and luciferase induction as a function of Hg(II) concentration was sigmoidal. Luciferase activity was detected in immobilized cells for more than 78 h after exposure of the cells to HgCl2. Addition of 1 mM D-cysteine to the pyruvate buffer increased luciferase induction more than 100-fold in the immobilized cell patches and 3.5-fold in a comparable suspension culture. The copolymer patches with immobilized cells were stable at -20 degrees C for at least 3 months, and the Hg(II)-induced luciferase activity after storage was similar to that of samples assayed immediately after coating. Patches stored desiccated at room temperature for 2 weeks showed lower mercury-induced luciferase activity when compared to freshly prepared patches, but they still had a considerable detection range of 1 to 10 000 nM HgCl2.

Blind and naïve classification of toxicity by fish chromatophores

“…Researchers from The University of Turku, Finland, whose technology was presented at the workshop, developed a whole-cell bacterial sensor for the detection of heavy metals (specifically mercury and arsenite) by measuring luciferase-catalyzed bioluminescence (Tauriainen et al, 1999;Ivask et al, 2001;Hakkila et al, 2003). A similar concept was utilized for designing a fibre-optic biosensor for mercury detection, based on genetically engineered E.coli MC 1061 containing the mercury-responding promoter to control the expression of luxCDABE from Photorhabdus luminescens (Green et al, 2002), as well as for developing a latex-coated mercury sensor using E.coli-harboring pRB28 with a mer-lux constract (Lyngberg et al, 1999(Lyngberg et al, , 2001). However, the use of bioluminescence-based techniques is not limited only to mercury and arsenite testing.…”

Microbial Cell Arrays

“…Luminescence was induced with 20 ng mL À1 Hg 2+ , and the array responded within 1 h. Mercury was also detected with similar immobilized or nonimmobilized constructs at lower concentrations, down to the parts per trillion scale, albeit not in an array format [97][98][99].…”

Spatial expression of a mercury-inducible green fluorescent protein within a nanoporous latex-based biosensor coating

“…As opposed to immobilized enzymes, living cells can generate the expensive but critical reaction components such as ATP and NADPH, but the immobilization process must maintain cell viability and reactivity for the desired period of time. Adhesive latex has been used as an immobilization support for the development of biosensors and biocatalysts containing whole cells [8][9][10] and is available in a variety of polymer particle sizes and formulations with diVerent coalescence characteristics [20][21][22][23]. These coatings are typically composed of one or more cell coat layers in which the cells are mixed with latex and spread as a 10-to 70-m thick sheet onto a polyester or metal substrate.…”