Quantification of endospores in ancient permafrost using time-resolved terbium luminescence.

We describe herein a simple procedure for quantifying endospore abundances in ancient and organic-rich permafrost. We repeatedly (10x) extracted and fractionated permafrost using a tandem filter assembly composed of 3 and 0.2 µm filters. Then, the 0.2 µm filter was washed (7x), autoclaved, and the contents eluted, including dipicolinic acid (DPA). Time-resolved luminescence using Tb(EDTA) yielded a LOD of 1.46 nM DPA (6.55 × 103 endospores/mL). In review, DPA/endospore abundances were ~2.2-fold greater in older 33 ky permafrost (258 ± 36 pmol DPA gdw-1; 1.15 × 106 ± 0.16 × 106 spores gdw-1) versus younger 19 ky permafrost (p = 0.007297). This suggests that dormancy increases with permafrost age.

Effects of terbium chelate structure on dipicolinate ligation and the detection of Bacillus spores.

Terbium-sensitized luminescence and its applicability towards the detection of Bacillus spores such as anthrax are of significant interest to research in biodefense and medical diagnostics. Accordingly, we have measured the effects of terbium chelation upon the parameters associated with dipicolinate ligation and spore detection. Namely, the dissociation constants, intrinsic brightness, luminescent lifetimes, and biological stabilities for several Tb(chelate)(dipicolinate)(x) complexes were determined using linear, cyclic, and aromatic chelators of differing structure and coordination number. This included the chelator array of NTA, BisTris, EGTA, EDTA, BAPTA, DO2A, DTPA, DO3A, and DOTA (respectively, 2,2',2″-nitrilotriacetic acid; 2,2-bis(hydroxymethyl)-2,2',2″-nitrilotriethanol; ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid; ethylenediamine-N,N,N',N'-tetraacetic acid; 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid; diethylenetriamine-N,N,N',N″,N″-pentaacetic acid; 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid; and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). Our study has revealed that the thermodynamic and temporal emission stabilities of the Tb(chelate)(dipicolinate)(x) complexes are directly related to chelate rigidity and a ligand stoichiometry of x=1, and that chelators possessing either aromaticity or low coordination numbers are destabilizing to the complexes when in extracts of an extremotolerant Bacillus spore. Together, our results demonstrate that both Tb(EDTA) and Tb(DO2A) are chemically and biochemically stable and thus applicable as respectively low and high-cost luminescent reporters for spore detection, and thereby of significance to institutions with developing biodefense programs.