Assessing CaDPA levels, metabolic activity, and spore detection through deuterium labeling.

Many strains among spore-forming bacteria species are associated with food spoilage, foodborne disease, and hospital-acquired infections. Understanding the impact of environmental conditions and decontamination techniques on the metabolic activity, viability, and biomarkers of these spores is crucial for combatting them. To distinguish and track spores and to understand metabolic mechanisms, spores must be labeled. Staining or genetic modification are current methods for this, however, these methods can be time-consuming, and affect the viability and function of spore samples. In this work, we investigate the use of heavy water for permanent isotope labeling of spores and Raman spectroscopy for tracking sporulation/germination mechanisms. We also discuss the potential of this method in observing decontamination. We find that steady-state deuterium levels in the spore are achieved after only ∼48 h of incubation with 30% D2O-infused broth and sporulation, generating Raman peaks at cell silent region of 2200 and 2300 cm-1. These deuterium levels then decrease rapidly upon spore germination in non-deuterated media. We further find that unlike live spores, spores inactivated using various methods do not lose these Raman peaks upon incubation in growth media, suggesting these peaks may be used to indicate the viability of a spore sample. We further observe several Raman peaks exclusive to deuterated DPA, a spore-specific chemical biomarker, at e.g. 988 and 2300 cm-1, which can be used to track underlying changes in spores involving DPA. In conclusion, permanent spore labeling using deuterium offers a robust and non-invasive way of labeling bacterial spores for marking, viability determination, and characterising spore activity.

Electrochemical and electrogenerated chemiluminescent studies of a trinuclear complex, [((phen)2Ru(dpp))2RhCl2]5+, and its interactions with calf thymus DNA.

The electrochemical behavior of a trinuclear ruthenium(II)-containing complex, [((phen)(2)Ru(dpp))(2)RhCl(2)](5+) (where phen = 1,10-phenanthroline, dpp = 2,3-bis(2-pyridyl)pyrazine), was studied in acetonitrile (MeCN) and aqueous solutions. In MeCN containing 0.10 M tetra-n-butylammonium perchlorate (TBAP), the complex displayed a reversible, overlapping Ru(II/III) redox process with E(1/2) = +1.21 V vs Ag/Ag(+) (10 mM), an irreversible reduction of Rh(III/I) at -0.73 V vs Ag/Ag(+), and two quasi-reversible dpp/dpp(-) couples with E(1/2) = -1.11 and -1.36 V vs Ag/Ag(+) at a Pt electrode with a scan rate of 50 mV s(-1). In 0.20 M Tris buffer solution (pH 7.4), an irreversible, overlapping Ru(II/III) oxidation at +1.48 V vs Ag/AgCl (3 M KCl), and an irreversible reduction of Rh(III/II) at -0.78 V vs Ag/AgCl were observed at a glassy carbon electrode with a scan rate of 50 mV/s. Investigations on the electrogenerated chemiluminescence (ECL) of the complex revealed that 2-(dibutylamino) ethanol (DBAE) was superior to tri-n-propylamine (TPrA) as an ECL coreactant within their entire concentration range of 10-100 mM in MeCN, and in aqueous media, as low as 1.0 nM of the complex can be detected using TPrA coreactant ECL. A maximum ECL emission of 640 nm, which is about 55 nm blue shift to its fluorescence, was observed in MeCN with DBAE as a coreactant. Interactions of the complex with calf thymus DNA (ctDNA) were conducted with a flow-cell based quartz-crystal microbalance, and a binding constant of 2.5 x 10(5) M(-1) was calculated on the basis of the Langmuir isotherm equation.