Capacitive sensing of N-formylamphetamine based on immobilized molecular imprinted polymers

A highly sensitive, capacitive biosensor was developed to monitor trace amounts of an amphetamine precursor in aqueous samples. The sensing element is a gold electrode with molecular imprinted polymers (MIPs) immobilized on its surface. A continuous-flow system with timed injections was used to simulate flowing waterways, such as sewers, springs, rivers, etc., ensuring wide applicability of the developed product. MIPs, implemented as a recognition element due to their stability under harsh environmental conditions, were synthesized using thermo-and UV-initiated polymerization techniques. The obtained particles were compared against commercially.,available MIPs according to specificity and selectivity metrics; commercial MIPs were characterized by quite broad cross-reactivity to other structurally related amphetamine-type stimulants. After the best batch of MIPs was chosen, different strategies for immobilizing them on the gold electrode's surface were evaluated, and their stability was also verified. The complete, developed system was validated through analysis of spiked samples. The limit of detection (LOD) for N-formylamphetamine was determined to be 10 mu M in this capacitive biosensor system. The obtained results indicate future possible applications of this MIPs-based capacitive biosensor for environmental and forensic analysis. To the best of our knowledge there are no existing MIPs-based sensors toward amphetamine-type stimulants (ATS)

CIRCADIAN RHYTHMICITY AND MELATONIN SENSITIVITY IN THE HUMAN GUT COMMENSAL BACTERIUM \u3ci\u3eKLEBSIELLA AEROGENES\u3c/i\u3e

While the expression of circadian rhythms is nearly universal among multicellular eukaryotic organisms, demonstration of this phenomenon in prokaryotes has been largely restricted to photosynthetic cyanobacteria until very recently. Further, growing interest in gastrointestinal microbiomes has revealed a complex temporal relationship between the gastrointestinal clock and the bacterial microbiome within. At least one member of the gut microbiome, Klebsiella (née Enterobacter) aerogenes, responds to the indoleamine hormone melatonin, secreted by the gastrointestinal system itself. Further research revealed that K. aerogenes also expresses a circadian rhythm in motility and gene expression that is temperature compensated. Although rhythmicity is unaltered by changes in temperature, cycles of ambient temperature entrain circadian rhythms in K. aerogenes. In this work I investigated new aspects of circadian rhythmicity in Klebsiella aerogenes. I characterized new clock-controlled genes and found that circadian rhythms in this bacterium rapidly decrease in amplitude following exposure to temperature cycles irrespectively of tested luciferase reporters. I discussed and explained the mechanisms of this damping. The next hypothesis I tested was whether this circadian rhythmicity of K. aerogenes persist in vivo within the gastrointestinal track of the host. To test this, antibiotic treated laboratory mice (a heterologous host for this bacterium) were infected with K. aerogenes. Then I determined whether the bacterium’s circadian rhythmicity was sustained by quantifying endogenous and infection bacterial DNA within the lumen of the gut. I found that K. aerogenes persisted within the gut for several days, and its abundance was rhythmic. Additionally, the quantity of total bacteria and enterobacteria was also rhythmic. Further, for the first time I characterized transcriptome of this bacterium as the culture matures. Moreover, I investigated the transcriptional changes induced by melatonin in K. aerogenes. I demonstrated that the majority of differentially expressed genes are growth stage specific. This indole molecule affects genes related to biofilm formation, fimbria biogenesis, transcriptional regulators, carbohydrate transport and metabolism, phosphotransferase system (PTS), stress response, metal ion binding and transport. It is likely that differential expression of biofilm and fimbria related genes is responsible for differences in macrocolony area. Additionally, melatonin potentially helps Klebsiella aerogenes in host colonization. These experiments in sum suggest a role of melatonin in modifying transcriptome of Klebsiella aerogenes and suggest its potential role in communication between the host and its commensal microbiota. Additionally, my work demonstrated rhythmicity of additional clock-controlled genes, improved experimental approach necessary to study circadian rhythmicity in K. aerogenes and helped to better understand this phenomenon

Sensitive flow-through immunoassay for rapid multiplex determination of cereal-borne mycotoxins in feed and feed ingredients

An easy-to-operate membrane-based flow-through test for multiplex screening of four mycotoxins (zearalenone, deoxynivalenol, aflatoxin B-1, and ochratoxin A) in a variety of cereal-based feed ingredients and compound feeds, such as wheat, barley, soybean, wheat bran, rice, rice bran, maize, rapeseed meal, and sunflower meal, and various types of complete feed (duckling feed, swine feed, broiler feed, piglet feed) was developed and validated. First, the antibodies were evaluated by enzyme-linked immunosorbent assay and then employed in the membrane rapid test. The cutoff levels for zearalenone, deoxynivalenol, aflatoxin B-1, and ochratoxin A were 50, 200, 1, and 10 mu g/kg, respectively, based on European regulations and consumers' requirements. As sample pretreatment, consecutive steps of extraction, dilution, solid-phase extraction by addition of C18 sorbent, and final filtration of supernatant were followed. Both the sample preparation and the analysis procedure were simple, cost-effective, and easy to perform on-site in a nonlaboratory environment. The impact of sample processing on the result of the experiment was investigated supported by experimental design. The validation procedure was performed on the basis of Commission Regulation 2006/401/EC. The numbers of false-positive and false-negative outcomes were < 5%, going along with the Commission Decision 2002/657/EC. Liquid chromatography-tandem mass spectrometry was performed as a confirmatory technique