A Chemistry Department Thesis Defense by Bronson Samel-Garloff (Koley Group)

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Herein we explore the characterization and application of micro-volume hydrogel based electrochemical analysis for monitoring the metabolic output of bacteria in complex multi-species biofilms. The main analytical challenges this method aims to address are quantification of very low amounts of analyte, direct analysis of the raw untreated sample after collection, and real-time monitoring of metabolic output by bacteria at biological temperature. This novel method is currently equipped to coulometrically measure the hydrogen peroxide and lactic acid produced by oral bacteria in dental plaque samples, which has potential application as a point-of-care screening device for early detection of dysbiosis within the oral microbiome. Thus, this method can facilitate prevention of oral diseases by providing patients and dentists with pertinent information on the balance of pathogenic and non-pathogenic species in the oral microbiome. The fundamental electrochemistry principles that govern this method will be explained, followed by the electrochemical cell design rationale. Other analytical methods used for studying the human microbiome, and biofilms in general, will also be discussed in terms of their respective advantages and limitations, and how the presented method can fill gaps in knowledge. The first study presented focused on the characterization of platinum black, an electrodeposited high surface area platinum catalytic towards the oxidation of hydrogen peroxide, for measuring from populations of hydrogen peroxide producing bacteria (green fluorescent protein labeled S. sanguinis) of less than one hundred cells on a 50 μL pH buffered hydrogel. The average number of cells added was quantified using confocal microscopy. The buffering characteristics of the hydrogel were measured using a novel method for quantifying the buffering capacity through monitoring the redox behavior of pyocyanin as it depended on the proton concentration within the hydrogel as it was titrated with acid. Coulometric quantification of hydrogen peroxide was achieved by integration of amperometric data after glucose feeding of the sample at 37 ℃. Sensor integrity and electrochemical cell connection was maintained at higher temperature in the presence of organic acid production based on the size-exclusive nature of the hydrogel polymer, the unique pH buffering nature of a morpholine type monomer within the hydrogel structure, and the relatively slow evaporation rate of water from within the hydrogel. Coulometric data yielding the moles of analyte measured were normalized by the average number of bacteria added to each experiment. In the second study presented, the working electrode material was composed of conductive carbon mixed into polydimethylsiloxane (PDMS), and the sensing material consisted of an electrodeposited layering of Prussian blue and nickel hexacyanoferrate crystal which is reduced to Prussian white in the presence of hydrogen peroxide. The electrochemical cell hydrogel medium was also augmented from a single buffering hydrogel to a layering of two unbuffered hydrogels for encapsulating the bacteria and a smaller buffered hydrogel between the encapsulated sample and the working electrode. Lactate sensing was also incorporated into the method by adding lactose oxidase enzymes to the buffering hydrogel where its activity was maintained by the high buffering capacity of the hydrogel. The hydrogen peroxide and lactate produced from dental plaque samples was measured in the same manner as in the first study, and the resulting data was normalized by the mass of dental plaque added. The mole fraction of hydrogen peroxide produced was found by dividing the moles of hydrogen peroxide from experiments without lactate oxidase by the moles of hydrogen peroxide and lactate from experiments with lactate oxidase, which was used as the primary metric for evaluating the state of the dental plaque sample. This method was validated using quantitative polymerase chain reaction experiments on genomic DNA extracted from the remaining dental plaque sample coupled with the respective electrochemical experiments. The genes quantified were spxB, gtfB and 16s which served as genetic markers for peroxide producing species, pathogenic species, and total bacteria respectively. Further normalization of electrochemical data by the genetic data was conducted to discern random error from actual fluctuations in dental plaque species composition. The limitations of this method, and potential means of further optimization with regards to the experiment enclosure, sensor design and hydrogel composition will be discussed. It is foreseeable that this method can be adapted with sensors for other analytes pertinent to a wider variety of complex biological samples such as soil, urine, and feces, which will be discussed as an avenue for future research with this method.