Strategy, Design, and Fabrication of Electrochemical Biosensors: A Tutorial.

Advanced healthcare requires novel technologies capable of real-time sensing to monitor acute and long-term health. The challenge relies on converting a real-time quantitative biological and chemical signal into a desired measurable output. Given the success in detecting glucose and the commercialization of glucometers, electrochemical biosensors continue to be a mainstay of academic and industrial research activities. Despite the wealth of literature on electrochemical biosensors, reports are often specific to a particular application (e.g., pathogens, cancer markers, glucose, etc.), and most fail to convey the underlying strategy and design, and if it is transferable to detection of a different analyte. Here we present a tutorial review for those entering this research area that summarizes the basic electrochemical techniques utilized as well as discusses the designs and optimization strategies employed to improve sensitivity and maximize signal output.

Structural Insights into the Substrate Range of a Bacterial Monoamine Oxidase.

Monoamine oxidases (MAOs) play a key role in the breakdown of primary and secondary amines. In eukaryotic organisms, these enzymes are vital to the regulation of monoamine neurotransmitters and the degradation of dietary monoamines. MAOs have also been identified in prokaryotic species, although their role in these organisms is not well understood. Here, we report the biophysical and structural properties of a promiscuous, bacterial MAO from Corynebacterium ammoniagenes (caMAO). caMAO catalyzes the oxidation of a number of monoamine substrates including dopamine and norepinephrine, as well as exhibiting some activity with polyamine substrates such as cadaverine. The X-ray crystal structures of Michaelis complexes with seven substrates show that conserved hydrophobic interactions and hydrogen-bonding pattern (for polar substrates) allow the broad specificity range. The structure of caMAO identifies an unusual cysteine (Cys424) residue in the so-called "aromatic cage", which flanks the flavin isoalloxazine ring in the active site. Site-directed mutagenesis, steady-state kinetics in air-saturated buffer, and UV-vis spectroscopy revealed that Cys424 plays a role in the pH dependence and modulation of electrostatics within the caMAO active site. Notably, bioinformatic analysis shows a propensity for variation at this site within the "aromatic cage" of the flavin amine oxidase (FAO) superfamily. Structural analysis also identified the conservation of a secondary substrate inhibition site, present in a homologous member of the superfamily. Finally, genome neighborhood diagram analysis of caMAO in the context of the FAO superfamily allows us to propose potential roles for these bacterial MAOs in monoamine and polyamine degradation and catabolic pathways related to scavenging of nitrogen.

Paper-Based Progesterone Sensor Using an Allosteric Transcription Factor.

Progesterone monitoring is an essential component of in vitro fertilization treatments and reproductive management of dairy cows. Gold-standard biosensors for progesterone monitoring rely on antibodies, which are expensive and difficult to procure. We have developed an alternative transcription factor-based sensor that is superior to conventional progesterone biosensors. Here, we incorporate this transcription factor-based progesterone sensor into an affordable, portable paperfluidic format to facilitate widespread implementation of progesterone monitoring at the point of care. Oligonucleotides labeled with a fluorescent dye are immobilized onto nitrocellulose via a biotin-streptavidin interaction. In the absence of progesterone, these oligonucleotides form a complex with a transcription factor that is fluorescently labeled with tdTomato. In the presence of progesterone, the fluorescent transcription factor unbinds from the immobilized DNA, resulting in a decrease in tdTomato fluorescence. The limit of detection of our system is 27 nm, which is a clinically relevant level of progesterone. We demonstrate that transcription factor-based sensors can be incorporated into paperfluidic devices, thereby making them accessible to a broader population due to the portability and affordability of paper-based devices.

A progesterone biosensor derived from microbial screening.

Bacteria are an enormous and largely untapped reservoir of biosensing proteins. We describe an approach to identify and isolate bacterial allosteric transcription factors (aTFs) that recognize a target analyte and to develop these TFs into biosensor devices. Our approach utilizes a combination of genomic screens and functional assays to identify and isolate biosensing TFs, and a quantum-dot Förster Resonance Energy Transfer (FRET) strategy for transducing analyte recognition into real-time quantitative measurements. We use this approach to identify a progesterone-sensing bacterial aTF and to develop this TF into an optical sensor for progesterone. The sensor detects progesterone in artificial urine with sufficient sensitivity and specificity for clinical use, while being compatible with an inexpensive and portable electronic reader for point-of-care applications. Our results provide proof-of-concept for a paradigm of microbially-derived biosensors adaptable to inexpensive, real-time sensor devices.

Deletion of PHO13, encoding haloacid dehalogenase type IIA phosphatase, results in upregulation of the pentose phosphate pathway in Saccharomyces cerevisiae.

The haloacid dehalogenase (HAD) superfamily is one of the largest enzyme families, consisting mainly of phosphatases. Although intracellular phosphate plays important roles in many cellular activities, the biological functions of HAD enzymes are largely unknown. Pho13 is 1 of 16 putative HAD enzymes in Saccharomyces cerevisiae. Pho13 has not been studied extensively, but previous studies have identified PHO13 to be a deletion target for the generation of industrially attractive phenotypes, namely, efficient xylose fermentation and high tolerance to fermentation inhibitors. In order to understand the molecular mechanisms underlying the improved xylose-fermenting phenotype produced by deletion of PHO13 (pho13Δ), we investigated the response of S. cerevisiae to pho13Δ at the transcriptomic level when cells were grown on glucose or xylose. Transcriptome sequencing analysis revealed that pho13Δ resulted in upregulation of the pentose phosphate (PP) pathway and NADPH-producing enzymes when cells were grown on glucose or xylose. We also found that the transcriptional changes induced by pho13Δ required the transcription factor Stb5, which is activated specifically under NADPH-limiting conditions. Thus, pho13Δ resulted in the upregulation of the PP pathway and NADPH-producing enzymes as a part of an oxidative stress response mediated by activation of Stb5. Because the PP pathway is the primary pathway for xylose, its upregulation by pho13Δ might explain the improved xylose metabolism. These findings will be useful for understanding the biological function of S. cerevisiae Pho13 and the HAD superfamily enzymes and for developing S. cerevisiae strains with industrially attractive phenotypes.