Development of enzymatic and transcription factor-based biosensors
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Sensor technologies have the potential to provide a wealth of opportunities for detection and monitoring across the landscape in clinical, food, environmental, and biothreat/biowarfare analyses. Immobilization of biosensors on or in a functional material is critical for subsequent device development and translation to wearable technology. Upon receptor discovery, integration of receptor and transducer determines the performance of the electrochemical biosensor. First, immobilization of biosensors on surfaces is a key step towards development of devices for real world applications. The preparation, characterization, and evaluation of a surface bound transcription factor – nucleic acid complex for analyte detection as an alternative to conventional systems employing aptamers or antibodies are described. The sensor consists of a gold surface modified with thiolated Cy5 fluorophore-labeled DNA and an allosteric transcription factor (TetR) linked to a quantum dot. Upon addition of anhydrotetracycline (aTc) – the target analyte – the TetR-QDs release from the surface-bound DNA, resulting in loss of the Förster resonance energy transfer (FRET) signal. The sensor responds in a dose-dependent manner over the relevant range of 0-200 µᴍ aTc with a limit of detection of 80 nᴍ. The fabrication of the sensor and the subsequent real-time quantitative measurements establish a framework for the design of future surface-bound, affinity-based biosensors using allosteric transcription factors for molecular recognition. Second, the development and assessment of an immobilized quantum dot - transcription factor - nucleic acid complex for progesterone detection as a first step toward such device integration are presented. The sensor is composed of a polyhistidine-tagged transcription factor linked to a quantum dot and a fluorophore-modified cognate DNA and embedded within a hydrogel as an immobilization matrix. The hydrogel is optically transparent, soft, and flexible as well as traps the quantum dot - transcription factor DNA assembly but allows free passage of the analyte, progesterone. Upon progesterone exposure, DNA dissociates from the quantum dot - transcription factor DNA assembly resulting in an attenuated ratiometric fluorescent output via Förster resonance energy transfer. The sensor performs in a dose-dependent manner with a limit of detection of 55 nM. Repeated analyte measurements are also similarly successful. Our approach combines a systematically characterized hydrogel as an immobilization matrix and a transcription factor – DNA binding as a recognition/ transduction element, offering a promising framework for future biosensor devices based upon an allosteric transcription factor. Lastly, the lack of available and functionally validated enzymes is prohibiting the development of redox-based sensors for important analytes. Nicotine is one such analyte of interest, is present in tobacco products, and is one of the most heavily used addictive stimulants. Today, cigarette smoking prevails in 1.1 billion people, causes about 8 million deaths a year worldwide (World Health Organization). It is the leading preventable cause of disease, disability, and death. Cigarette smoking takes a tremendous economic toll, costing the United States nearly 170 billion annually on healthcare. Thus, the detection, quantification, and monitoring of nicotine, the substance responsible for the dependence-forming properties of tobacco product use, is of substantial interest. Herein, the development and assessment of an electrochemical nicotine biosensor, using a known nicotine catabolizing redox enzyme are presented.