Electrochemical Impedance Biosensor for Glucose Detection Utilizing a Periplasmic E. coli Receptor Protein

Abstract

We report the development of a reagentless electrochemical impedance biosensor for glucose that employs the D-glucose/galactose receptor from E. coli for direct glucose detection. The biological platform for this sensor is an Au surface to which the protein is immobilized through formation of an Au-S bond to a genetically engineered cysteine residue at the N-terminus. The impedance signal detects the extensive ligand-induced domain motion within the protein upon glucose binding. We show the applicability of impedance spectroscopy in conjunction with periplasmic binding proteins as a general method for detecting small molecules.For control of diabetes mellitus, maintenance of blood glucose concentrations within the normal physiological range is critical to minimizing diabetic complications. As a result, accurate in vivo and in vitro measurement of glucose concentration in physiological fluids has long been a central goal of biosensor research. Traditionally, glucose biosensors employ indirect detection of the products of the glucose oxidase or glucose dehydrogenase reactions, although direct detection has also been reported. In general, glucose oxidase has been the favored biological recognition element due to the high binding specificity, high turnover rate, and high stability of this enzyme. However, indirect detection of hydrogen peroxide, or other products, often suffers from low selectivity arising from chemical interference by other easily reducible substances, such as ascorbate and urate. Direct detection suffers from two general drawbacks that limit the efficiency with which electrons can be collected: proteins are usually electrically insulating, and the reaction site may be located far from the collection electrode. 1 In addition, electron transfer proteins are also required. To circumvent these problems, the current report focuses on direct glucose detection by electrochemical impedance using the D-glucose/galactose receptor ͑GGR͒ from E. coli.The potential use of the bacterial periplasmic binding protein superfamily ͑bPBP͒ in biosensors has arisen due to their solubility, stability and ability to reversibly bind a large variety of small ligands including sugars, amino acids, and inorganic ions. 2 These proteins comprise a large family of functionally similar receptors with a two-domain structure that exhibit a large hinge-twist conformational change upon ligand binding. Whereas the proteins are open and flexible when the ligand is absent, they are more structurally compact and closed when the ligand is bound. This large amplitude motion can be exploited for biosensor development of small molecules. [3][4][5][6] The GGR protein is specific for glucose or galactose and binds these ligands in the micromolar range, which is more sensitive than that needed for blood glucose. A number of groups have detected sugars by labeling this protein with fluorescent tags in sites where the chemical environment is altered during ligand binding. 7-10 Two recent studies have used surface plasmon resonance ͑SPR͒ methods w...

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