Biosensing and electrochemical properties of flavin adenine dinucleotide (FAD)-Dependent glucose dehydrogenase (GDH) fused to a gold binding peptide

Orientated Immobilization of FAD-Dependent Glucose Dehydrogenase on Electrode by Carbohydrate-Binding Module Fusion for Efficient Glucose Assay

“…This discrepancy may result from the high retention rate of GDH with the correct conformation achieved by CBM binding as previously reported [ 13 , 32 ]. In addition, we compared the sensitivity, linear range, and response time of the biosensors developed in this study with several previously reported glucose sensors, as shown in Table 1 [ 26 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 ], which revealed that the performances of all these sensors are comparable ( Table 1 ).…”

“…The results revealed that higher reproducibility was obtained with the GDH-NL-CBM2/NC/S-MWNT/GCE (relative standard deviation (RSD) < 5%) compared with the GA/CS/GDH/S-MWNT/GCE (RSD < 10%) (Figure 8a,b), but the two electrodes prepared in this study exhibited a comparable performance to that of other electrodes previously reported (Table 1). In addition, the stability was evaluated by CV in different concentrations of glucose (5,10,15,20,25,30,40, and 50 mM; in PBS (0.1 M citric acid/0.2 M Na 2 HPO 4 buffer, pH 6.0)) in a week, and the redox peak currents induced by 20 mM glucose in seven successive days were compared, as shown in Figure 8c,d. The results showed that the current response of the oxidation peak remained almost at 98.6% of the initial response for the GDH-NL-CBM2/NC/S-MWNT/GCE, while for the GA/CS/GDH/S-MWNT/GCE electrode, a considerable increase (83%) of the current density was observed at day 2 compared with that at day 1, and in the six successive days, the change of its current response was negligible, and approximately 98% of its current response at day 2 was retained.…”

“…Culture supernatants containing the heterologously expressed enzymes were collected by centrifugation at 10,000× g for 10 min and filtered through a 0.22 µm polyethersulfone (PES) filter membrane (Millipore GmbH, Eschborn, Germany). Then, the enzymes in the supernatants were purified using His60 Ni Superflow Resin & Gravity Columns (Takara), according to the manufacturer's instructions and eluted with a series of imidazole concentration gradients (5,10,20,30,40,50,60, 100, and 250 mM). The protein solution was concentrated using 30-kDa cutoff ultrafiltration tubes (Pall Corp., Port Washington, NY, USA) and transferred to citric acid/Na 2 HPO 4 (pH 6.0).…”

“…In addition, the enzyme was added to citric acid/Na 2 HPO 4 buffer (pH 3.0-8.0) for different time periods (0.5, 1, 2, 5, and 10 h) to determine the pH stability of GDH. The enzyme in citric acid/Na 2 HPO 4 buffer (pH 5.0) was incubated at different temperatures (24,37,40,50,60, and 70 • C) for different time intervals (0.5, 1, 2, 5, and 10 h) to determine the temperature stability of GDH. Kinetic analyses of the catalytic reactions were performed under standard GDH activity determination conditions using various concentrations of glucose.…”

Direct Electrochemical Enzyme Electron Transfer on Electrodes Modified by Self-Assembled Molecular Monolayers

“…Lee and associates demonstrated controlled DET of bacterial FAD-GDH from Burkholderia cepacia on three different SAM-modified electrodes, on which catalytic current density decreases with increasing SAM chain length [118]. A glucose biosensor based on FAD-GDH was reported recently by introducing a gold-binding peptide (GBP) for enzyme immobilization on a screen-printed electrode (SPE) [24]. GBP composed of 12 amino acids exhibiting a strong binding affinity to the gold electrode surface was fused to the enzyme terminus, thereby determining the enzyme orientation on the electrode.…”