Third-Generation Biosensor for Lactose Based on Newly Discovered Cellobiose Dehydrogenase

Abstract

The present paper describes the principle and characteristics of a biosensor for lactose based on a third-generation design involving cellobiose dehydrogenase. As resulted from a previous comparative study (submitted manuscript), the novelty of this lactose biosensor is based on highly efficient direct electron transfer between two newly discovered cellobiose dehydrogenases (CDH), from the white rot fungi Trametes villosa and Phanerochaete sordida, and a solid spectrographic graphite electrode. CDH was immobilized on the electrode surface (0.073 cm2) by simple physical adsorption, and the CDH-modified electrode was next inserted into a wall-jet amperometric cell connected on-line to a flow injection setup (0.5 mL x min(-1)). The P. sordida CDH-based lactose biosensor, proved to be the better one, has a detection limit for lactose of 1 microM, a sensitivity of 1100 microA x mM(-1) x cm(-2), a response time of 4 s (the time required to obtain the maximum peak current), and a linear range from 1 to 100 microM lactose (correlation coefficient 0.998). The simplicity of construction and analytical characteristics make this CDH-based lactose biosensor an excellent alternative to previous lactose biosensors reported in the literature or commercially available. The CDH-lactose sensor was used to quantify the content of lactose in pasteurized milk, buttermilk, and low-lactose milk, using the standard addition method. No effects of the samples matrixes were observed. The operational stability of the sensor was tested for 11 h by continuous injection of 100 microM lactose (290 injections). The final signal of the sensor was maintained at 98% of its initial signal, with a low standard deviation of 1.72 (RSD 2.41%).

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“…Optimization studies were also performed with a CtCDH modified electrode Operational stability studies at an applied potential of 0.5 V were performed for 72 hours with GOx and CtCDH modified electrodes (using Os(bpy) 2 PVI) in glucose saturated buffer (Figure 4). Both electrodes displayed immediate decreases in response, in contrast to previous reports where CtCDH modified anodes maintained their stability for up to 11 hours [38,41]. A half-life of ca.…”

Modulation of the Silica Sol–Gel Composition for the Promotion of Direct Electron Transfer to Encapsulated Cytochrome c

“…Accordingly, several strategies for the immobilization of enzymes have been assayed: covalent binding 15,16 , physical adsorption [17][18][19][20][21] or encapsulation within polymeric matrices 22 .…”

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