A miniaturized fiberless optical sensor integrated in an automated sequential injection (SI) manifold for mesofluidic handling of sample, conditioning and regeneration solutions is herein proposed for monitoring glucose (as a model analyte) in human serum. The optofluidic biosensor capitalizes on the co-immobilization of Prussian Blue (PB) and glucose oxidase (GOx) on a polyester film working concomitantly as a chemo- and bioreceptor. The oxidation of β-glucose at the receptor surface by GOx yields hydrogen peroxide whereby reoxidizing the reduced form of PB (the so-called Prussian White) so as to generate a deep blue color. The change in the optical properties of the film was continuously monitored by red paired emitter-detector diodes (PEDDs). A full factorial design followed by a Doehlert matrix-based response surface was exploited for multivariate optimization of the optofluidic PB-GOx-PEDD biosensor. The most significant variables influencing sensor's response were the current powering the light emitting diode (LED) emitter and the surface concentration of GOx. The optosensor was proven rugged as the response varies by merely 5% from the optimal value whenever the GOx concentration increases or decreases by 17% or the current powering the LED by 18.5%. Under the optimized physicochemical conditions, the limits of detection and quantification at the 3s(blank) and 10s(blank) levels, respectively, were estimated to be 23.8μmolL(-1) and 79.3μmolL(-1), respectively, with a dynamic working range spanning from 0.1 to 2.5mmolL(-1) of glucose. The trueness of the biosensor measurements was assessed with certified pathological and physiological human serum materials and compared against the spectrophotometric Trinder method. The devised enzymatic biosensor is affordable (less than 0.2€), sturdy, and versatile inasmuch as the chemical composition of the receptor and pair of LEDs might be customized at will.
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