Self-validating lab-on-a-chip for monitoring enzyme-catalyzed biological reactions

[Display omitted] •A lab-on-a-chip with dual detection of enzymatic reactions is presented.•A biofunctionalized mixer/reactor, and a measurement chamber is integrated.•Optical (colorimetric) and electrochemical (amperometric) detection approaches are implemented.•Glucose is detected in only 60s at concentrations in the micromolar range.•The dual detection allows for self-validation, ideal for biomedical applications. This paper reports on a miniaturized and highly integrated lab-on-a-chip (LoC) combining both optical and electrochemical transduction modes for self-validating determination of biological analytes that undergo enzyme-catalyzed-reactions. This LoC monolithically integrates a biofunctionalized microfluidic mixer, also working as bioreactor, together with a measurement chamber, both fabricated in polydimethylsiloxane (PDMS) and applied for enzyme-mediated detection of glucose in continuous flow regime. The measurement chamber combines a multiple internal reflection (MIR) photonic cuvette and a tailor-made electrochemical cell, where gold electrode areas are defined by a silicon oxide passivation layer. Having separated mixing/reaction and measurement chambers greatly facilitates the functionalization process during the microsystem fabrication and the further calibration/rinsing steps carried out during the microsystem performance. A cascade bi-enzymatic reaction involving glucose oxidase (GOx) and horseradish peroxidase (HRP) together with azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) mediator is applied to carry out the glucose dual detection. The optical and electrochemical properties of the ABTS enable simultaneous absorbance and amperometric measurements, without cross-talk. The system requires low sample volumes below 15μL and presents tunable analytical properties that can be adjusted by varying the flow rate. Linear ranges extending up to 1.6mM and 2mM glucose and limits of detection of 0.23±0.02mM and 0.064±0.001mM are achieved with the optical and electrochemical detection approaches, respectively, when operating simultaneously at 10μL/min flow rate. The high degree of integration results in a minimization of dead volumes, reagent consumption and response time, providing with a high performance self-validating structure ideal for biomedical applications.