Microfluodic Chip Electrochemical Detection Of Glucose With Monolithic Immobilized Enzyme

Glucose biosensor is widely used for glucose determination in clinical practice, and miniaturization and portability of the detection system is highly demanded. The occurance of microfluidic technology offers a good opportunity for the development of small biosensor device with less sample and reagent consumption. On the basis of existing research, the established electrochemical flow analytical system was improved in this paper. A monolithic immobilized enzymatic microreactor was successfully prepared and integrated to the microfluodic chip electrochemical detection cell, and determination of glucose in human plasma was demonstrated.Firstly, an open-tubular immobilized enzymatic microreactor was prepared by immobilization of glucose oxidase onto inner capillary surface by covalent binding. This microreactor was connected with a microfluidic electrochemical detection cell fabricated by photolithography. The fluid was driven by gravity instead of syringe pump, and the continuous detection of glucose content was achieved. The Michaelis-Menten constant (Km) of enzyme was 9.37 mmol/L, and a good linearity (R=0.9988) was obtained in the range of 0.5-6 mmol/L with a detection limit of 0.02 mmol/L(S/N=3) and a relative standard deviation (RSD) of 1.6%(n=11) at the potential of +0.5 V vs. Ag/AgCl. P-benzoquinone (p-BQ) was introduced as an electron mediator for lowering the electrochemical interference due to other electrochemically active species such as ascorbic acid and uric acid. With 2.5 mmol/L p-BQ, at +0.3 V vs. Ag/AgCl, a linear response (R=0.9985) was obtained in the range of 1.0-10 mmol/L with RSD of 4.5%(n=11). The system was simplified comparing to the system driven by a syringe pump, and the detection limit and reproducibility were also improved.A one-step procedure for the immobilization of glucose oxidase in fused-silica capillary was successfully achieved based on enzyme entrapment in a polyacrylamide-based monolithic capillary column. The microreactor thus prepared was connected to the chip electrochemical detection cell for flow detection of glucose. The Michaelis-Menten constant (Km) of enzyme was 1.64 mmol/L, and at +0.5 V vs. Ag/AgCl, a good linearity (R=0.9984) was obtained in the range of 0.05～2 mmol/L with a detection limit of 0.01 mmol/L(S/N=3) and a relative standard deviation (RSD) of 3.8%(n=11).Comparing to the system with open-tubular immobilized enzymatic microreactor, the Michaelis-Menten constant (Km) was decreased significantly, and sensitivity and detection were also improved.