First-generation enzyme-based biosensors

Enzymes were historically the first molecular recognition elements [2-6] included in biosensors and continue to be the basis for a significant number of pubUcations in this field. Amperometric biosensors based on glucose oxidase (GOx) are the most famous example of biosensors applied to medical diagnostic. The three modes of oxidation reactions that occur in redox enzyme-based biosensors, like GOx, are referred to as first, second, and third generation as follows [7] ... 

Enzyme sensors are based primarily on the immobilization of an enzyme onto an electrode, either a metallic electrode used in amperometry (e.g., detection of the enzyme-catalyzed oxidation of glucose) or an ISE employed in potentiometry (e.g., detection of the enzyme-catalyzed liberation of hydronium or ammonium ions). The first potentiometric enzyme electrode, which appeared in 1969 due to Guilbault and Montalvo [140], was a probe for urea with immobilized urease on a glass electrode. Hill and co-workers [141] described in 1986 the second-generation biosensor using ferrocene as a mediator. This device was later marketed as the glucose pen . The development of enzyme-based sensors for the detection of glucose in blood represents a major area of biosensor research. 

The first system, devised in the University of Porto, Portugal, was a sensor array for the measurement of creatinine in urine [18]. It consisted of a creatinine iminohydrolase enzyme immobilized by entrapment using a chitosan membrane onto a nonactin ISE. Catalytic hydrolysis by the enzyme generated ammonium ion which was then directly detected. The system was completed with ISEs for ammonium, potassium, sodium and calcium which allowed to correct for any endogenous ammonium (by the first ISE) or for alkaline and alkaline-earth interference in the ammonium-based biosensor (the other three). Linear response ranges were between 0.1 and 10 mM, what permitted the resolution of the multicomponent determination by PLS method. Comparison with reference Jaffe method showed a satisfactory correlation, although the slope of obtained vs. reference values was rather low the obtained value was 0.87, whereas theoretical value should be 1.0. 

An interesting feature of DET biosensors is a simple and robust electrode architecture (e.g., no leaking of soluble redox mediators). Additionally, many interfering substances affecting the detection in first- and second-generation biosensors do not interfere with biosensors based on DET. The number of possible analytes is of course restricted to the number of available enzymes, but by use of modem protein engineering techniques the spectmm of analytes will be broadened in future. An example is the modification of the substrate specificity of cellobiose... 

---