Routine Applications of Enzyme Electrodes

Routine application of enzyme electrodes is presently restricted to the determination of low-molecular metabolites.

Redox enzymes catalyzing the reactions between small molecules are characterized by the catalytic center buried well within the protein. Communication between this center and an electrode serving as a source or sink for electrons is frequently poor or nonexistent. Heterogeneous electron transfer rates for this type of enzymes are vanishingly low so that the enzyme redox activity must be coupled to the electrode using cosubstrates or mediators (first and second generation).

The most relevant fields of practical application of enzyme electrodes are medical diagnostics, followed by process control, food analysis, and environmental monitoring [24]. The first commercial enzyme electrode-based analyzer (Yellowsprings 1975) was developed to meet the high demand for glucose determination in the blood of diabetic patients. Since 1975, analyzers for about 12 different analytes have been commercialized. As compared to conventional enzymatic analysis, the main advantages of such analyzers are the extremely low enzyme demand (a few milliunits per sample), their simplicity of operation, the high sample throughput rate, and high analytical quality.

The selective determination of blood glucose is of extraordinary importance for the screening and treatment of diabetes. In the medical field, a trend toward handheld devices based on disposable glucose enzyme electrodes for home control of diabetes and for on-site monitoring of surgery and exercise is evident.

Personal blood glucose meters are based on disposable (screen-printed) enzyme electrode test strips. Such single-use disposable strips are mass-produced by the thick-film (screen-printing) microfabrication technology. Each strip contains the printed working and reference electrodes, with the working one coated with the necessary reagents (i.e., enzyme, mediator, stabilizer, linking agent).

The first product was a pen-style device (the Exatech), launched by Medisense Inc. in 1987, that relied on the use of a ferrocene-derivative mediator [25]. Various commercial strips and pocket-sized test meters, for self-monitoring of blood glucose - based on the use of ferricyanide or ferrocene mediators - have since been introduced (Table 4.4). The Johnson & Johnson Company, which is currently the market leader in blood glucose self-testing, launched its first biosensor product - the Fast Take™ system, in April 1998. The biggest suppliers to this market all now sell biosensor-based products.

Tab. 4.4 Glucose sensors for patient self-control

Electron acceptor

Enzyme

Company

Product

O2

GOD

BST

Glucometer 3000

Ferricyanide

GOD

Bayer

Glucometer Elite

Glucometer Elite XL

(PQQ)GDH

Roche

ACCUcheck Advantage

(NAD)GDH

Abbott

SOFT-Tact

LifeScan

One Touch Ultra

Ferrocene adsorbed

GOD

Abbott

Precision QID

Exac Tech

Os Redox polymer

(PQQ)GDH

TheraSense

FreeStyle

The handheld devices for self-control of blood glucose are based on the combination of the enzymes glucose oxidase and PQQ-dependent or NAD+-dependent glucose dehydrogenase (GDH) with different redox mediators. Whenever GOD is still dominating, both GDHs present the advantage of abolishing parasitic oxygen reactions.

For glucose measurement by the first device of the electrochemical meters - the Medisense glucose pen - the sample is put directly on the surfaces of the enzyme electrode. The glucose-converting reaction requires the dissolution of the oxidized ferrocene mediator within the electrode-near layer in order to reoxidize the GOD, which is adsorbed at the carbon electrode. The electrode current is generated by the anodic oxidation of the enzymatically reduced mediator.

However, the majority of commercial glucose meters perform the sample application by filling a microreaction chamber using capillary forces. The reagents are dissolved from the walls of the chamber by the inflowing sample liquid, and the formation of the reduced mediator is quantified by the amperometric electrode.

The capillary-based meters do not fulfill the IUPAC definition in the strict sense because the enzyme is not in direct contact with the electrochemical transducer but is trapped in a confined space.

These arrangements prevent misfunctions of the device by inappropriate sample application such as insufficient volume or mechanical stress of the sensor and allow the decrease of the sample volume from almost 20 ||L to a minimum of 300 nL. Furthermore, the inconvenience of taking 10 to 20 |L of blood by punching the finger is avoided because the sample can be taken almost painlessly with the recently introduced Free Style (Thera Sense) device. The integration of the sample transport into the device by a vacuum pump is realized in the Soft-Tact (Abbott). It represents a further breakthrough because the handling and the reliability of the whole measuring is improved.

Even less painful is the iontophoretic detection with the Gluco Watch™ automatic glucose biographer from Cygnus. This device controls blood glucose over a range of 2.2 to 22.2 mmol L-1 (40-400 mg dL-1) for up to 12 h using a single-point calibration. The automatic, frequent, and noninvasive measurements provide more information about glucose levels than the current standard of care.

Tighter glycemic control through continuous in vivo monitoring is desired for triggering proper alarm in cases of hypo- and hyperglycemia and for making valid therapeutic decisions. Most of the recent attention has been given to the development of subcutaneously implantable needle-type electrodes. Such devices are designed to operate for a few days, for example, the recently introduced CGMS unit of Minimed. Inc. (Sylmar), which offers 72 h of subcutaneous monitoring with measurement of tissue glucose every 5 min.

For a continuous in vivo monitoring of glucose, long-term stability, selectivity and sensitivity in the mMolar range is needed. In vivo devices are mediatorless because of potential leaching and toxicity of the mediator. Extremely fast-responding glucose sensors with submicrometer tip diameter have been constructed by using platinum-deposited, flame-etched carbon fiber electrodes additionally coated with glucose oxidase [26].

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