**3. Kinetic modelling of NADH oxidation at chemically modified electrodes.**

324 Dehydrogenases

voltage of approximately 0.3 V.

shown below in Figure 2.

interest are voltammograms a and e. These show the direct oxidation of NADH (e) at the glassy carbon electrode and the cyclic voltammogram (a) of the immobilised species. Addition of NADH to the solution causes an increase in 1. Note that the signal due to the oxidation of NADH now occurs at the potential of the mediator resulting in a decrease in the operating

Among the mediators used so far are quinones, diimines, ferrocene, thionine oxometalates, polymetallophthalocyanines, ruthenium complexes, pyrroloquinoline quinone, fluorenones, and quinonoid redox dyes such as indamines, phenazines, phenoxazines and phenothiazines.11 A generalised reaction schematic for the mediated oxidation of NADH is

**Figure 2.** Scheme showing the mediated oxidation of NADH. In this context, the mediator reacts with NADH in a chemical step to oxidise it (a) and is itself reduced. The mediator is then itself reoxidised at the electrode surface (step b). The chemical oxidation of NADH by this mechanism bypasses the

To design an NADH sensor, the mediator is normally immobilized on the electrode surface or within the electrode material. Mediators can be immobilized by chemisorption by covalent attachment directly electrode surface or by electrochemical polymerisation of the mediators at the electrode surface or, alternatively via covalently attached/physically entrapment in polymers, incorporation in carbon paste grown at electrode surface or deposited on the electrodes by drop coating. The method that is ultimately chosen to produce the modified electrode depends upon the method of mass production (for commercial sensors) and the materials used in the device. For example, in sensors that utilise screen printed carbon based electrodes (*screen-printing technology is a kind of low-cost thick film technology which allows to deposit thick films, a few to hundreds micrometers and is well suited for mass production and portable devices. Such a micro fabrication route offers high-volume production of extremely inexpensive and yet highly reproducible disposable enzyme electrodes – this will be discussed further in the text*) it is convenient to incorporate the mediator directly into the carbon ink particularly if the mediator contains delocalised aromatic rings, as found in quinine and phenoxazine dye based mediators, which form strong chemisorbed bonds with the carbon and graphite plates. The mediator loading, activity stability etc can all be investigated using conventional electrochemical techniques such as DC cyclic voltammetry.

problems associated with the direct oxidation at a bare electrode surface.

In the scientific literature, NADH oxidation at chemically modified electrodes is most commonly suggested to occur via a two-step reaction mechanism. In the first step, NADH forms a charge transfer complex with the oxidised form of the mediator bound to the electrode surface. In the second step, electron exchange takes lace and the complex breaks down producing NAD+ and a reduced mediator site. Because the electrode is polarised, the reduced mediator site is reoxidised in a non-rate limiting electron exchange to the bulk electrode material. This scheme is shown in Figure 3. The important kinetic constants are also represented.

$$\begin{array}{c} \mathsf{k\_{\mathcal{O}lo}}\\ \mathsf{NADH} \leftarrow \mathsf{Medidator\_{\{\mathcal{O}lo}}} \end{array} \xrightarrow[\begin{array}{c} \mathsf{K\_{\mathcal{I}lo}}\\ \mathsf{K\_{\mathcal{I}I}} \end{array} \xrightarrow[\begin{array}{c} \mathsf{[\ \mathsf{NADH}\leftarrow\mathsf{Medidator]}\ ]\\ \mathsf{NAD}\leftarrow\mathsf{Med}\end{array} \begin{array}{c} \mathsf{k\_{\mathcal{I}I}}\\ \mathsf{NAD}\leftarrow\mathsf{Med}\mathsf{Ator}\_{\{\mathsf{NAD}\}} \end{array} \begin{array}{c} \mathsf{k\_{\mathcal{I}I}}\\ \mathsf{NAD}\leftarrow\mathsf{Med}\mathsf{Ator}\_{\{\mathsf{NAD}\}} \end{array} \end{array} \begin{array}{c} \mathsf{k\_{\mathcal{I}I}}\\ \mathsf{NAD}\leftarrow\mathsf{Med}\mathsf{Ator}\_{\{\mathsf{NAD}\}} \end{array}$$

**Figure 3.** Two-step mechanism commonly proposed for the oxidation of NADH at chemically modified electrodes. The mediator and NADH form a charge transfer complex that dissociates to give rise to the reduced mediator and biologically active NAD+.

For this type of mechanism, the catalytically limiting current (ik) observed under controlled hydrodynamic conditions can be expressed as: ik = nFAKcatCNADH/KM where CNADH is the bulk concentration of NADH and represents the concentration of binding sites in the immobilised film. This type of model assumes that the rate of electron transfer between the mediating species in the film and the NADH is sufficiently fast so as not to be rate limiting, the NADH freely diffuses into the film whereupon it adsorbs to the catalytic site and it undergoes oxidation to NAD+. Also, the expression for *ik* is valid only for thin films where the concentration of NADH is insufficient to saturate the binding sites.[12]

Due to the formation of the charge-transfer complex, this reaction scheme is commonly analysed using Michaelis-Menten kinetics. From figure 3 it is possible to construct the following kinetic argument.

This is now a straight line plot. From the slope of such a plot, values of *k+2* can be calculated. By extrapolation of zero NADH concentration, i.e. the intercept, values of *KM* can be estimated. Values for *kOb*s can be obtained from Koutecky-Levitch plots under steady state

Amperometric Glucose Sensors for Whole Blood Measurement Based on Dehydrogenase Enzymes 327

very interesting effects that are not possible using other printing methods. Because of the simplicity of the application process, a wider range of inks and dyes are available for use in screen printing than for use in any other printing process. The major chemicals used include screen emulsions, inks, and solvents, surfactants, caustics and oxidizers used in screen

Screen printing consists of three elements: the screen which is the image carrier; the squeegee; and ink. The screen printing process uses a porous mesh stretched tightly over a frame made of wood or metal. Proper tension is essential to accurate colour registration. The mesh is made of porous fabric or stainless steel mesh. A stencil is produced on the screen either manually or photochemically. The stencil defines the image to be printed in other

 Screen printing ink is applied to the substrate by placing the screen over the material. Ink with a paint-like consistency is placed onto the top of the screen. Ink is then forced through the fine mesh openings using a squeegee that is drawn across the screen, applying pressure thereby forcing the ink through the open areas of the screen. Ink will pass through only in areas where no stencil is applied, thus forming an image on the printing substrate. The diameter of the threads and the thread count of the mesh will determine how much ink is deposited onto the substrates. Figure 4 shows an example of an image for an electrochemical cell consisting of a working, a counter and a reference element. This type of structure can be

**Figure 4.** Example of a screen printed electrochemical cell (screen image) showing the Reference element (A), the working electrode (B) and the counter electrode (C). Comercially available screen printed electrodes for research purposes can be obtained from DropSens, Edificio CEEI - Parque Tecnológico de Asturias - 33428 Llanera (Asturias) Spain. http://www.dropsens.com/en/screen\_printed\_electrodes\_pag.html

Many factors such as composition, size and form, angle, pressure, and speed of the blade (squeegee) determine the quality of the impression made by the squeegee. At one time most blades were made from rubber which, however, is prone to wear and edge nicks and has a tendency to warp and distort. While blades continue to be made from rubbers such as neoprene, most are now made from polyurethane which can produce as many as 25,000

If the item was printed on a manual or automatic screen press the printed product will be placed on a conveyor belt which carries the item into the drying oven or through the UV

impressions without significant degradation of the image.

printing technologies this would be referred to as the image plate.

easily produced by screen printing.

reclamation.

$$k\_{\text{obs}} = \frac{k\_{\star 2}}{k\_M + [NADH]} \tag{59}$$

$$\frac{1}{\frac{1}{k\_{\text{obs}}}} = \frac{K\_M}{k\_{\star 2}} + \frac{[NADH]}{k\_{\star 2}} \tag{6.9}$$

**Scheme 1.**

oxidation conditions as described by Compton and Hancock.[12] Typically, values of kObs tend to be in the range 10-3 to 10-1 cm s-1. Thus it is possible using such laboratory techniques to ensure that the surface coverage of mediator (moles/cm2) is optimised to achieve favourable measurement linearity and speed of response.
