大川 祐輔

The transient responses of enzyme monolayer- and bilayer-modified electrodes were theoretically analyzed. The response was numerically simulated with parameters used in the foregoing paper. The transient process consists of three elementary steps: enzymatic reaction step, substrate and mediator consumption step, and charge-carrying mediator accumulation step. Contributions of the parameters to these elementary steps and the overall response were examined. Experimental observations are successfully interpreted in terms of the theoretical results.

Setting temperature and setting time were obtained readily with good reproducibility. Setting speed was defined from these two values. The relationship between the concentration (C), the cooling temperature (T) and the setting time (t) of the gelatin solution was studied using one IAG-gelatin (M-8929). The relation of these three factors could be expressed empirically as: lnt=a+b (C/T) from which can be found the characteristic point of the gelation. It seems that this value is related to the factors controlling the gelation. We could not find the characteristic point of the gelation for gelatin containing little high molecular weight components, resulting from ultrasonic wave irradiation.

Different types of gelatin were irradiated with ultrasonic waves to investigate the process of ultrasonic degradation of gelatin. The physical characteristsics of irradiated gelatins were also studied. The kinetics of the ultrasonic degradation of fraction H (the highest molecular weight component in the gelatin) were studied and found to be first order. The ultrasonic degradation constant k of fraction H varied with type of gelatin, concentration and quantity of gelatin solution. Values of k for lime-processed gelatins were about the same as each other, whereas the k for acid-processed gelatins were different from one another. Fraction H of the acid-processed gelatins was more degradable than those of lime-processed gelatins. There was a negative relationship between Log(k) and the concentration of the gelatin solution. Correlation of Log(k) and the quantity of sample solution was also negative. The effect of irradiation time on the physical characteristics of the gelatin was also observed. The jelly strength of the irradiated gelatins had a peak during irradiation, whereas the viscosity decreased monotonously.

A Langmuir-Blodgett (LB) film consisting of octadecylamine as the binding site for the enzyme molecule, an amphiphilic ferrocene derivative as the electron mediator, and octadecanol as the matrix for these molecules was deposited on a hydrophobized tin oxide (SnO2) electrode. Glucose oxidase (GOx) was then covalently immobilized on the film via glutaraldehyde, and the GOx electrode thus obtained worked as an amperometric glucose sensor based on the oxidation of hydrogen peroxide liberated by the enzymatic reaction. The sensitivity of this sensor was about twice as high as that of a GOx electrode prepared with the same LB film as above but without the ferrocene. The sensitizing effect of ferrocene was attributed to its capability of electron mediation between hydrogen peroxide and the SnO2 electrode.

A flow-through electrolytic cell (electrochemical filter; ECF) was newly designed for the elimination of electroactive interferents with electrochemical biosensors. The ECF has been combined with a glucose oxidase monolayer electrode in a flow injection analysis (FIA) setup. The addition of ferricyanide to the carrier buffer drastically increased the elimination efficiency for L-ascorbic acid and the biosensor output. Glucose content in soft drinks has been determined without any pretreatment.

The ascorbic acid radical produced by the anodic oxidation of ascorbic acid was detected by uv absorption using a spectroelectrochemical method during the voltammetric measurements. The radical generation was further confirmed by the measurement of the half-wave potential for the cathodic reduction and the pH dependence of the decay rate using a rotating ring-disc electrode.

An enzyme-based amperometric biosensor for hydrogen peroxide was developed, employing a horseradish peroxidase (HRP) monolayer covalently attached to a tin oxide electrode and a dissolved electron mediator. The sensor can determine hydrogen peroxide at levels down to 10-8 M and can be applied to a flow system. Stability of the electrode, kinetics of the surface process, and the efficiencies of different mediators were studied. As an extension, glucose oxidase (GOx) was chemically bound to the HRP-modified electrode. A GOx/HRP bilayer-modified electrode thus obtained exhibits much better performance in glucose detection limit, sensitivity, and response speed than previously reported GOx monolayer-modified electrodes.