Sanjay Upadhyay

Needle-Type Ag ∕ AgI Reference Electrode with a Stagnant Electrolyte Layer and an Active Liquid Junction

A needle-type micro liquid-junction reference electrode for general use was developed based on the novel use of a thin-film Ag/AgI electrode. The internal iodide ion concentration was maintained at a constant value determined by the solubility constant of AgI. Iodide ions in an external solution that might penetrate into the interior were eliminated. They were electrochemically converted into iodine molecules and trapped in a starch matrix that filled in the liquid junction. The sensitivities of the bare Ag/AgI electrode to interferents were 4.2, 2.8, and -6.1 mV/dec for L-ascorbic acid (50-200 μM), uric acid (50-200 μM), and NaCI (50-200 mM), respectively. The internal filling solution was rapidly exchanged, within several minutes, for step changes of the NaCI concentration which expanded 1 order of magnitude. When the liquid junction was active, changes that occurred in the electrode potential when the external iodide ion concentration changed by 1 order of magnitude in the nanomolar range were suppressed within several millivolts. When the liquid-junction reference electrode was used with a pH-indicator electrode in solutions containing different concentrations of various interferents, the achieved precision was ±0.01 pH unit. The pH measurement could also be conducted in human serum solutions of only several microliters.

Behavior of a Microelectrode with a Concentrated Enzyme-Immobilized Layer

The behavior of electrodes with a highly concentrated enzyme-immobilized layer was examined. The enzyme-immobilized layer was formed by placing a droplet of an enzyme solution on the working electrode surrounded by a super-hydrophobic layer and shrinking it by evaporation. A significant increase in the output current was observed compared electrodes fabricated by a conventional method. The current density increased by decreasing the electrode size and increasing enzyme loading. The sensor fabricated by this method was incorporated in a micro-flow channel. With smaller electrodes, the current density and the conversion efficiency increased and the flow dependence became weaker. The sensitivity and the conversion efficiency could also be improved by placing two same electrodes in the flow channel.

Freeze-Dried Matrix as an Alternative to Solution Mixing for Enzyme Analysis in a Micro Flow Channel

A freeze-dried matrix packed in a micro flow channel was used to analyze an enzymatic reaction rapidly without using external pumps. The very porous and fine precipitated matrix facilitated the permeation of a sample solution into the flow channel and the dissolution of the components. The amount of products produced as a result of the enzymatic reactions was sufficient and a significant increase in the resulting sensor output was observed. The applicability of the device was demonstrated in the determination of the activities of enzymes, the analysis of enzyme kinetics, and the monitoring of a multi-step reaction. This can be a basic technology in conducting microfluidic diagnostic systems with higher functions including the analysis of enzymatic reactions.

Microfluidic system for the determination of the activities of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase

A microfludic system for the analysis of glutamic oxaloacetic transaminase (GOT) or glutamic pyruvic transaminase (GPT) activity was developed. The system consisted of a glass substrate with a micro amperometric L-glutamate sensor and a polydimethylsiloxane (PDMS) substrate with a Y-shaped flow channel. A sample solution containing either of the enzymes and a solution containing the substrates for the enzymes were introduced from two injection ports. When the flows were stopped, the two solutions mixed immediately within seconds by diffusion. L-glutamate produced by the enzymatic reaction of GOT or GPT was detected by using the amperometic L-glutamate sensor. Current increase was observed immediately after mixing and the initial slope of the response curve depended linearly on the activity of GOT or GPT. The response was enhanced as the volume of the mixing channel increased. The influence of interferents was negligible, because the activity was determined by the rate of the current increase.