Evaluation of Enzyme Cascade Electrode Reactions at Nanoscale Area
Authors: Ariga Kotoko , Tsujimura Seiya , Mikawa Tsutomu 
Published: December 2024

Introduction

Enzymes are promising catalysts with high selectivity and low environmental load. Especially, redox enzymes are attracting attentions because of their applicability to electrodes for biosensors and biofuel cells. For example, wearable lactate biosensors which can detect lactate in sweat and use the reaction to drive itself have been developed recently[1]. Various biosensors are already available today. However, most of them use single-enzyme electrodes. Compared to single-enzyme electrodes, multi-enzyme electrodes are expected to improve the performance of conventional biosensors and biofuel cells by realizing detection of multiple biomarkers and increased energy density. This study uses the multi-enzyme system involving lactate oxidase (LOx), pyruvate decarboxylase (PDC), and aldehyde dehydrogenase (ALDH) as the reaction model. In this system, four electrons are produced during one lactate molecule is converted into acetate via pyruvate, which is double the number of what are produced by an LOx single-enzyme electrode used in conventional lactate biosensors. This study aims to improve the current response of bioelectrodes by causing enzyme cascade reactions within nanoscale pores of MgO-templated carbon (MgOC). MgOC is a porous carbon material, which is believed to enhance the efficiency of cascade reactions by providing high surface area and control the enzyme activity by changing the solution environment within the nanoscale pores[2]. The prepared electrodes were evaluated electrochemically to investigate how enzyme cascade and the use of MgOC affect the electrode performance.

Experimental

The ink containing MgOC was dropped to glassy carbon (GC) electrodes to make MgOC electrodes. Subsequently, solution of 1,2-naphtoquinone, the mediator, was added to the MgOC surface. Finally, the mixture of certain ratios of enzymes, crosslinker, Mg-salt, and thiamine pyrophosphate (TPP) was dropped. The latter two were added to promote enzyme reactions. Using the MgOC electrode as a working electrode, three-electrode measurement system was constructed. The measurement solution was 0.1 M phosphate buffer solution (pH 7.0) containing about 15 mM of L-lactate as the substrate. Chronoamperometry (CA) was carried out with an MgOC electrode and a flat GC electrode immobilized with the same amount and ratio of enzymes to see how the use of MgOC affect the current response. Then, the enzyme cascade effect was evaluated by measuring CA of single-enzyme (LOx) electrodes and multi-enzyme (LOx+PDC+ALDH) electrodes. Furthermore, solution environment dependence of the cascade effect was measured by changing the concentration of enzymes and ammonium sulfate ((NH4)2SO4), a kosmotropic salt which is known to salt out enzymes.

Results and Discussion

The current density observed with the MgOC electrode immobilized with LOx, PDC, and ALDH was about 6.8 times higher than that obtained from the GC electrode also dropped with the three kinds of enzymes. This result indicates the use of MgOC as the electrode surface material is beneficial for enzyme cascade systems because of its higher surface area. We also consider that MgOC has the effect to promote product transfer in cascade systems by containing enzymes in nanoscale pores. The relationship between the pore size of MgOC and the performance of enzyme cascade electrodes will be explored in future experiments.
Enzyme cascade effect was evaluated by comparing the current density of single-enzyme MgOC electrodes dropped with only LOx and multi-enzyme MgOC electrodes with LOx, PDC, and ALDH. The figure shows the results. The multi-enzyme electrodes exhibited about 1.98 times higher current density than the single-enzyme electrodes. It implies that the enzyme cascade reactions caused by LOx, PDC, and ALDH increased the number of electrons produced in the system. In this experiment, enzyme solution contained 10 mg/mL of each enzyme and 1.5 mol/L of (NH4)2SO4. When the (NH4)2SO4 concentration was raised to 3.0 mol/L, the current density increased by 2.40 times from single-enzyme to multi-enzyme. On the other hand, it rose by 2.51 times with 5 mg/mL of enzymes and 3.0 mol/L of (NH4)2SO4. Judging from the reaction scheme, the current increase should not be more than double. The increase reached around 2.5 times probably because the forward enzyme reactions were promoted to balance the equilibrium which was displaced by the addition of PDC and ALDH. The enzyme cascade effect was enhanced with increased (NH4)2SO4 concentration, but was slightly affected by the enzyme concentration. These results indicate kosmotropic salts like (NH4)2SO4 shorten the distance between enzymes by salting them out, which can positively impact the cascade reactions. The relationship between enzyme cascade effect and (NH4)2SO4 concentration will be further investigated in future studies.
[1] I. Shitanda, et al., ACS Sens.
8(2023), 6, 2368-2374.
[2] S. Tsujimura, K. Murata, and W. Akatsuka, J. Am. Chem. Soc.
136 (2014) 14432-14437.

Figure 1