Real‐Time Monitoring of Mass‐Transport‐Related Enzymatic Reaction Kinetics in a Nanochannel‐Array Reactor

Abstract

To understand the fundamentals of enzymatic reactions confined in micro-/nanosystems, the construction of a small enzyme reactor coupled with an integrated real-time detection system for monitoring the kinetic information is a significant challenge. Nano-enzyme array reactors were fabricated by covalently linking enzymes to the inner channels of a porous anodic alumina (PAA) membrane. The mechanical stability of this nanodevice enables us to integrate an electrochemical detector for the real-time monitoring of the formation of the enzyme reaction product by sputtering a thin Pt film on one side of the PAA membrane. Because the enzymatic reaction is confined in a limited nanospace, the mass transport of the substrate would influence the reaction kinetics considerably. Therefore, the oxidation of glucose by dissolved oxygen catalyzed by immobilized glucose oxidase was used as a model to investigate the mass-transport-related enzymatic reaction kinetics in confined nanospaces. The activity and stability of the enzyme immobilized in the nanochannels was enhanced. In this nano-enzyme reactor, the enzymatic reaction was controlled by mass transport if the flux was low. With an increase in the flux (e.g., >50 microL min(-1)), the enzymatic reaction kinetics became the rate-determining step. This change resulted in the decrease in the conversion efficiency of the nano-enzyme reactor and the apparent Michaelis-Menten constant with an increase in substrate flux. This nanodevice integrated with an electrochemical detector could help to understand the fundamentals of enzymatic reactions confined in nanospaces and provide a platform for the design of highly efficient enzyme reactors. In addition, we believe that such nanodevices will find widespread applications in biosensing, drug screening, and biochemical synthesis.

Nanoconfinement Effects: Glucose Oxidase Reaction Kinetics in Nanofluidics

“…Recently, we used porous anodic alumina (PAA) with a size‐defined nanochannel array to investigate both the confinement of enzyme reaction kinetics and the molecule diffusion behavior. We found that the reaction kinetics changes from a kinetically controlled process at low transport rates of the substrate to a diffusion‐limited one at higher substrate transport rates 8. In that report, the size of the nanochannels was relatively large (200 nm in diameter), and thus the confinement effect of the enzyme reaction kinetics was not that significant.…”

“…Herein, four nanochannels with a length of 1.2 cm, a width of 200 μm and heights of 55, 80, 110 or 140 nm are fabricated and used as the confined enzyme reactors. Since substrate transport considerably affects the enzyme reaction kinetics,8, 18 a constant flow rate of the glucose substrate was kept during the measurements (for the detailed process, see the Supporting Information, Table S1).…”

“…Nanostructured materials are interesting building blocks to mimic the confinement conditions of living systems, and the constructured nanomaterials/biomolecules hybrids are promising for bioanalysis, bioelectronics and biofuel cells 3–8. Examples of such nanostructures include zeolite structures within microchips,4 mesoporous materials,5 and layered materials 6.…”

Nanostructured Membranes for Enzyme Catalysis and Green Synthesis of Nanoparticles

“…In particular, glucose oxidase has demonstrated a sevenfold reduction in activity upon covalent immobilization on a porous alumina support compared with the activity in the homogeneous (bulk) phase. [21] Further to reports on catalase immobilization on various supports, [22] our approach allows for the tuning of product yield through variation of residence time (t) in membrane pores. The residence time was calculated as t = V/(AJ V ), where V is the membrane volume, A the external area (33.2 cm 2 ), and J V the permeation flux (cm 3 cm 2 s À1 ) through volume V. In addition, V = eAL, where e is the porosity (70 % average, from manufacturer's data) and L the membrane thickness (125 mm).…”

Engineering a NO‐Regulated Nanofluidic Sensor through the Cyclization Reaction Strategy

“…Problems with the instability of biological ion channels underi nv itro conditions, still trouble their utilizationi nr eal environments. [5] Thus,f abricatingasmart artificial channel with the ability of mimicking the biological process of NO-regulated ion transport is highly demanded, which will greatly expand the applications of gas-drivens ystems for real-world applications.…”