Formation and Characterization of a Stable Monolayer of Active Acetylcholinesterase on Planar Gold

Abstract

Enzyme activity is the basis for many biosensors where a catalytic event is used to detect the presence and amount of a biomolecule of interest. To create a practical point-of-care biosensor, these enzymes need to be removed from their native cellular environments and immobilized on an abiological surface to rapidly transduce a biochemical signal into an interpretable readout. This immobilization often leads to loss of activity due to unfolded, aggregated, or improperly oriented enzymes when compared to the native state. In this work, we characterize the formation and surface packing density of a stable monolayer of acetylcholinesterase (AChE) immobilized on a planar gold surface and quantify the extent of activity loss following immobilization. Using spectroscopic ellipsometry, we determined that the surface concentration of AChE on a saturated Au surface in a buffered solution was 2.77 ± 0.21 pmol cm −2 . By calculating the molecular volume of hydrated AChE, corresponding to a sphere of 6.19 nm diameter, divided by the total volume at the AChE−Au interface, we obtain a surface packing density of 33.4 ± 2.5% by volume. This corresponds to 45.1 ± 3.4% of the theoretical maximum monolayer coverage, assuming hexagonal packing. The true value, however, may be larger due to unfolding of enzymes to occupy a larger volume. The enzyme activity and kinetic measurements showed a 90.6 ± 1.4% decrease in specific activity following immobilization. Finally, following storage in a buffered solution for over 100 days at both room temperature and 4 °C, approximately 80% of this enzyme activity was retained. This contrasts with the native aqueous enzyme, which loses approximately 75% of its activity within 1 day and becomes entirely inactive within 6 days.

Gold Nanoparticles Are an Immobilization Platform for Active and Stable Acetylcholinesterase: Demonstration of a General Surface Protein Functionalization Strategy

“…The activity remained the same over the course of three weeks (after which the experiment was ended), demonstrating that binding to the AuNP imparted stability on the enzyme. We have previously reported that native aqueous AChE completely loses activity after 6 days of storage at 4 °C but remains stable when adsorbed onto a flat macroscopic Au surface . The data presented in Figure B corroborates the observation that immobilization onto a scaffold of some kind, regardless of its size, imparts stability on the enzyme’s function.…”

“…We have previously reported that native aqueous AChE completely loses activity after 6 days of storage at 4 °C but remains stable when adsorbed onto a flat macroscopic Au surface. 31 The data presented in Figure 4B corroborates the observation that immobilization onto a scaffold of some kind, regardless of its size, imparts stability on the enzyme's function. This is significant since it shows that the act of immobilizing proteins onto a platform is not only key to integrating proteins into inorganic materials but is also in fact beneficial to the stability and function of any device using this strategy over time.…”

“…Further insights into the relationship between the changes in structure and the activity were investigated using Michaelis–Menten kinetics, which measures enzyme activity as a function of substrate concentration. This was performed on both the AuNP–AChE construct and native aqueous AChE. , The data were fit to the Michaelis–Menten model (eq ): ν = 0.25em V Max [ S ] K normalM + [ S ] where ν is the initial rate of reaction, V Max is the maximum achievable rate, [ S ] is the concentration of the substrate, and K M is the Michaelis constant, which is equal to [ S ] at (1/2) V Max . The activity is normalized by the amount of protein in the sample resulting in an activity unit of U mg –1 , which allows for a direct comparison to be made between bound AChE and native aqueous AChE.…”

“…AChE was purified using a previously reported procedure. , Briefly, 6.5 mg of AChE solid was dissolved in 650 μL of running buffer (10 mM phosphate, 400 mM NaCl, pH 7) and injected into a Superdex 200 size exclusion column. AChE was collected as a single peak and concentrated with Amicon Ultra 0.5 mL 10k spin filters at 14,000 g .…”

“…We have previously studied the effects on the activity and structure of adsorbing a monolayer of the enzyme acetylcholinesterase (AChE) onto a planar gold surface, observing that although the enzyme retained approximately 10% of its activity, it subsequently maintained that activity over 100 days in buffer solution at 4 °C . This was particularly striking when contrasted to the native aqueous enzyme, which was observed to become completely inactive after 6 days in the same buffer solution, demonstrating that immobilization onto a surface imparted stability on the structure and function of the enzyme.…”