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

Immobilizing enzymes onto abiological surfaces is a key step for developing protein-based technologies that can be useful for applications such as biosensors and biofuel cells. A central impediment for the advancement of this effort is a lack of generalizable strategies for functionalizing surfaces with proteins in ways that prevent unfolding, aggregation, and uncontrolled binding, requiring surface chemistries to be developed for each surface–enzyme pair of interest. In this work, we demonstrate a significant advancement toward addressing this problem using a gold nanoparticle (AuNP) as an initial scaffold for the chemical bonding of the enzyme acetylcholinesterase (AChE), forming the conjugate AuNP–AChE. This can then be placed onto chemically and structurally distinct surfaces (e.g., metals, semiconductors, plastics, etc.), thereby bypassing the need to develop surface functionalization strategies for every substrate or condition of interest. Carbodiimide crosslinker chemistry was used to bind surface lysine residues in AChE to AuNPs functionalized with ligands containing carboxylic acid tails. Using amino acid analysis, we found that on average, 3.3 ± 0.1 AChE proteins were bound per 5.22 ± 1.25 nm AuNP. We used circular dichroism spectroscopy to measure the structure of the bound protein and determined that it remained essentially unchanged after binding. Finally, we performed Michaelis–Menten kinetics to determine that the enzyme retained 18.2 ± 2.0% of its activity and maintained that activity over a period of at least three weeks after conjugation to AuNPs. We hypothesize that structural changes to the peripheral active site of AChE are responsible for the differences in activity of bound AChE and unbound AChE. This work is a proof-of-concept demonstration of a generalizable method for placing proteins onto chemically and structurally diverse substrates and materials without the need for surface functionalization strategies.