Gold Nanoparticles
Are an Immobilization Platform
for Active and Stable Acetylcholinesterase: Demonstration of a General
Surface Protein Functionalization Strategy
posted on 2022-12-12, 20:33authored byPaul R. Handali, Lauren J. Webb
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.