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

Abstract

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.

Quantifying Bound Proteins on Pegylated Gold Nanoparticles Using Infrared Spectroscopy

“…We attempted to move BSA into HPW with Amicon MWCO filters for more representative calibration standards, but this resulted in BSA and AuNP-N 3 /COOH precipitating from the solution. It is important to note that this did not occur when buffer-exchanging AuNP-bound BSA, indicating that covalent binding to the AuNP-N 3 /COOH imparted further stability to the BSA. , BSA is a representative case, as precipitation in the absence of buffer salts is a likely occurrence for many proteins of interest. This issue of buffer salts interfering with measurements can be circumvented by using different peaks in the IR spectrum (as will be discussed later), and thus, the instability of a protein in HPW does not affect generalizability of this method.…”

“…To see if this analytical procedure can be used on actual protein–NP complexes, we used EDC/NHS cross-linking chemistry, a commonly used bioconjugation reaction, ,− to bind BSA and Cyt C to AuNP-N 3 /COOH (making AuNP-BSA and AuNP-Cyt C), and analyzed the samples using IR. To validate our results, we also performed amino acid analysis (AAA) on these bioconjugated constructs.…”

“…AuNPs with SH-C11-(OEG) 4 -X (X = N 3 , COOH) ligands (hereafter called “AuNP-N 3 /COOH”) were synthesized using a previously published procedure. , Briefly, an aqueous solution of HAuCl 4 (0.142 M, 1 mL) was added to a solution of TOAB (0.030 M, 10 mL) and stirred for 30 min until the solution turned dark brown. An aqueous solution of NaBH 4 (1.5 M, 1 mL) was quickly added and left to stir overnight, turning dark purple minutes after the addition of NaBH 4 .…”

“…Nanoparticles (NPs) have been shown to be effective scaffolds on which stable and active proteins and enzymes can be immobilized. − Characterization of proteins grafted onto NPs is crucial to the development of these protein-based nanomaterials. Some common and important characterization parameters include activity, stability, and amount of bound protein. , Out of these important characteristics, the quantification of the amount of bound protein is one of the most important since it determines the efficiency of binding and the effects of binding on protein activity. − We recently surveyed the currently available methods to determine this quantity on NPs and the few available methods often require expertise and instrumentation that are not readily available .…”

“…Infrared (IR) spectroscopy is a technique often used to characterize protein–NP materials. ,− For example, IR spectroscopy has been used to quantify ligands on NPs, − although it is often difficult to accommodate high limits of detection and variability in absolute absorbance between scans. However, including an internal standard in the measurement can significantly increase consistency, and using attenuated total internal reflection (ATR) IR to measure dry samples can increase the sensitivity.…”