Surface Modification of Gold Nanoparticles and their Applications as Mass Tags for Protein Marker Detection in Laser Ionization Mass Spectrometry

Abstract

Introduction: Gold nanoparticles (AuNPs) have been extensively applied for biomarker detection in bioanalytical studies over the last decade based on their plasmonic properties. Recent work by our group utilized antibody-conjugated AuNPs as mass tags for indirect antigen detection on animal tissue and protein chips via laser ionization MS, where explosive ionization of AuNPs occurs upon irradiation to generate abundant reporter ions for strong MS signal amplification [1]. The analytical performance of antibody-conjugated AuNP mass tags may be further improved by optimizing their stability in biological media to increase sensitivity and reduce noise. Here, the surface modification of AuNP mass tags using polyethylene glycol (PEG) was investigated to determine its significance in terms of S/N for protein marker detection. [1] https://www.sciencedirect.com/science/article/pii/S0003267019300248 Methods: The study was performed using HPV18-E7 cervical cancer marker protein as a model antibody-antigen system.To create antibody-conjugated AuNPs, citrate-stabilized AuNPs were first synthesized via seed mediated growth. Citrate-stabilized AuNPs were functionalized via ligand exchange with a mixture of bifunctional PEG containing either i) carboxylate or ii) anti-HPV18-E7 detection antibody on one end, and a thiol group on the other for AuNP binding. The analytical performance of the anti-HPV18-E7-AuNPs conjugates for LI-MS were characterized via solid-phase sandwich immunoassay using HPV18-E7 recombinant protein and standard immunoassay protocols, followed by analysis via MALDI-TOF MS instrument. Each sample well was rasterized to generate reporter Au+, Au2+ and Au3+ ions, whose intensities were summed to determine the quantity of protein immobilized. Preliminary Data: Surfaces of 20nm AuNPs were modified with various ratios of 5kDA carboxylate PEG stabilizer (cPEG) and antibody-functionalized PEG (Ab-PEG) to study the influence of PEG stabilization for AuNP mass tags. The total mass of PEG added to functionalize 100uL of AuNPs was fixed at 34.5ug, experimentally determined to be sufficient to stabilize the AuNPs during ligand exchange. On the other hand, the ratio of stabilizer-to-antibody PEG was varied from 100, 134, 202, 406, 1018, and 2038 cPEG per Ab-PEG. For the sandwich immunoassay, 5.9 fmol HPV18E7 recombinant protein was incubated on the substrate to measure the total gold ion intensity generated by AuNP mass tags. For the blank, a solution of blocking buffer was applied instead to measure the background noise and therefore degree of non-specific binding. For a fixed mass of PEG, it was observed that a decrease in the ratio of cPEG:Ab-PEG yielded an exponential increase in S/N from 4.38 at a cPEG:Ab-PEG ratio of 2038:1 to an S/N of 169 at a ratio of 202:1, a 50 times improvement. Raising the quantity of Ab-PEG and therefore degree of surface functionalization beyond this ratio however, yielded a 50% decrease in S/N to 82. These results suggest that there is an optimal degree of surface functionalization for AuNP-based detection probes. Given that 20nm AuNPs have a surface area of 1256nm 2, this approximates to 6 antibodies attached per NP, based on the binding density of ~1 PEG/nm 2 [2]. Further antibody conjugation of AuNPs may lead to undesired non-specific binding between AuNPs with each other or with the substrate, leading to decrease S/N. It is anticipated that optimization of surface functionalities of AuNP mass tags will allow for detection limits in the low attomole range and perhaps even lower when AuNPs of larger sizes are applied for LI-MS analysis. [2] https://link.springer.com/article/10.1007/s13404-011-0015-8 Novel Aspect: Adjustment of surface functionalization of antibody-conjugated AuNPs is critical for improving S/N in LI-MS based detection and other analytical applications.