The ability to evaluate antibody immobilization onto gold nanoparticles is critical for assessing coupling chemistry and optimizing the sensitivity of nanoparticle-enabled biosensors. Herein, we developed a fluorescence-based method for directly quantifying antibodies bound onto gold nanoparticles. Antibody-modified gold nanoparticles were treated with KI/I2 etchant to dissolve the gold nanoparticles. A desalting spin column was used to recover the antibody released from the nanoparticles, and NanoOrange, a fluorescent dye, was used to quantify the antibody. We determined 309 ± 93 antibodies adsorb onto a 60 nm gold nanoparticles (2.6 × 10(10) NP mL(-1)), which is consistent with a fully adsorbed monolayer based on the footprint of an IgG molecule. Moreover, the increase in hydrodynamic diameter of the conjugated nanoparticle (76 nm) compared to that of the unconjugated nanoparticle (62 nm) confirmed that multilayers did not form. A more conventional method of indirectly quantifying the adsorbed antibody by analysis of the supernatant overestimated the antibody surface coverage (660 ± 87 antibodies per nanoparticle); thus we propose the method described herein as a more accurate alternative to the conventional approach.

A simple, rapid, and sensitive immunoassay has been developed based on antigen-mediated aggregation of gold nanoparticles (AuNP) and surface-enhanced Raman spectroscopy (SERS). Central to this platform is the extrinsic Raman label (ERL), which consists of a gold nanoparticle modified with a mixed monolayer of a Raman active molecule and an antibody. ERLs are mixed with sample, and antigen induces the aggregation of the ERLs. A membrane filter is then used to isolate and concentrate the ERL aggregates for SERS analysis. Preliminary work to establish proof-of-principle of the platform technology utilized mouse IgG as a model antigen. The effects of membrane pore diameter and AuNP size on the analytical performance of the assay were systematically investigated, and it was determined that a pore diameter of 200 nm and AuNP diameter of 80 nm provide maximum sensitivity while minimizing signal from blank samples. Optimization of the assay provided a detection limit of 1.9 ng/mL, 20-fold better than the detection limit achieved by an ELISA employing the same antibody-antigen system. Furthermore, this assay required only 60 min compared to 24h for the ELISA. To validate this assay, mouse serum was directly analyzed to accurately quantify IgG. Collectively, these results demonstrate the potential advantages of this technology over current diagnostic tests for protein biomarkers with respect to time, simplicity, and detection limits. Thus, this approach provides a framework for prospective development of new and more powerful tools that can be designed for point-of-care diagnostic or point-of-need detection.

Antibody-modified gold nanoparticles (AuNPs) are central to many novel and emerging biosensing technologies due to the specificity provided by antibody-antigen interactions and the unique properties of nanoparticles. These AuNP-enabled assays have the potential to provide significant improvements in sensitivity and multiplexed analysis compared to conventional immunoassays. However, a major challenge for these AuNP platform technologies is the synthesis of stable antibody-AuNP conjugates that resist aggregation in high salt environments and biological matrices. Moreover, synthetic strategies to form stable conjugates often require different solution conditions, e.g., pH, for each unique antibody. Herein we describe our effort to develop an approach to chemically modify lysine residues on antibodies to facilitate the formation of stable antibody-AuNP conjugates over a wide pH range. In this work, we systematically investigated the immobilization of native and chemically modified antibodies to 60 nm citrate-capped AuNPs as a function of pH and evaluated the stability of the antibody-AuNP conjugate in a saline environment. We have developed a method to chemically modify the lysine residues on an antibody prior to conjugation to the AuNP that results in stable conjugates over a wide pH range (6.0-8.5). Amino acid analysis and zeta potential measurements of native and modified antibodies reveal that the requisite modification correlates with the number of lysine residues, and a reduction in positive charge contribution from protonated lysine is required to form stable, pH-independent conjugates. Furthermore, we demonstrate that the chemically modified antibodies maintain antigen-binding capabilities. We apply this novel conjugation strategy to develop a surface-enhanced Raman spectroscopy (SERS)-based assay for the accurate subtyping of avian influenza viruses.

Dynamin plays an important role in clathrin-mediated endocytosis (CME) by cutting the neck of nascent vesicles from the cell membrane. Here through using gold nanorods as cargos to image dynamin action during live CME, we show that near the peak of dynamin accumulation, the cargo-containing vesicles always exhibit abrupt, right-handed rotations that finish in a short time (~0.28 s). The large and quick twist, herein named the super twist, is the result of the coordinated dynamin helix action upon GTP hydrolysis. After the super twist, the rotational freedom of the vesicle drastically increases, accompanied with simultaneous or delayed translational movement, indicating that it detaches from the cell membrane. These observations suggest that dynamin-mediated scission involves a large torque generated by coordinated actions of multiple dynamins in the helix, which is the main driving force for vesicle scission.