Characterizing the Surface Coverage of Protein–Gold Nanoparticle Bioconjugates

• Published in: Bioconjugate chemistry 2018-08, Vol.29 (8), p.2691-2700
• Main Authors: Kozlowski, Rachel, Ragupathi, Ashwin, Dyer, R. Brian
• Format: Article
• Language: English
• Subjects: Attachment; Binding sites; Biosensors; Biotechnology; Catalysis; Curvature; Cysteine - chemistry; Dihydrofolate reductase; Drug delivery; Drug delivery systems; Dynamic Light Scattering; Electrophoresis, Polyacrylamide Gel; Enzymatic activity; Enzyme activity; Escherichia coli - enzymology; Fluorescence; Gel electrophoresis; Gold; Gold - chemistry; Light scattering; Metal Nanoparticles - chemistry; Mutants; Mutation; Nanoparticles; Optimization; Photon correlation spectroscopy; Proteins; Scattering; Sodium lauryl sulfate; Spectrometry, Fluorescence; Spectrophotometry; Spectrophotometry, Ultraviolet; Surface Properties; Tetrahydrofolate Dehydrogenase - chemistry; Tetrahydrofolate Dehydrogenase - genetics; Ultraviolet radiation
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/acs.bioconjchem.8b00366

Functional enzyme–nanoparticle bioconjugates are increasingly important in biomedical and biotechnology applications such as drug delivery and biosensing. Optimization of the function of such bioconjugates requires careful control and characterization of their structures and activity, but current methods are inadequate for this purpose. A key shortcoming of existing approaches is the lack of an accurate method for quantitating protein content of bioconjugates for low (monolayer) surface coverages. In this study, an integrated characterization methodology for protein–gold nanoparticle (AuNP) bioconjugates is developed, with a focus on site-specific attachment and surface coverage of protein on AuNPs. Single-cysteine-containing mutants of dihydrofolate reductase are covalently attached to AuNPs with diameters of 5, 15, and 30 nm, providing a range of surface curvature. Site-specific attachment to different regions of the protein surface is investigated, including attachment to a flexible loop versus a rigid α helix. Characterization methods include SDS-PAGE, UV–vis spectrophotometry, dynamic light scattering, and a novel fluorescence-based method for accurate determination of low protein concentration on AuNPs. An accurate determination of both protein and AuNP concentration in conjugate samples allows for the calculation of the surface coverage. We find that surface coverage is related to the surface curvature of the AuNP, with a higher surface coverage observed for higher surface curvature. The combination of these characterization methods is important for understanding the functionality of protein–AuNP bioconjugates, particularly enzyme activity.