Employing defined bioconjugates to generate chemically functionalised gold nanoparticles for in vitro diagnostic applications

Novel methods for introducing chemical and biological functionality to the surface of gold nanoparticles serve to increase the utility of this class of nanomaterials across a range of applications. To date, methods for functionalising gold surfaces have relied upon uncontrollable non-specific adsorption, bespoke chemical linkers, or non-generalisable protein–protein interactions. Herein we report a versatile method for introducing functionality to gold nanoparticles by exploiting the strong interaction between chemically functionalised bovine serum albumin (f-BSA) and citrate-capped gold nanoparticles (AuNPs). We establish the generalisability of the method by introducing a variety of functionalities to gold nanoparticles using cheap, commercially available chemical linkers. The utility of this approach is further demonstrated through the conjugation of the monoclonal antibody Ontruzant to f-BSA–AuNPs using inverse electron-demand Diels–Alder (iEDDA) click chemistry, a hitherto unexplored chemistry for AuNP–IgG conjugation. Finally, we show that the AuNP–Ontruzant particles generated via f-BSA–AuNPs have a greater affinity for their target in a lateral flow format when compared to conventional physisorption, highlighting the potential of this technology for producing sensitive diagnostic tests.

BSA-TCOX characterisation TCO:BSA ratio was calculated according to the following equation, where BSA = 1: PeakcenterM represents the MALDI peak center of the modified BSA, and PeakcenterN represents the MALDI peak center of native BSA. The value of 399.5 represents the molecular weight (in g/mol) of the TCO moiety when attached to BSA.

BSA-TCO26-ONT-AuNP pH optimisation
Solutions of BSA-TCO26-AuNPs (108 µL, OD = 1.1) were prepared in triplicate in the following buffers; 20 mM phosphate buffer pH = 6.0, 7.0, 8.0, and 20 mM carbonate buffer pH = 9.0, 10.0. All buffers contained 0.05% tween 20. To these solutions ONT (12 µL, 300 nM in d.d. H2O) was added and the samples incubated at 21 °C for 16 hours. The particle solutions were subsequently centrifuged (5000 r.c.f, 10 minutes) to pellet the particles, and then washed (6 x 1 mL carbonate storage buffer). After the final wash the particles were resuspended into carbonate storage buffer, briefly vortexed and sonicated, and stored at 4 °C. Binding to HER2 was assessed using a sandwich LFIA as detailed in the methods section.

ONT-AuNP pH optimisation
Solutions of ONT (213 µL, 0.59 µM in BBS pH = 8.0 -10.0) were prepared in duplicate and added to sample tubes. To each sample, 40 nm citrate-capped AuNPs (50 µL, OD = 5, BBS solutions) were added and the solutions left to incubate at 21 °C for 1 hour, before the UV-Vis spectra were obtained on a plate reader.

BSA-TCO26-ONT-AuNP concentration optimisation
To a solution of BSA-TCO26-AuNPs (135 µL, OD = 1.1 in storage buffer), ONT-Tet (15 µL, 125 -8000 nM in d.d H2O) was added and the solution left to incubate at 21 °C for 16 hours. The particle solution was subsequently centrifuged (5000 r.c.f, 10 minutes) to pellet the particles, and then washed (6 x 1 mL carbonate storage buffer). After the final wash the particles were resuspended into carbonate storage buffer, briefly vortexed and sonicated, and stored at 4 °C. Binding to HER2 was assessed using a sandwich LFIA as detailed in the methods section.

ONT-AuNP concentration optimisation
To a solution of 40 nm citrate AuNPs (135 µL, OD = 1.1 in pH = 7.0 phosphate buffer), ONT (15 µL, 125 -8000 nM in d.d H2O) was added and the solution left to incubate at 21 °C for 16 hours. Blocking buffer (50 µL) was added and the solution incubated at 21 °C for 30 minutes. The particle solution was subsequently centrifuged (5000 r.c.f, 10 minutes) to pellet the particles, and then washed (6 x 1 mL carbonate storage buffer). After the final wash the particles were resuspended into carbonate storage buffer, briefly vortexed and sonicated, and stored at 4 °C. Binding to HER2 was assessed using a sandwich LFIA as detailed in the methods section.

BSA-TCO26-ONT-AuNP time optimisation
To a solution of BSA-TCO26-AuNPs (135 µL, OD = 1.1 in storage buffer), ONT-Tet (15 µL, 400 nM in d.d H2O) was added and the solution left to incubate at 21 °C for 1 -32 hours. The particle solution was subsequently centrifuged (5000 r.c.f, 10 minutes) to pellet the particles, and then washed (6 x 1 mL carbonate storage buffer). After the final wash the particles were resuspended into carbonate storage buffer, briefly vortexed and sonicated, and stored at 4 °C. Binding to HER2 was assessed using a sandwich LFIA as detailed in the methods section.

ONT-AuNP time optimisation
To a solution of 40 nm citrate AuNPs (135 µL, OD = 1.1 in pH = 7.0 phosphate buffer), ONT (15 µL, 400 nM in d.d H2O) was added and the solution left to incubate at 21 °C for 1 -32 hours. Blocking buffer (50 µL) was added and the solution incubated at 21 °C for 30 minutes. The particle solution was subsequently centrifuged (5000 r.c.f, 10 minutes) to pellet the particles, and then washed (6 x 1 mL carbonate storage buffer). After the final wash the particles were resuspended into carbonate storage buffer, briefly vortexed and sonicated, and stored at 4 °C. Binding to HER2 was assessed using a sandwich LFIA as detailed in the methods section

Antibody per particle quantification
To a solution of BSA-TCO26-AuNPs (280 µL, 0.166 nM, OD = 1.1111, phosphate buffer pH = 7.0 0.05% tween 20), ONT-Tet (20 µL, 600 nM, d.d. H2O) was added. A control solution of the same volume and concentration of particles was prepared, and d.d. H2O was added in the place of ONT-Tet. A standard curve (in triplicate) of ONT-Tet (0 -150 nM, phosphate buffer pH = 7.0 0.05% tween 20) was also prepared. Each solution was incubated at 21 °C for 16 hours. The samples were centrifuged (5000 r.c.f, 10 minutes), and 100 µL of the supernatant from each sample and standard was added to a black 96 well plate. To each sample and standard, a working CBQCA reagent solution (0.4 mM ATTO-TAG CBQCA, 1.33 mM KCN, Invitrogen™) was added. The plate was covered and incubated at 21 °C for 1 hour with constant shaking (500 rpm). The fluorescence emission was read (lex/ lex = 465 / 550, SpectraMax M5, Molecular Devices). The controls were subtracted, and the antibody concentration in the supernatant was determined from interpolation of the standard curve. The number of IgG ligands per particle was determined according to the following equation:

Nanoparticle stability test
Serial dilutions of BSA and GSH were prepared (150 µL, 2.0 ×10 -3 -2000 µM in storage buffer) in Eppendorf tubes. To these solutions, BSA-Biotin-AuNPs were added (300 µL), and the mixtures incubated at 21 °C for 5 hours. The particles were pelleted via centrifugation (5000 r.c.f, 10 minutes), and the supernatant from each sample was removed. The concentration of BSA-Biotin in the supernatant was deduced using ELISA. Briefly, a NeutraAvidin coated 96 well plate (Pierce) was blocked with 2% BSA in PBST (0.1% tween 20) for 1 hour at 21 °C, and then washed with PBST. The Supernatant (100 µL) from each sample was added to the plate, along with a standard curve of BSA-Biotin (0.137 -100 nM in storage buffer), which was incubated at 21 °C for 90 minutes followed by washing with PBST. Streptavidin-HRP (100 µL, 1 in 80,000 dilution in PBST + 2% BSA, Abcam) was added to the plate, which was incubated at 21 °C for 90 minutes followed by washing with PBST. Visualization of the formed complex was achieved using OneStep Ultra TMB (Thermo Scientific™) according to the manufacturer's instructions. The plate was read at 450 nm using a SpectraMax M5 plate reader (Molecular Devices). Lateral flow strips These images are the raw data for the LFIA plot in Fig. 7 of the manuscript.