Accelerated colorimetric immunosensing using surface-modified porous monoliths and gold nanoparticles

Abstract

A rapid and sensitive immunoassay platform integrating polymerized monoliths and gold nanoparticles (AuNPs) has been developed. The porous monoliths are photopolymerized in situ within a silica capillary and serve as solid support for high-mass transport and high-density capture antibody immobilization to create a shorter diffusion length for antibody-antigen interactions, resulting in a rapid assay and low reagent consumption. AuNPs are modified with detection antibodies and are utilized as signals for colorimetric immunoassays without the need for enzyme, substrate and sophisticated equipment for quantitative measurements. This platform has been verified by performing a human IgG sandwich immunoassay with a detection limit of 0.1 ng ml^-1. In addition, a single assay can be completed in 1 h, which is more efficient than traditional immunoassays that require several hours to complete.

Keywords: colorimetric assay; gold nanoparticles; immunoassay; porous monoliths.

Figure 1.

The sandwich immunosensing strategy using porous monolith and AuNPs probes. (a) The monoliths are photopolymerized within silica capillary forming globule clusters with a ∼1 μm diameter. (b) The AuNPs that are used in the tests are ∼13 nm in diameter, as verified via TEM images. (c) (I) The monoliths are first modified with monoclonal caHIgG and the nonspecific binding sites are eliminated by surface-attached BSA. (II) AuNP colloids are conjugated with aHIgG, followed by (III) incubating with different concentrations of HIgG. (IV) The HIgG–aHIgG–AuNPs complex is then injected into a silica capillary and (V) bound to caHIgG on the monolith surfaces. The amount of HIgG can be determined via the color variance of the bound AuNPs (red) label on the monolith (white) using a smart phone or a desktop scanner.

Figure 2.

Immobilization of antibody on the thiolated monolith surface. Cysteamine is introduced to the poly(GMA-co-EDMA) monoliths containing oxirane moieties to open the oxirane ring and transform it into a thiol group. When the caHIgG is added, the cysteine sulfhydryl group of the caHIgG antibody can react with the thiol group on the monolith surface to form the intermolecular disulfide bridges between the antibody and the monolith.

Figure 3.

The visualization of AuNPs conjugated with antibodies via UV–Vis spectra. (I) The UV–Vis spectra of AuNPs have an absorbance peak at 520 nm. (II) After adding 50 mM NaCl solution to the 1 nM AuNPs colloid, the AuNPs aggregated and the absorbance peak was red-shifted to ∼625 nm. (III) However, when we modified the aHIgG antibodies on the AuNPs surface with a ratio of 10:1, the aHIgG created a steric distance, and it possessed the same surface charges as the adjacent AuNPs to avoid substantial aggregation.

Figure 4.

Porous monolith-based colorimetric immunoassay combining AuNPs probes for the detection of HIgG. (a) (I) The caHIgG-conjugated poly(GMA-co-EDMA) monolith is white. (II), (III) When 0.1 and 1 nM HIgG–aHIgG–AuNPs mixtures are injected and captured by the caHIgG-monolith surfaces, the length of the monolith segments is increased and their color changes from white to dark red. The higher concentration of HIgG leads to longer segments of monoliths that turn red. (b) A detection limit of ∼0.1 ng ml⁻¹ is obtained using this immunoassay platform. The error bars represent the standard deviation from the measurements (n = 5). A linear correlation existed over the range of 0.1 ng ml⁻¹–10 μg ml⁻¹ (R² = 0.98).