Biotinylated Au Nanoparticle-Based Artificial Antibody for Detection of Lysozyme by the Lateral Flow Immunoassay and Enzyme-Linked Immunosorbent Assay

Abstract

Antibody-based immunoassays such as the lateral flow immunoassay (LFIA) and enzyme-linked immunosorbent assay (ELISA) are currently indispensable analytical methods widely used in many fields, mainly due to the high sensitivity and specificity of antibodies. However, the high cost of monoclonal antibodies and their susceptibility to high temperature limit the application of immunoassays. Previously, we developed a class of gold nanoparticle (AuNP)-based artificial antibodies, called Goldbody. Goldbodies not only bind antigens as specifically as monoclonal antibodies do but also have far better stability than monoclonal antibodies. To take advantage of the excellent specificity, stability, and easy functionalization of Goldbodies and use them for the substitution of monoclonal antibodies in immunoassays, herein, we synthesize a biotinylated anti-lysozyme Goldbody and successfully construct a competitive LFIA for the detection of lysozyme in the range of 17.98–226.49 ng·mL–1. At the same time, the biotinylated anti-lysozyme Goldbody can easily replace the detection antibody in the commercial BA (biotinylated antibody)-ELISA kit for the detection of lysozyme with a lower detection limit of 0.34 ng·mL–1 and a wider detection range of 0.89–20 ng·mL–1 compared with the commercial BA-ELISA kit. In addition, the biotinylated anti-lysozyme Goldbody has good thermal stability in both assays and can accurately detect spiked samples even after pretreatment at 100 °C, demonstrating the high potential of Goldbodies as a good replacement of monoclonal antibodies in immunoassays.

Anti‐Carbonic Anhydrase Goldbodies by Conformational Reconstruction of the Complementary‐Determining Regions of Phage‐Displayed Antibodies

“…A common merit of Goldbodies is their excellent thermal stability, which is due to the stable and rigid core of AuNPs, which replace the protein skeletons [11,31–35] . As demonstrated by the SPR binding results presented in Figure 6, Gb‐CA05 and Gb‐CA06 maintain their binding capabilities with CA even after preincubation at 100 °C for 30 min.…”

“…As shown in Figure 3A, with the increase of P05 numbers conjugated on one AuNP, the binding signal of AuNP‐xP05 (where x represents the number of P05 conjugated per AuNP) with CA gradually increased and reached the peak value at 15 P05 per AuNP, and then decreased continuously. This peptide density effect is a typical phenomenon for conformational engineering of protein fragments on NPs [11,33–36] . Generally, at low peptide density, the conjugated peptides have more space to move more freely on the surface of AuNPs, thus cannot maintain their native conformation.…”

“…This peptide density effect is a typical phenomenon for conformational engineering of protein fragments on NPs. [11,[33][34][35][36] Generally, at low peptide density, the conjugated peptides have more space to move more freely on the surface of AuNPs, thus cannot maintain their native conformation. While when the peptide density is too high, peptides are squeezed in a crowded space, thus cannot adopt their native conformation either.…”

“…Till now, several Goldbodies have been developed by grafting CDRs onto AuNPs, in our group [11,[33][34][35][36] as well as in other independent group. [37] Though CDRs are mostly in loop conformation (some may contain one or two circles of helix), which is relatively more flexible than the α-helix and β-sheet conformation, and is almost indistinguishable from the unfolded conformation by common circular dichroism (CD) spectra.…”

Redox-Concatenated Aptamer Integrated Skin Mimicking Electrochemical Patch for Noninvasive Detection of Cortisol

“…Additionally, electrode surfaces where the aptamer is immobilized play a key role in enhancing the wearable biosensor's performance. − By altering the surface of the electrode, it is feasible to improve the biosensor’s overall performance by increasing the loading density of the biorecognition reagent on electrodes for enhanced interaction with analytes . Many innovative strategies ranging from using nanoparticles, hierarchical, ,, or templated nanostructures or template-based surface texture , have been used to achieve high analytical sensitivity in biosensors . A 2-fold strategy of integrating the high surface-to-volume ratio and redox-active biorecognition reagents such as redox aptamers has the potential to alleviate the performance challenges of wearable biosensors.…”