Dual Electrophoresis Detection System for Rapid and Sensitive Immunoassays with Nanoparticle Signal Amplification

Abstract

An electrophoretic technique was combined with an enzyme-linked immunosorbent assay (ELISA) system to achieve a rapid and sensitive immunoassay. A cellulose acetate filter modified with polyelectrolyte multilayer (PEM) was used as a solid substrate for three-dimensional antigen-antibody reactions. A dual electrophoresis process was used to induce directional migration and local condensation of antigens and antibodies at the solid substrate, avoiding the long diffusion times associated with antigen-antibody reactions in conventional ELISAs. The electrophoretic forces drove two steps in the ELISA process, namely the adsorption of antigen, and secondary antibody-labelled polystyrene nanoparticles (NP-Ab). The total time needed for dual electrophoresis-driven detection was just 4 min, nearly 2 h faster than a conventional ELISA system. Moreover, the rapid NP-Ab electrophoresis system simultaneously achieved amplification of the specific signal and a reduction in noise, leading to a more sensitive NP-Ab immunoassay with a limit of detection (LOD) of 130 fM, and wide range of detectable concentrations from 0.13 to 130 pM. These results suggest that the combination of dual electrophoresis detection and NP-Ab signal amplification has great potential for future immunoassay systems.

Optical Glass Microsphere Enabled Rapid Single‐Molecular Fluoroimmunoassay

“…[ 6a,c,7b,21 ] In addition, the immunoassay constructed through the centrifugation‐associated microfluidics also had been reported to shorten the assay time to 30 min; [ 23 ] the immunoassay implemented in the capillary has been proposed to cut the diffusion time and to achieve the shortened assay time of 16 min; [ 24 ] the printed paper‐immunoassay has been fabricated to have the sensitive and rapid immunoassay for the inspiration of the paper fiber in 51 min; [ 18 ] the dual electrophoresis had been coupled with ELISA to accelerate the movement of the reagent to have the rapid immunoassay in 4 min. [ 25 ] Nevertheless, the immunoassay could be completed in minutes and ultrasensitive detection at a single‐molecular level is still scarce, which would be a powerful tool in the monitoring of low‐abundance cytokines and the diagnosis of the diseases at the early stage. Especially, it would be a powerful complement to highly sensitive real‐time quantitative polymerase chain reactions to sensitively inspect the pathogens and viruses alive like severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐19).…”

Poly(amino acid) Multilayers Modified Dendritic Mesoporous Silica Nanoparticles Achieve Effective Enzyme Stability for Ultrasensitive Immunoassay

“…Poly­(γ-glutamic acid) (PGA), a water-soluble negatively charged poly­(amino acid) with excellent biocompatibility, has broad prospects in biomedical applications. − Besides, polylysine (PL) is also one of the commonly used cationic polyelectrolytes to modify nanomaterials and immobilize bioactive molecules. , Therefore, PGA and PL multilayers are expected to encapsulate enzyme markers with a great stability in DMSN for signal amplification. Moreover, our group has demonstrated polyelectrolyte multilayers could contribute to the development of a variety of highly sensitive ELISA biosensors. ,, …”

“…Moreover, our group has demonstrated polyelectrolyte multilayers could contribute to the development of a variety of highly sensitive ELISA biosensors. 3,26,27 In this study, we developed a signal-amplified nanoparticle composed of a horseradish peroxidase (HRP)-loaded DMSN modified with poly(amino acid) multilayers (PAMs) (defined as DSHP) for an ultrasensitive ELISA system (Scheme 1). The DSHP preparation and Ab 2 adsorption were carried out under moderate conditions to maintain the HRP activity and the Ab 2 capture ability.…”