Hydrophilic Polyelectrolyte Multilayers Improve the ELISA System: Antibody Enrichment and Blocking Free

Abstract

Abstract:In this study, polyelectrolyte multilayers were fabricated on a polystyrene (PS) plate using a Layer-by-Layer (LbL) self-assembly technique. The resulting functional platform showed improved performance compared with conventional enzyme-linked immunosorbent assay (ELISA) systems. Poly(diallyldimethylammonium chloride) (PDDA) and poly(acrylic acid) (PAA) were used as cationic and anionic polyelectrolytes. On the negatively-charged (PDDA/PAA) 3 polyelectrolyte multilayers the hydrophilic PAA surface could efficiently decrease the magnitude of the noise signal, by inhibiting nonspecific adsorption even without blocking reagent adsorption. Moreover, the (PDDA/PAA) 3 substrate covalently immobilized the primary antibody, greatly increasing the amount of primary antibody adsorption and enhancing the specific detection signal compared with a conventional PS plate. The calibration curve of the (PDDA/PAA) 3 substrate showed a wide linear range, for concentrations from 0.033 to 33 nM, a large specific signal change, and a detection limit of 33 pM, even though the conventional blocking reagent adsorption step was omitted. The (PDDA/PAA) 3 substrate provided a high-performance ELISA system with a simple fabrication process and high sensitivity; the system presented here shows potential for a variety of immunosensor applications.

Blocking-Free ELISA Using a Gold Nanoparticle Layer Coated Commercial Microwell Plate

“…After confirming that the GNP layer didn’t cause an adverse impact, we tried to explore whether it had positive effects on the ELISA. Considering there was no improvement in sensitivity of the ELISA, we decided to verify if introduction of the GNP layer can simplify procedures of the ELISA, for example, eliminating the blocking step that requires 2 h. Because PEI and citrate-GNPs were both hydrophilic and some literature has reported how the hydrophilic nature of some polymer-modified microplates can directly decrease nonspecific protein adsorption without any blocking step [ 18 ], we preformed the following exploration.…”

“…It is known that the properties of a substrate are a critical factor in influencing protein adsorption in ELISA [ 18 , 19 ]. Therefore, substrate surface modifications could be the most valid means for exhibiting improvements over conventional ELISA.…”

“…This method lacks exploration in improving blocking efficiency to decrease unspecific adsorption. Other researchers have made use of a layer-by-layer (LbL) self-assembly technique to fabricate polyelectrolyte multilayers on a polystyrene (PS) plate, which could inhibit nonspecific adsorption, even without blocking reagent adsorption [ 18 ]. Nonetheless, with the physical adsorption of antibodies on the functionalized surface of the plate, linearity of the calibration curve was poor and the greatest R 2 value was only 0.92.…”

The synergistic interference effect of silica nanoparticles concentration and the wavelength of ELISA on the colorimetric assay of cell toxicity

“…Although this is a simple and repeatable method 8 , it has limitations such as nonspecific adsorption, relatively poor sensitivity, and low surface to volume ratio. These limitations in some cases can challenge, and invalidate the evaluation process basis 9 . Previous studies have shown that certain conditions, such as antioxidants, change of pH range, reducing agents, temperature variation, and the type of solvents, are the disruption factors on regeneration formazan and MTT test 6 , 10 – 12 .…”

Preparation and Characterization of AgNPs In Situ Synthesis on Polyelectrolyte Membrane Coated Sericin/Agar Film for Antimicrobial Applications

“…In this technique, two oppositely charged polyelectrolytes are alternately deposited on a support surface via van der Waals forces and electrostatic attractions [ 29 ]. Polyacrylic acid (PAA) and poly (dimethyldiallylammonium chloride) (PDDA) are two common polymers used to form PEM on the surface of materials [ 30 , 31 ]. PEM could not only provide more silver ion binding sites for the synthesis of high-density AgNPs, but also prevent AgNPs from oxidization or falling off to make the synthesized AgNPs more stable.…”