Facile Preparation of Glycoprotein-Imprinted 96-Well Microplates for Enzyme-Linked Immunosorbent Assay by Boronate Affinity-Based Oriented Surface Imprinting

Abstract

Molecularly imprinted polymers (MIPs), as inexpensive and stable substitutes of antibodies, have shown great promise in immunoassays. Glycoproteins are of significant diagnostic value. To facilitate the application of MIPs in clinical diagnostics, a general and facile imprinting method toward glycoproteins oriented for an enzyme-linked immunosorbent assay (ELISA) in the form of a 96-well microplate is essential but has not been fully explored yet. In this study, a new method called boronate affinity-based oriented surface imprinting was proposed for facile preparation of glycoprotein-imprinted microplates. A template glycoprotein was first immobilized by a boronic acid-modified microplate through boronate affinity binding, and then, a thin layer of polyaniline was formed to cover the microplate surface via in-water self-copolymerization. After the template was removed by an acidic solution, 3D cavities that can rebind the template were fabricated on the microplate surface. Using horseradish peroxidase (HRP) as a model target, the effects of imprinting conditions as well as the properties and performance of the prepared MIPs were investigated. α-Fetoprotein (AFP)-imprinted microplate was then prepared, and thereby, a MIP-based ELISA method was established. The prepared MIPs exhibited several highly favorable features, including excellent specificity, widely applicable binding pH, superb tolerance for interference, high binding strength, fast equilibrium kinetics, and reusability. The MIP-based ELISA method was finally applied to the analysis of AFP in human serum. The result was in good agreement with that by radioimmunoassay, showing a promising prospect of the proposed method in clinical diagnostics.

Gold nanoparticles and polyethylene glycols functionalized conducting polyaniline nanowires for ultrasensitive and low fouling immunosensing of alpha-fetoprotein

“…4B) with a limit of detection (LOD) of 0.007 pg mL À 1 (3s, n¼10). The LOD is much lower than that achieved by previously reported AFP immunosensors, such as those using localized surface plasmon resonance (LSPR) based on gold nano-mushroom (Li et al, 2015b), surface-enhanced Raman scattering (SERS) based on gold nanoparticle-embedded metal-organic framework (Hu et al, 2014), and enzyme-linked immunosorbent assay (ELISA) based on molecularly imprinted polymers (MIPs) (Bi and Liu, 2014). This low LOD was mainly attributed to the good electron transfer capability of AuNPs/PANI and the excellent biocompatibility of PEG, which can provide a suitable micro-environment for the immobilized AFP antibody to exhibit better binding affinity.…”

“…Thus, detecting AFP in human serum is of great significance for the diagnosis and management of HCC (Wang et al, 2014a). Various methods have been developed for the detection of AFP, such as chemiluminescence (Fu et al, 2006), enzyme-linked immunosorbent assay (Bi and Liu, 2014), surface plasmon resonance (Chang et al, 2009) and radioimmunoassay (Shafik et al, 2014). Besides, electrochemical immunosensors, possessing high sensitivity, rapid detection, low cost and ease miniaturization, have aroused great interests and have been applied in the detection of tumor markers (Gao et al, 2013;Wang et al, 2014a).…”

Selective extraction and enrichment of glycoproteins based on boronate affinity SPME and determination by CIEF-WCID

“…A C C E P T E D ACCEPTED MANUSCRIPT 4 It is well documented in the literature that boronic acids are important ligands for specific recognition, isolation, and enrichment of cis-diol-containing biomolecules such as glycoproteins and saccharides [15,16]. Due to these merits, boronic-acid-functionalized materials have increasingly attracted attention in recent years [16][17][18].…”

Selective Enrichment and MALDI-TOF MS Analysis of Small Molecule Compounds with Vicinal Diols by Boric Acid-Functionalized Graphene Oxide

“…Biomolecules with vicinal diols comprise an important family of compounds in life-science research, and some of them have attracted a great deal of attention, such as glycoproteins and glycopeptides in proteomics [21], nucleosides in metabolomics [22], and saccharides in glycomics [23]. However, many important biological molecules with vicinal diols are often present in low abundance, and their analysis suffers from serious interference by other highly abundant substances in the samples.…”