Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP

Abstract

Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.

Molecularly Imprinted Polymer Based Sensors for Medical Applications

“…In a molecularly imprinted polymer, specific sites can be created for various templates such as amino acids [43], proteins [44,45,46], enzymes [47,48], hormones [49,50,51], antibodies [52,53,54], nucleic acids [55,56], bacteria [57], viruses [58,59,60], drugs [61,62], metal ions [63,64,65,66], toxins [67], antibiotics [68] and pesticides [69,70] and so on. Furthermore, MIPs are easy to synthesis, highly stable, cost-effective, and user-friendly.…”

Introduction to nanoMIPS Prepared by Solid-Phase approach as an Alternative for Antibodies in the pseudo-ELISA Diagnostic Assay

“…Superior of MIPs compared with natural antibodies is also reflected in the types of the analytes that MIPs can be produced for such as toxins and some pesticides which prove to be difficult to produce antibodies for. In effect, the plastic antibodies/imprinted polymers in various formats (nanoparticle or film/microparticles) have successfully been produced against a wide range of targets such as: ions (phosphate [5], carbonate [6], copper ions [7], small molecules (caffeine [8], cocaine [9], melamine [10], ascorbic acid [11], sugars [12], fentanyl [13], doxorubicin [14,15], phosphorganic pesticides [16], explosives [17], etc), peptides (vancomycin [18][19][20], peptides/protein epitopes [21,22]), proteins (trypsin [21,23,24], acetylcholinesterase [25]), E. coli [26].…”

Electrochemical Chiral Recognition of Tryptophan Isomers Based on Nonionic Surfactant-Assisted Molecular Imprinting Sol–Gel Silica

“…Molecular imprinting technology, offering a synthetic route to artificial recognition systems, has attracted great concerns because of its importance in separation, − chemo/biosensors, , catalysis, and biomedicine, which forms the selective recognition sites to template (typically the target analytes) in a polymer matrix after removing the template out of the polymer matrix. Traditional molecular imprinting polymers (MIP) are organic polymers obtained via free radical polymerization of functional monomer in organic solvents.…”