A Direct Comparison of Antibody and nanoMIP Affinities using Surface Plasmon Resonance and Electrochemical Techniques: A Haemoglobin Model

Abstract

This study presents, for the first time, a direct quantitative comparison between the binding affinities and selectivity of antibodies and their molecularly imprinted polymer (nanoMIP) counterparts for a target protein antigen. NanoMIPs were synthesized upon protein functionalised magnetic nanoparticle (MNP) using bovine haemoglobin as target protein. This solid-phase synthesis process gave nanoMIP yields of 10 ±2 mg produced in less than 1 hr. Physical characterization of nanoMIPs by dynamic light scattering (DLS) revealed an average particle diameter of 121 ± 53 nm, consistent with nanoparticle tracking analysis (NTA) results, confirming uniform particle formation and comparable concentrations to antibody preparations.Antibody and nanoMIP affinities were characterized using surface plasmon resonance (SPR) the current gold-standard technique as well as using a newly developed electrochemical method based on electrochemical impedance spectroscopy (EIS). This dual approach enables direct comparison and standardization of nanoMIPs as synthetic alternatives to conventional antibodies. NanoMIP binding affinities of 34.7 pM ± 2 pM (EIS) and 3.06 pM (SPR) were obtained, with selectivity factors of 130:1 and 1000:1 (target : non-target), respectively. In contrast, the corresponding polyclonal antibody for haemoglobin (pAb) demonstrated contrasting affinities of 51.9 pM ± 0.74 pM (EIS) and 48.7 nM (SPR) and with substantially lower selectivity ratios of 14:1 and 10:1. These results indicate that whereas the two sensor techniques are ideal for nanoMIP characterisation, further harmonisation is required for antibody binding characterisation. We demonstrate that the developed nanoMIPs not only match but can surpass traditional animal-derived antibodies in both affinity and molecular discrimination. Overall, these findings highlight nanoMIPs to be a robust and reproducible alternative to antibodies, offering superior selectivity and comparable affinity for next-generation bioanalytical and diagnostic applications.