Electrochemical measurement of antibody-antigen recognition biophysics: Thermodynamics and kinetics of human chorionic gonadotropin (hCG) binding to redox-tagged antibodies

• Published in: Journal of electroanalytical chemistry (Lausanne, Switzerland) Switzerland), 2018-06, Vol.819, p.533-541
• Main Authors: Partington, Lee I., Atkin, Stephen L., Kilpatrick, Eric S., Morris, Samuel H., Piper, Mark, Lawrence, Nathan S., Wadhawan, Jay D.
• Format: Article
• Language: English
• Subjects: Adsorption; Antibodies; Antigens; Binding sites; Biophysics; Chemoreception biophysics; Electrodes; Electroimmunoassay; Frumkin isotherm; Hydrophobicity; Immunoglobulins; Kinetics; Molecular wire; Organic chemistry; Partially blocked electrodes; Rate constants; Surface chemistry; Thermodynamics; Urine
• ISSN: 1572-6657; 1873-2569
• DOI: 10.1016/j.jelechem.2018.02.062

The thermodynamics and kinetics of antigen binding under diffusive conditions to an electrode surface modified with ferrocene-tagged antibodies is studied within this work, and realised experimentally for the case of human chorionic gonadotropin (hGC) as the antigen with monoclonal anti-hCG antibodies immobilised on an electrode surface via a molecular wire platform. The formation of the antigen-antibody complex is monitored through the blocking of the ferrocene voltammetry, thereby enabling the fractional coverage of the electrode binding sites to be unravelled as a function of time. It is found that, at low antigen concentrations, a Frumkin adsorption isotherm fits the data, with repulsive interactions between bound antigens playing a significant rôle, with an affinity constant that is an order of magnitude larger than in the case of an untagged antibody, suggesting that the chemical hydrophobicity of the redox tag may encourage stronger binding. Comparison of the experimental temporal data with relevant diffusion-adsorption models under activation control allows for the extraction of the kinetic parameters; at zero coverage, the rate constants for adsorption and desorption are, respectively, larger and smaller than the untagged antibody. The kinetic study enables the confirmation that this type of platform may be utilised for rapid (15 min) and quantitative electroimmunoassay. This is validated through proof-of-concept analytical measurements, yielding a limit of detection around 25 mIU mL−1 (corresponding to 2.7 ng mL−1) – a value used clinically for urine hCG measurements corresponding to around four weeks of gestational age. [Display omitted] •Highly sensitive route for electroimmunoassay via recognition of the extent of antigen binding.•Experimental data fit a Frumkin adsorption model at low antigen concentration.•Electrochemical determination of rate and equilibrium constants.•Repulsive lateral interactions on bound antigen demonstrated.