Antibody-Ferrocene Conjugates as a Platform for Electro-Chemical Detection of Low-Density Lipoprotein

Abstract

Low-density lipoprotein (LDL) is a cardiac biomarker identified in the pathology of cardiovascular disease (CVD). Typically, the level of LDL is calculated using the Friedewald relationship based on measured values of total cholesterol, high-density lipoproteins (HDL), and triglycerides. Unfortunately, this approach leads to some errors in calculation. Therefore, direct methods that can be used for fast and accurate detection of LDL are needed. The purpose of this study was to develop an electrochemical platform for the detection of LDL based on an antibody-ferrocene conjugate. An anti-apolipoprotein B-100 antibody labeled with ferrocene was covalently immobilized on the layer of 4-aminothiophenol (4-ATP) on the surface of gold electrodes. Upon interaction between LDL and the antibody-ferrocene conjugate, a decrease in the ferrocene redox signal registered by square wave voltammetry was observed, which depends linearly on the concentration from 0.01 ng/mL to 1.0 ng/mL. The obtained limit of detection was equal to 0.53 ng/mL. Moreover, the satisfied selectivity toward human serum albumin (HSA), HDL, and malondialdehyde-modified low-density lipoprotein (MDA-LDL) was observed. In addition, the acceptable recovery rates of LDL in human serum samples indicate the possible application of immunosensors presented in clinical diagnostics.

Keywords: electrochemical detection; ferrocene–antibody conjugates; immunosensor; low-density lipoprotein (LDL).

Scheme 1

Schematic illustration of the conjugation procedure between the AbM-anti-apoB and ferrocene carboxylic N-hydroxysuccinimide ester (ferrocene-NHS).

Scheme 2

Schematic representation of the electrochemical immunosensor preparation steps: (a) bare Au, (b) Au/4-ATP, (c) Au/4-ATP/AbM-anti-apoB-Fc, (d) Au/4ATP/AbM-anti-apoB-Fc/BSA.

Figure 1

Cyclic voltammograms curves registered for (a) bare gold electrode surface and (b) immunosensor at the final step of modification: Au/4-ATP/AbM-anti-ApoB-Fc/BSA. Supporting electrolyte: 0.1 M sodium phosphate buffer, 0.15 M Na₂SO₄, pH 7.0. Scan rate: 0.1 V/s.

Figure 2

(A) Cyclic voltammograms of the immunosensor Au/4-ATP/AbM-anti-ApoB-Fc/BSA recorded at various scan rates: 0.05, 0.1, 0.20, 0.3, 0.4, 0.5 V/s. Supporting electrolyte: 0.1 M sodium phosphate buffer + 0.15 M Na₂SO₄, pH 7.0. (B) Plots of the oxidation and reduction peak current of ferrocene attached to antibodies vs. scan rates.

Figure 3

Optimization of the incubation time (15, 30, 45, 60 min) of LDL solution of 0.5 mg/mL on AbM-anti-apoB-Fc-modified gold electrodes registered using square wave voltammograms in buffer (pH = 7.0).

Figure 4

(A) Square wave voltammograms recorded for immunosensor upon contact with LDL standard solution with different concentrations (0, 0.01, 0.05, 0.1, 0.5, 1.0 ng/mL). The measuring parameters of SWV: step potential = 5 mV; square wave frequency = 10 Hz; amplitude = 50 mV. (B) The relationship between relative intensity of ferrocene redox current vs. log concentration of LDL (ΔI = I_n – I, where I_n is the peak current value registered at particular “n” concentrations of LDL and I is the peak current value measured in buffer free of LDL (n = 5)). Measuring conditions: 0.1 M sodium phosphate buffer, 0.15 M Na₂SO₄, pH 7.0.

Figure 5

Results of (A) reproducibility, (B) repeatability, (C) selectivity of immunosensor with 0.01 ng/mL LDL with different interferents at a concentration of 0.5 ng/mL (high-density lipoprotein (HDL), human serum albumin (HSA), and malondialdehyde-modified low-density lipoprotein (MDA-LDL)), and (D) stability studies registered within 14 days, obtained for the platform based on antibody–ferrocene conjugates.