A Novel Strategy for Selective Thyroid Hormone Determination Based on an Electrochemical Biosensor with Graphene Nanocomposite

Abstract

This article presents a novel and selective electrochemical bioassay with antibody and laccase for the determination of free thyroid hormone (free triiodothyronine, fT3). The biosensor was based on a glassy carbon electrode modified with a Fe₃O₄@graphene nanocomposite with semiconducting properties, an antibody (anti-PDIA3) with high affinity for fT3, and laccase, which was responsible for catalyzing the redox reaction of fT3. The electrode modification procedure was investigated using a cyclic voltammetry technique, based on the response of the peak current after modifications. All characteristic working parameters of the developed biosensor were analyzed using differential pulse voltammetry. Obtained experimental results showed that the biosensor revealed a sensitive response to fT3 in a concentration range of 10-200 µM, a detection limit equal to 27 nM, and a limit of quantification equal to 45.9 nM. Additionally, the constructed biosensor was selective towards fT3, even in the presence of interference substances: ascorbic acid, tyrosine, and levothyroxine, and was applied for the analysis of fT3 in synthetic serum samples with excellent recovery results. The designed biosensor also exhibited good stability and can find application in future medical diagnostics.

Keywords: antibodies; laccase; nanomaterials; thyroid hormones; voltammetry.

Figure 1

Chemical structure of (a) fT3 and (b) T4 hormones.

Figure 2

(a) Schematic representation of the biosensor’s working principle; (b) schematic representation of the main steps of the preparation of the biosensor for the determination of fT3; (c) proposed, specified mechanism of the electrode modification with the antibody. GCE—glassy carbon electrode; Lac—laccase.

Figure 2

(a) Schematic representation of the biosensor’s working principle; (b) schematic representation of the main steps of the preparation of the biosensor for the determination of fT3; (c) proposed, specified mechanism of the electrode modification with the antibody. GCE—glassy carbon electrode; Lac—laccase.

Figure 3

Representative SEM image of the Fe₃O₄@graphene nanocomposite.

Figure 4

(a) Representative CV scans of GCE/Fe₃O₄@graphene/Ab/Lac (green line) in the presence of 200 μM fT3, and GCE/Fe₃O₄@graphene/Ab/Lac in PBS buffer (black line); inset: CV scan of a bare GCE in the presence of fT3; (b) GCE/Fe₃O₄@graphene/Ab/Lac in 200 μM fT3; all measurements were performed under applied potential in the range of −0.2–1.3 V, scan rate 50 mV s⁻¹, vs. Ag/AgCl.

Figure 5

Scheme of the proposed mechanism of the redox enzyme-catalyzed fT3 oxidation.

Figure 6

(a) Representative DPV scans for different concentrations of fT3 in the range of 10–200 μM vs. Ag/AgCl; (b) linear relationship between current and fT3 concentration (10–200 μM).

Figure 7

Influence of interfering substances (50 μM) on fT3 detection.

Figure 8

Representative CV scans of GCE/Fe₃O₄@graphene/Ab/Lac in the presence of 200 μM fT3 after 21 days. Potential range: −0.2–1.3 V, scan rate: 50 mV s⁻¹, vs. Ag/AgCl electrode, 40 cycles.