Three-dimensional gold nanoparticles/prussian blue-poly(3,4-ethylenedioxythiophene) nanocomposite as novel redox matrix for label-free electrochemical immunoassay of carcinoembryonic antigen

Abstract

We present a novel label-free electrochemical immunosensor for detecting carcinoembryonic antigen (CEA) based on three-dimensional (3D) gold nanoparticles/prussian blue-poly(3,4-ethylenedioxythiophene) (AuNPs/PB-PEDOT) nanocomposite, which was firstly synthesized by a simple redox reaction between the PB precursors and EDOT in an aqueous solution, followed by the electrochemical reduction of HAuCl₄. The 3D nanocomposite not only possessed large surface area and favorable microenvironment, but also exhibited remarkable conductivity, stability and excellent biocompatibility. In addition, PB showed excellent redox properties. Then AuNPs/PB-PEDOT was used as both electron mediators and 3D matrices in the fabrication of immunosensor. FT-IR spectra were employed to confirm the formation of PB, PEDOT and PB-PEDOT. Significantly, the AuNPs/PB-PEDOT exhibited a 3D and hierarchically porous nanostructure, while the PB-PEDOT showed core-shell structure. The AuNPs/PB-PEDOT modified immunosensor showed good linearity with the concentration of CEA ranging from 0.05 to 40 ng mL⁻¹, and the detection limit was 0.01 ng mL⁻¹. Moreover, the prepared electrode displayed good selectivity, high stability and good repeatability, and showed great potential for application in real sample analysis.

Introduction

Carcinoembryonic antigen (CEA) as a clinical tumor marker, which could express in lung cancer, ovarian carcinoma, breast cancer, cystadenocarcinoma and others [1], [2]. The level of CEA monitoring could help detect, diagnose, and manage some types of cancer [3], [4]. Therefore, it is necessary to establish a highly sensitive, specific and accurate measurement for the determination of CEA. Recently, electrochemical immunosensors have attracted great interest in many areas, such as clinical diagnostics, environmental control and biochemical studies [5], [6], due to their low-cost, fast analytical time, excellent selectivity and high sensitivity [7], [8]. Among all types of the electrochemical immunosensors, the label-free electrochemical immunosensors exhibit several unique advantages, for instance, they could be directly used to monitor the binding process of antibody-antigen reaction and avoid disturbances from conjugated markers or handle with hazardous materials [9]. The primary challenge of label-free electrochemical immunosensors is to fabricate a suitable bridge which possesses excellent biocompatibility, remarkable conductivity and stability, and large active surface area between the protein and the electronic transducer.

Among the electrochemical tags, Prussian blue (PB), as a well-known “artificial peroxidase”, has attracted tremendous attention due to the excellent redox-active, electrochemical properties, electrochromic, photo-physical and magnetic properties [10], [11]. However, the PB usually shows poor conductivity [12] and low stability [13], which severely restrains the performance of the constructed immunosensor. To overcome these shortages, many conducting materials, such as conducting polymers (CPs) [14], [15] were frequently used for the PB-conducting polymers nanocomposite preparation to improve the conductivity of PB. Meanwhile, the stability of the PB could be increased by the introduction of conducting polymers supports due to the physical interaction between them [16], [17]. Generally, PB can be synthesized through two different chemical routes [18], [19]: Fe³⁺ + [Fe^II(CN)₆]⁴⁻ or Fe²⁺ + [Fe^III(CN)₆]³⁻. Unfortunately, the reaction between the two agents was too fast, thereby making it difficult to control the product, which resulted in its poor repeatability for its application in sensing [18], [20]. To address this problem, recently, Zou et al. synthesized polypyrrole-prussian blue-graphene oxide nanocomposite via spontaneous redox reactions for high-performance supercapacitors [21]. Li et al. fabricated prussian blue-polyaniline-multiwalled carbon nanotubes nanocomposite for in vivo determination of glucose in rat brains [14]. In these redox reaction systems, pyrrole or aniline was used as weak reducing agent and Fe³⁺ was slowly reduced to Fe²⁺, resulting in the formation of PB nanoparticles in the presence of [Fe^III(CN)₆]³⁻. These slow redox reactions provide a simple way for the controllable preparation of PB-conducting polymers.

Recently, the synthesis of CPs with various nanostructures also has been extensively studied owing to the fact that nanostructural CPs usually provide different or even superior electric or electrochemical properties compared to traditional bulk CPs [22], [23], [24]. Among numerous CPs, Poly(3,4-ethylenedioxythiophene) (PEDOT) is attractive material for its good biocompatibility, conductive stability, relatively low production cost and good environmental stability [25], [26]. Until now, various 0D, 1D and 2D PEDOT nanomaterials have been studied in electrochemical immunosensor. For example, Jun et al. fabricated 0D PEDOT nanodots by using electropolymerization method for enhanced cell capturing [27]. Xie et al. reported a 1D functionalized PEDOT nanowire device by using a simple electric-filed-assisted method for label free protein detection [28]. Guo et al. reported a 2D PEDOT nanocomposite via a simple one-step reaction for the detection of E. coli O157:H7 [29]. Although various 0D, 1D, 2D PEDOT nanomaterials have been applied for electrochemical immunosensor, to the best of our knowledge, only few studies about 3D nanostructures PEDOT materials were applied for electrochemical immunosensor. Compared with 0D, 1D and 2D PEDOT, 3D nanostructural PEDOT shows high specific surface areas and fast electron transport kinetics. In this background, we attempt to fabricate an electrochemical immunosensor based on 3D structural PB-PEDOT nanocomposite.

Compared with physical adsorption and covalent coupling methods, the use of AuNPs to immobilize the antibody shows many advantages. For example, AuNPs can firmly adsorb antibody because of their large specific surface area, good biocompatibility and high surface free energy [30]. Moreover, the functional groups such as amino group (−NH₂), mercapto group (−SH) on the antibody have a high affinity for Au [31]. The method also can avoid the complex cross-linking process.

In this work, we designed a new label-free electrochemical immunosensor for the detection of CEA based on AuNPs decorated PB-PEDOT nanocomposite with 3D hierarchically porous structure and excellent redox-active property. A simple and green redox route was demonstrated for the syntheses of PB-PEDOT nanocomposite which was obtained by mixing Fe³⁺, [Fe(CN)₆]³⁻ and EDOT aqueous solution without applying any additional reductants or surfactants. During the preparation process, EDOT acts as the reductant, and Fe³⁺ act as the oxidant. The 3D structural PB-PEDOT nanocomposite not only possesses remarkable conductivity and stability, but also has excellent biocompatibility. Moreover, the fabricated nanocomposite with the porous hierarchical structure can provide large surface area, favorable microenvironment and more efficient reactivity sites. Then, AuNPs were loaded on PB-PEDOT nanocomposite by a simple electrodeposition method, which were used to immobilize the CEA antibody. The fabricated immunosensor showed high sensitivity, selectivity, and reproducibility and could be used for the detection of CEA in real samples with satisfactory results.