Phytic acid functionalized antifouling conducting polymer hydrogel for electrochemical detection of microRNA

Highlights

• Conducting polymer hydrogel PANI/PA was prepared by one-step copolymerization way.

• The PANI/PA hydrogel demonstrated antifouling property.

• The low fouling biosensor has been constructed for MicroRNA24.

• The biosensor has shown excellent sensing performances with wide linear range and low sensing limit.

Abstract

A rapid, sensitive and low-fouling sensor for application to detect clinical biomarkers directly in biological media is highly desirable. Herein, we report a versatile strategy to prepare polyaniline (PANI) based conducting polymer hydrogel through the assembly of PANI and phytic acid (PA) by dynamic boronate bond. PANI/PA, a conductive hydrogel with high specific area and multiple pore structures have demonstrated excellent antifouling ability and electrochemical property. The electrochemical biosensors for microRNA can be developed by the immobilization of DNA probes onto PANI/PA interface (microRNA24 is used as a model case), and redox currents of PANI were utilized as the sensing signals to assay DNA/RNA hybridization reaction. Owing to the typical characteristics of PANI/PA hydrogel, the biosensor has shown excellent sensing performances with wide linear range (1.0 fM-1.0 pM), low sensing limit (0.34 fM) and efficient ability to detect microRNA mismatches. Given the facile processability, excellent stability and good antifouling property of the PANI/PA hydrogel, the proposed hydrogel-based biosensor may find broad applications in clinical diagnostics and biomedical devices.

Introduction

Disease diagnostic methods are required to be rapid, sensitive, specific and economical [1,2]. MicroRNAs (miRNAs), approximately 19–23 nucleotides, are a large group of small RNAs related to tumour transformation [3], stem cell regeneration [4] and viral spread [5]. MiRNAs are also emerging biomarkers that can show the occurrence of disease and predict the prognosis of the disease. For the assay of miRNAs, their properties, such as short sequence, high similarity in size and low concentration, cause great difficulty using conventional strategies including Northern blotting [6,7] and polymerase chain reaction (PCR) [8]. Lots of new technologies, such as colorimetric assay [9,10], fluorescence technology [11,12] and electrochemical assay [[13], [14], [15]], have been established to improve the sensitivity and applicability of the measurement. Among these ways, electrochemical sensors have received more and more attentions due to their merits (low test cost, simple instrument operation, fast test data and high sensitivity) [16].

Due to the serious nonspecific adsorption and biological penetration of complex biological media, the practical clinical application of biosensors still remain enormous challenges [2]. Therefore, depressing nonspecific protein adsorption on the sensor interfaces is very important for the application of biosensors in complex samples, and many attempts have been made to solve the problem. It is well known that surface hydrophilization is crucial to the improvement of interfacial antifouling ability [17]. The self-assembled antifouling monolayer [18] was utilized to modify the surface of the polymer layer [19] and the polymer brush [20] to inhibit the adhesion of nonspecific proteins. For example, a label-free electrochemical DNA sensor based on self-assembled antifouling peptide layer was constructed for the direct assay of breast cancer marker BRCA1 in human serum samples [21]. Polyethylene glycol (PEG), a hydrophilic polymer grafted onto the surface of polyaniline (PANI), has been used to determine DNA in human serum [22]. However these antifouling interfaces have many preparation steps and the process is relatively complicated. Therefore, it is of great significance to develop antifouling materials that can deposit antifouling coatings directly through simple, scalable and economical synthesis methods.

Conductive polymer hydrogels, owing to their excellent biocompatibility, high electrical conductivity, three-dimensional nanostructures and large specific surface area, have been employed as bases for the preparation of electrochemical sensors [23]. Hydrogels are polymeric networks that have a high level of hydration and three-dimensional (3D) microstructures bearing similarities to natural tissues [24]. Hydrogels based on conducting polymers (e.g., polythiophene, polyaniline, and polypyrrole) represent a novel polymeric material platform that synergizes the advantages of both organic conductors and hydrogels [25,26]. Among numerous conducting polymer hydrogels, PANI-based hydrogels have received extensive concerns because they own lots of promising properties including excellent mechanical properties, label free, high electronic conductivity, reversibility in redox and good biocompatibility [[27], [28], [29], [30]]. In addition, the amino group of PANI (-NH₂) can be easily modified with biomacromolecules, making it very desirable for biosensor construction. Moreover, the antifouling material is inspired by hydrophilicity to construct the interface of the biosensor. Phytic acid (PA) is an ecologically friendly, renewable and abundant biomass, which is easily gained from grains [[31], [32], [33]]. The six phosphate groups supply all kinds of feasible crosslinking sites that may “suture” many conducting polymers sheets, forming the three-dimensional assembly body [34]. In previous reports, PA was used to construct a super-hydrophilic interface. For instance, Zhao’s group reported the use of PA to construct a super-hydrophilic coating in a one-step assembly process [35]. Herein, a conductive polymer hydrogel with good antifouling property was synthesized by one-step electrochemical method with 3-aminobenzene boric acid (ABA) as crosslinking agent, PA doped PANI.

In this work, we aim to construct a novel PANI/PA conducting polymer hydrogel by one-step electrochemical copolymerization way. Boronic acid groups were covalently inserted into PANI chains through aggregating of 3-aminophenylboronic acid (ABA) and aniline [30,36]. Boronic acid as the functional group has been chosen to crosslink PA and PANI at molecular level to form PANI/PA hydrogel [30]. The gelation mechanism of PANI/PA hydrogel is illustrated in Fig. 1. The resulting PANI/PA conducting polymer hydrogel, combining strong hydrophilicity and biocompatibility of PA with the good electrochemical property of PANI, supply a good base for the development of antifouling electrochemical sensors. MiRNA24 was used to be a model case, which plays important roles in cancer cell proliferation and differentiation. As shown in Scheme 1, the DNA probes were attached onto the surface of the synthesized PANI/PA hydrogel by covalent bond modification to construct an electrochemical biosensor with both antifouling property and good selectivity for miRNA24.