Antifouling zwitterionic peptide hydrogel based electrochemical biosensor for reliable detection of prostate specific antigen in human serum

Highlights

• The designed zwitterionic peptide hydrogel exhibited good antifouling property.

• The peptide hydrogel containing functional groups was suitable for biosensor construction.

• The peptide hydrogel based electrochemical biosensor was capable of detecting PSA in real samples.

Abstract

An electrochemical biosensor based on the antifouling zwitterionic peptide hydrogel (CFEFKFC) and the poly(3,4-ethylenedioxythiophene) (PEDOT) was fabricated to accurately detect prostate specific antigen (PSA) in complex human serum. The electrode was modified with the conducting polymer PEDOT and gold nanoparticles (AuNPs) in sequence through electrodeposition, and then the designed zwitterionic peptide hydrogel prepared through self-assembly was immobilized onto the modified electrode surface via the Au–S bond. The zwitterionic peptide hydrogel with cysteine terminal is easy for immobilization onto the gold surface, and it is also suitable for the immobilization of biomolecules such as PSA antibody in this work, through the formation of covalent amide bonds. The peptide hydrogel possessed excellent antifouling property, and it was able to effectively prevent the adsorption of nonspecific proteins, cells and other biomolecules. The developed antifouling biosensor showed a linear response range from 0.1 ng mL⁻¹ to 100 ng mL⁻¹, with a low limit of detection down to 5.6 pg mL⁻¹. These results encourage the wide use of zwitterionic peptide hydrogels as antifouling materials in various sensing and bio-sensing devices.

Introduction

In the early diagnosis, prostate specific antigen (PSA), as a cancer biomarker, can be used for primary screening for prostate cancer [1,2]. The concentration of PSA is below 4.0 ng mL⁻¹ in serum of normal human, and people are at increased risk of being diagnosed with prostate cancer when the concentration of PSA exceeds the threshold [3]. At present, the commonly utilized methods for the specific determination of PSA include surface plasmon resonance technique [4], electrochemical detection [5], electrochemiluminescence analysis [6], amperometric immunoassay [7] and so on. Among them, the electrochemical analysis has drawn substantial attention owing to its sensitivity, convenience, and low cost [[8], [9], [10]]. Nevertheless, the absorption of an enormous number of nonspecific proteins and cells on the interface of electrochemical sensors poses a serious biofouling problem when those biosensors performed in real complex samples [11]. Therefore, it is necessary to explore biosensors with antifouling capability for target detection in complex biological environments.

To date, various materials have been applied to prevent nonspecific adsorption of proteins and other biomolecules onto the sensing surfaces of electrochemical devices [[12], [13], [14]]. Typically, poly (ethylene glycol) (PEG) and its derivatives are extensively adopted for the construction of antifouling surfaces [15]. However, PEG is vulnerable to oxidative degradation, which affect its long-term practical application [16]. Zwitterionic polymers can combine with water molecules to form a tightly bound hydration layer, which possess resistance ability of protein adsorption [17]. Furthermore, zwitterionic polymers simultaneously contain the same account of cationic and negative groups and can be electrically neutral [18], and the charges can attract water molecules to enhance their hydration [19]. For example, Liu et al. have proved that the zwitterionic polymers formed brushes exhibited stronger hydration than the PEG brushes [20].

Zwitterionic peptides, composed of amino acid residues with positive and negative charges, are similar to zwitterionic polymers and can be neutral in charge [21]. Additionally, zwitterionic peptides have excellent biocompatibility compared with other zwitterionic polymers. Consequently, peptides with different composition and chain length consist of natural and synthetic amino acids can form diverse sequences of peptides with various antifouling capabilities [22,23]. For example, Chen's group has proved that EK amino acid residues presented by alternating or mixed charges were effective for the construction of almost nonfouling peptides [24]. Ye et al. have reported that the p-EK peptide self-assembled to thiolated hyaluronic acid (HA)-modified gold substrate, which exhibited excellent antifouling performance [25].

Hydrogels have been widely investigated in biosensor [26], wound healing [27], tissue engineering [28], drug delivery [29], antimicrobial materials [30] and implantable medical devices [31] due to their advantages including favorable hydrophilicity and biocompatibility. Peptide hydrogels prepared by self-assembly are formed into stable structures through various interactions, such as the hydrogen bond, electrostatic interaction, π-π stacking, and hydrophobic and hydrophilic interactions [32,33]. In addition, peptide hydrogel with a high water content can facilitate to form a hydration layer that effectively avoids the adhesion of nonspecific biomolecules [34]. Therefore, they are potentially suitable for the construction of electrochemical biosensors with antifouling abilities [35,36]. Nevertheless, little related work has been done to explore the application of zwitterionic peptide hydrogel in antifouling sensors.

Herein, the zwitterionic peptide hydrogel with a sequence of CFEFKFC was successfully prepared and adopted for the development of low fouling biosensors. Peptide hydrogels and the conducting polymer PEDOT were modified onto the electrode surface to fabricate the antifouling electrochemical biosensor. As shown in Scheme 1, the electrode was sequentially modified with PEDOT and gold nanoparticles (AuNPs) by electrodeposition, and then the zwitterionic peptide hydrogel with terminal cysteine (C) thiol groups were attached to the AuNPs via the Au–S bonds. After the further immobilization of anti-PSA antibodies, the antifouling biosensor for PSA detection in complex biological environments was developed.