A low fouling electrochemical biosensor based on the zwitterionic polypeptide doped conducting polymer PEDOT for breast cancer marker BRCA1 detection
Introduction
Biosensors have attracted much attention since their invention for applications in clinical disease diagnosis, food detection and environmental supervision [1], [2], [3]. Electrochemical biosensors are low cost and simple to operate and have become important in supporting high levels of assay sensitivity [4], [5]. Real clinical applications of electrochemical biosensors can be challenging because nonspecific adsorption of proteins in serum samples or other complex media can produce large background signal and limit accuracy and precision. An effective way to resolve this issue is to integrate recognition into the sensing surface that is highly antifouling [6]. Previous studies have demonstrated that various low-fouling functional materials are highly hydrophilic and/or zwitterionic, such as poly(ethylene glycol) (PEG), oligo(ethylene glycol) (OEG), and non-ionic or zwitterionic polymers [6], [7], [8], [9]. PEG and OEG motifs have been the most widely utilized antifouling polymers but they are subject to oxidation in the presence of transition-metal ions and oxygen, both of which are present in most biochemically relevant conditions [10]. Zwitterionic polymers, such as poly(carboxybetaine) and poly(sulfobetaine) have good antifouling performance even in real media due to their strong hydration capacity via electrostatic interactions [11], [12]. Jiang et al. reported that phosphorylcholine self-assembled monolayers have strong resistance to protein adsorption. Both experimental and molecular simulation techniques have proved that having balanced charge and minimized dipole are two key factors for their antifouling behavior [13]. However, the time-consuming and complex synthesis procedure seriously restrict their application.
An ideal antifouling interface should have the following characteristics: high antifouling property, good biocompatibility, convenient electrode integration and being easily modifiable with biomolecules [14], [15], [16]. Polypeptides with specific sequences are new group of antifouling materials that have been attracting attention recently [17]. Polypeptides are composed of natural amino acids, which have naturally superior biocompatibility and exist as zwitterionic molecules in biological systems [18]. Recent studies have shown that a series of polypeptides have antifouling property. For example, Jiang’s group has reported that the inclusion of an additional linker of four residues in length (-PPPPC) of a rigid, hydrophobic nature, is a better choice for forming peptide self-assembled monolayers with well-ordered structures. The experimental results proved that EKEKEKE-PPPPC-Am forms a secondary structure and shows excellent antifouling ability for fibrinogen and lysozyme [19]. Su’s group synthesized a zwitterionic polypeptide (CRERERE) that demonstrated relatively high antifouling property in single protein and real media, compared to an amphiphilic, non-ionic polypeptide (CYSYSYS) [17]. The molecular structures of the amphiphilic peptide and the zwitterionic peptide are shown in Fig. S1 of the supporting materials. Following the general design principles for producing antifouling materials with excellent hydrophilicity and electrical neutrality [20], we designed a polypeptide sequence (CPPPPNQNQNQNQ) that has been used to construct a sensing surface to explore the protein-resistance ability in this work.
It should be noted that polypeptides, which are not conductive, can increase the impedance of the electrode and decrease its sensitivity. It is expected that the combination of polypeptides with conducting polymers, may contribute to a solution to this problem. Recently, conducting polymers, like polyaniline, polypyrrole, and polythiophene, have emerged as promising materials in a variety of applications such as biosensors, antistatic coatings, supercapacitors, chemical sensors, electronic devices, light emitting diodes, and batteries [21], [22], [23], [24]. Amongst these polymers, poly(3,4-ethylenedioxythiophene) (PEDOT) is considered one of the most stable and successful commercially available conducting polymers. PEDOT has drawn considerable attention for the development of chemo/bio-sensors because of its high conductivity, great environmental stability, biocompatibility, low toxicity, and well-defined chemical structure [25], [26], [27]. For instance, PEDOT composite modified electrodes have been successfully used for the detection of heavy metals such as Hg, Cd, Pb, Cu and metalloid As. As an adsorbent in the preconcentration process, PEDOT-based materials can synergistically enhance the conductivity and stability of composites that play an important role in the adsorption and enrichment of heavy metals [28], [29]. PEDOT-based materials have been widely utilized for the sensitive and selective determination of carcinoembryonic antigens, alpha fetoproteins, the influenza A virus, C-reactive proteins, DNA sequences and glucose due to the electrostatic properties, biocompatibility and easy immobilization of biomolecule [14], [30], [31], [32]. However, due to high background signal and delayed responses produced by nonspecific protein adsorption, performing assays in complex media using PEDOT sensors has been challenging.
The breast cancer susceptibility gene (BRCA1) is an important tumour marker for breast cancer, ovarian cancer, pancreatic cancer and colon cancer [33]. Medical science has now proven that BRCA1 plays an important role in several cellular pathways that support genomic stability, such as DNA mending, protein ubiquitination, chromatin remodeling, and apoptosis [34]. Therefore, the reliable detection of BRCA1 at low concentrations is important for multiple research fields including diagnostics and forensics. In this work, an electrochemical DNA biosensor was developed for quantitative analysis of BRCA1 in complex samples. First, a novel conducting polymer nanocomposite of PEDOT doped with a polypeptide sequence was prepared through a simple electrodeposition method. The nanocomposite integrated the suitable compatibility, excellent conductivity, and stability of PEDOT with the strong antifouling ability and biocompatibility of the polypeptide, which results in an ideal substrate for a biosensor. The polypeptide used in this study can act as a bifunctional material that not only inhibits the nonspecific adsorption of proteins but also allows for the usage of its carboxylic to fix DNA probes (terminated with amino group), as shown in Scheme 1. The proposed PEDOT/PEP-based biosensor showed good analytical performance (ultrahigh sensitivity, low detection limit and antifouling ability) and proved to be capable of successfully detecting BRCA1 even in 1% (V/V) human serum samples.
Section snippets
Regents
3,4-Ethylenedioxythiophene (EDOT), LiClO4, methylene blue (MB) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimde (NHS) were purchased from Aladdin Reagents (Shanghai, China). The polypeptides used in this work (CPPPPNQNQNQNQ, purity > 99%) were both synthesized and purified by Bankpeptide biological technology Co. Ltd. (Hefei, China). The structures of the polypeptide sequences are shown in Fig. S1. Bovine serum albumin (BSA), adenosine triphosphate (ATP),
Characteristics of the modified electrodes.
Morphology and microstructure have a significant impact on the performance of conducting polymers [39]. The surface morphologies and microstructures of the PEDOT/LiClO4 and PEDOT/PEP films were characterized by SEM with an acceleration voltage of 5.0 kV. PEDOT/LiClO4 and PEDOT/PEP nanocomposites have very distinct morphologies. Fig. 1A and B show that the PEDOT/LiClO4 composite is relatively flat with only slight roughness. Interestingly the PEDOT/PEP nanocomposite has a highly porous network
Conclusion
Novel PEDOT/PEP nanocomposites with 3D porous morphology, good biocompatible and excellent hydrophilicity were successfully prepared by electrochemical polymerization. By using the prepared PEDOT/PEP nanocomposites, a sensitive and low fouling DNA biosensor was constructed through the immobilization of capture DNA. Owing to the good conductivity, excellent hydrophilicity and antifouling property of the PEDOT/PEP composite, the immobilized capture DNA retains its high binding affinity, and the
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This research was supported by the National Natural Science Foundation of China (21705088), Shandong Key Laboratory of Biochemical Analysis (SKLBA2008), Shandong Province Agricultural Application Technology Innovation Project (SD2019NJ001-2), and the Qingdao Agricultural University High-level Talent Project (663/1117025).
Note: All human serum (or plasma) experiments were conducted according to the ethical guidelines of Qingdao Agricultural University and the “3R” principle. All experiments and
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These authors contributed equally to this work.