Disposable and cost-effective label-free electrochemical immunosensor for prolactin based on bismuth sulfide nanorods with polypyrrole
Graphical abstract
Introduction
Electrochemical immunosensors are emerging as important bioanalytical sensing tools for the detection of different kinds of biomarkers for numerous disorders and because of their significant role in the development of point-of-care devices. These immunosensors exhibit several advantages, including low cost, short assay time, portable screening, miniaturization, and sensitivity, because of their highly specific antibody-antigen recognition and electrochemical transduction ability. Of note, the electrochemical sensing properties of an immunosensor relies on the well-constructed immobilization of bio-recognizing elements on the surface of the electrode as it enhances the transfer of electrons between the transducer and electroactive species [1]. Thus, the electrochemical immunosensor method was applied for the detection of the peptide hormone prolactin (PRL). PRL is a naturally occurring single-chain polypeptide hormone with 199 residues containing three disulfide bonds and has a molecular weight of approximately 23 kDa [2]. PRL regulates the metabolism, pancreatic development, and immune system. Significantly, PRL stimulates milk secretion in mammals, including humans, and plays a key role in the production of progesterone (P4) [3], [4]. The normal levels of PRL in the serum of pregnant women, non-pregnant women, and are 10–209, 2–29, and 2–18 ng mL−1, respectively [5]. However, high serum levels of PRL (>20 ng/mL) indicate hyperprolactinemia and hormonal changes, inducing important side effects, such as galactorrhea, infertility, hypogonadism, and menstrual disturbances, in women [6]. Therefore, the quantification of PRL has received significant attention for the early treatment of PRL-related diseases [7], [8], [9], [10]. To date, various immunoassay methods have been developed including (electro) chemiluminescence, fluorescence, enzyme-linked immunosorbent (ELISA), and electrochemical immunoassay [11], [12], [13], [14]. However, electrochemical immunoassays have more advantages than other techniques. Several electrochemical immunoassays have been established using different electrocatalysts for the detection of PRL. For instance, streptavidin-functionalized magnetic particles have been used for the voltammetric determination of PRL [4], and polymers incorporated nanogold particles [5], and multi-walled carbon nanotubes (MWCNT) [1] have been employed for immunosensing PRL. Interestingly, the conducting polymer poly (ethylene-dioxythiophene) (PEDOT) with AuNPs covered carbon nanotube screen-printed carbon electrodes have been applied for the simultaneous determination of PRL and human growth hormone [15]. Li et al. have reported a sandwich-type biosensor with carbonaceous nanomaterials and antibody-coated gold nanoparticles for PRL [16]. Sun et al. have fabricated a poly (pyrrolepropionic acid)/MWCNT composite modified electrode [17], and Beitollahi et al. have reported amperometric immunosensing of PRL constructed with nano-Au monolayer and ionic liquid carbon paste electrode [3].
In recent decades, metal chalcogenides have been extensively applied for the detection of various biomarkers. Importantly, bismuth has been widely applied for the fabrication of different kinds of nanomaterials because it is an inexpensive, non-toxic, and diamagnetic heavy metal. Recently, bismuth-containing materials have attracted considerable attention for biomedical, chemical, electronic, and optical applications because of their high stability, safety, large surface area, great versatility in terms of size, shape, and porosity, and cost-effective synthesis processes [18]. Among these materials, Bi2S3 is an excellent semiconducting material owing to its unique physicochemical properties, quantum confinement effect, and direct bandgap of 1.3–1.7 eV. Furthermore, Bi2S3 has been extensively applied in various fields including X-ray computed tomography imaging [19], gas sensors [20], lithium-ion batteries [21], thermoelectric cooling methods based on the Peltier effect [22], [23], photodiode arrays, and photovoltaic converters [22], and other fields [24]. Moreover, it has been broadly applied in biomedical applications because bismuth is biologically non-reactive and less toxic than other metals such as silver [25]. In addition, different types of Bi2S3 structures, including nanosheets [26], nanotubes [27], and nanorods [28], [29], [30], and nanoflower-like structured Bi2S3@MoS2 [31] have been used in biosensor applications. The abovementioned nanoflower-like structured Bi2S3@MoS2 has been employed in photoelectrochemical (PEC) nanosensors for the biosensing of different analytes including amyloid-β proteins [30], sulfadimethoxine [32], [33], microRNA [31], methylated DNA [28], and avian leukosis virus (ALVs-J) [34]. Previously, an immunosandwich-type biosensor was reported based on Bi2S3 nanorods for the PEC detection of ALVs-J [34], and Zhu et al. have reported an impedimetric DNA biosensor based on polyaniline-capped Bi2S3 nanocomposites for biomolecule detection [35].
To date, polypyrrole (PPy) emerging as important conducting polymer material in the field of electrochemical sensors and biosensors because of its stability, conductivity, and biocompatibility under physiological pH conditions [36]. It has several important characteristics such as stability in air and aqueous media and dopant-mediated tunable physical properties [37]. PPy can be easily modified by the covalent attachment of redox groups and proteins [38]. In particular, during the fabrication of enzyme biosensors, PPy can offer a suitable environment for the entrapment of biomolecules [39]. There are two important factors in the development of biosensors based on PPy: (i) immobilization of biomolecules that can specifically bind to the analyte molecules and (ii) application of the suitable electrochemical technique for the detection of the analyte [40]. Earlier, the combination of PPy with different types of nanomaterials has been employed for biosensing [41]. Previously, B. Long et al. reported the amorphous Bi2S3–PPy hollow spheres were used for the twin applications of Li-sulfur (Li-S) batteries and sodium (Na)-ion batteries (SIBs) [42]. H. Liang et al. reported the Bi2S3–PPy yolk-shell composite was used for the energy storage (Na and Li storage) application [43]. M.E. Rincon reported the electropolymerization of Py and its co-deposited Bi2S3 nanoparticles on chemically deposited Bi2S3 substrates that were used for the photovoltaic application [44]. V. Hebbar et al. reported the combination of polyvinyl alcohol (PVA): PPy composite containing various weight percentages of Bi2S3 particles were prepared by in-situ oxidation subsequently solution casting technique. The Bi2S3 filled PVA: PPy materials were used for the conductivity study [45]. Conversely, Q. Zhu et al. reported the polyaniline (PA) with Bi2S3 nanorods composited were modified with ionic liquid carbon paste electrode, which is used as an impedimetric biosensor for the detection of DNA [35]. In comparison, the PPy has a lot of works for immunosensor applications [37], [38], [40] rather than PA because the PPy can offer a proper environment for the entrapment of biomolecules. The Bi2S3/PPy has been integrated advantages such as the low cost, ease of fabrication, high surface area, excellent electronic conductivity, and good biocompatibility make the electrode favorable for biomolecules immobilization and detection. Electrochemical experiments showed that the composite film had excellent electrocatalysis toward the electroactive molecules due to the synergistic effect of the highly conductive PPy and Bi2S3. Recently, Bi2S3 with their composite materials were used for the various biosensing applications. Zhou et al reported the Bi2S3@MoS2 nanoflower on cellulose fibers combined with octahedral CeO2 for dual-mode microfluidic paper-based MiRNA141 sensors [46]. The core–shell structured Au@Bi2S3 nanorods have been prepared and applied as DNA immobilization matrix for electrochemical biosensor fabrication. The addition of metal (Au) controlled the aggregation of Bi2S3 during the synthesis process and effectively promote electron transfer kinetics and electrochemiluminescence signal intensity [47]. Veeralingam et al. reported the 2D-Bi2S3 incorporated PVDF/PPy nanofibers as a versatile platform for ultrasensitive pressure, strain, and temperature sensing. Nanoengineering of 2D materials into polymer networks has been studied to enhance the electrocatalytic properties and the dimensionality of the composite. The synergistic properties of Bi2S3 nano sticks embedded in PVDF/PPy matrix exhibited the high-performance multifunctional sensor [48]. Recently, the AgPt@Pt core–shell nanoparticles loaded polypyrrole nanosheet modified electrode used as label-free immunosensor for the detection of prostate-specific antigen [49]. However, no one reported the electrochemical immunosensing of PRL using Bi2S3/PPy composite.
In this study, Bi2S3 nanorods were synthesized using a hydrothermal technique. To improve the conducting capability, PPy was prepared by the oxidative chemical polymerization method. The combination of Bi2S3/PPy composite was prepared through the facile sonochemical approach. The Bi2S3/PPy composite was modified with MPA by physical adsorption between the MPA (–COOH) and PPy (–NH) to obtain a strong surface, and the results showed that the negatively charged MPA could be adsorbed on the surface of Bi2S3/PPy modified electrode and remained their hybridization activity. Consequently, EDC/NHS enabled the –COOH group activation in MPA, which is firmly attached with the anti-PRL molecules. The anti-PRL molecule's non-covalent interaction with the antigen PRL and form the antibody-antigen immunocomplex. Due to this interaction, protein molecules form an insulating layer on the electrode surface that enhances the resistance of the electrode. Therefore, the peak current density of the electrode will decrease after the complex formation. The reported immunosensor has various advantages including low cost, simple preparation, and good biocompatibility.
Section snippets
Materials
Bismuth nitrate pentahydrate (Bi(NO3)3·5H2O) was obtained from Bay Carbon/USA. Pyrrole (Py, C4H5N), sodium sulfide hydrate (Na2S·xH2O), iron chloride (FeCl3) (97%), ethanol, and acetone were purchased from Sigma/USA. Monosodium dihydrogen phosphate (NaH2PO4), potassium chloride (KCl), and potassium dihydrogen phosphate (KH2PO4) were obtained from JT Baker/USA. A recombinant prolactin antibody and prolactin human recombinant were procured from R&D/USA, and N-hydroxysulfosuccinimide sodium salt
Characterization of Bi2S3/PPy
XRD was used to investigate the crystalline behavior and electronic states of the Bi2S3/PPy composite. The XRD patterns of Bi2S3, PPy, and Bi2S3/PPy are shown in Fig. 2a. The XRD pattern of Bi2S3 shows high-intensity diffraction peaks at 15.74, 17.70, 22.43, 23.76, 24.95, 27.19, 28.62, 31.86, 32.99, 33.96, 35.56, 36.62, 39.12, 39.92, 42.70, 43.66, 45.66, 46.61, 49.23, 51.39, 52.74, 53.84, 59.03, 62.66, and 71.96° corresponding to the (0 2 0), (1 2 0), (2 2 0), (1 0 1), (1 3 0), (0 2 1), (2 1 1), (2 2 1), (3 0
Conclusion
We developed a novel label-free immunosensor using a Bi2S3/PPy-modified SPE for the first time to determine PRL. The base material Bi2S3/PPy of the sensor has a high surface area, high catalytic activity, strong electron-transport properties, and good conductivity. Bi2S3/PPy was simply modified with MPA to introduce COOH groups, and EDC/NHS was used to activate the terminal COOH groups on the electrode surface. The construction of the immunosensor was performed under the following optimal
CRediT authorship contribution statement
Rajalakshmi Sakthivel: Writing – original draft, Writing – review & editing, Methodology, Formal analysis, Data curation, Visualization. Lu-Yin Lin: Writing – review & editing, Methodology, Funding acquisition. Tzung-Han Lee: Investigation, Formal analysis, Data curation, Validation. Xinke Liu: Writing – review & editing, Conceptualization, Methodology, Project administration, Funding acquisition. Jr-Hau He: Methodology, Funding acquisition, Writing – review & editing. Ren-Jei Chung:
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.
Acknowledgement
The authors are grateful for the financial supports of this research by the Ministry of Science and Technology of Taiwan (MOST 106-2221-E-027-034; MOST 109-2222-E-027-004), the National Taipei University of Technology – Shenzhen University Joint Research Program (NTUT-SZU-108-05 (2019005); NTUT-SZU-109-02 (2020009); NTUT-SZU-110-09 (2021009)); in part from the Guangdong Province Key Research and Development Plan (2020B010174003); and the International Distinguished Visiting Professor support
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These authors contribute equally to this paper.