Application of green synthesized WO3-poly glutamic acid nanobiocomposite for early stage biosensing of breast cancer using electrochemical approach

Biopolymer films have drawn growing demand for their application in the point of care domain owing to their biocompatibility, eco-friendly, and eligibility for in vivo analyses. However, their poor conductivity restricts their sensitivity in diagnostics. For high-quality electrochemical biosensor monitoring, two vital factors to be greatly paid attention are the effective merge of amplification modifiers with transducing surface and the superior linking across the recognition interface. Here, we introduce an enzyme-free electrochemical biosensor based on electrosynthesized biocompatible WO3/poly glutamic acid nano-biocomposites to address the hardships specific to the analysis of circulating proteins clinical samples. In addition to its green synthesis route, the poor tendency of both components of the prepared nano-biocomposite to amine groups makes it excellent working in untreated biological samples with high contents of proteins. Several electrochemical and morphological investigations (SEM, EDX, and dot mapping) were fulfilled to gain a reliable and trustful standpoint of the framework. By using this nanobiosensor, the concentration of HER-2 was detectable as low as 1 fg mL−1 with a wide linear response between 1 ng mL−1 and 1 fg mL−1. Meanwhile, the protocol depicted ideal specificity, stability, and reproducibility for the detection of HER-2 protein in untreated serum samples of breast cancer patients.

www.nature.com/scientificreports/ (Ag/AgCl). All the experimental performances including preparations, depositions, and measurements, proceeded in ambient conditions. To homogenize the solutions, an ultrasonic bath (Transsonic, model 420) was employed. The pH of solutions was measured by a pH meter (Corning, model 120). A magnetic stirrer model Heidolph was applied for the blending of solutions. The scanning electron microscope (SEM), Energy-dispersive X-ray spectroscopy (EDX), and dot blotting imaging experiments were implemented on Tescan, model MIRA3.
Electrode preparation steps. Before preparation, the glassy carbon electrode (GCE) was polished physically and electrochemically using alumina powder and PBS solution (pH = 7.4) followed by washing with ultrapure water. Afterward, a precursor solution was manufactured by dissolving 1 g Na 2 WO 4 powder and 0.11 g glutamic acid powder into the 10 mL PBS (pH 7.4). After sonication for 30 min, the pH of the solution was adjusted to 7.4. The WO 3 /p-Glu nanocomposite was then electrochemically synthesized using a three-electrode system at ambient temperature. The potential of the working electrode was scanned between − 1 and 2.5 V (0.1 V s −1 ) vs. reference electrode. As a result, H + ions required for electrografting of WO 3 were produced by applying high positive potentials (up to 2.5 V) resulting in a desirable high current of about 0.001 A. As described by previous studies, p-Glu and WO 3 nanostructures were generated around 2 V 34,46 and − 0.6 V 47,48 , respectively. After electrodeposition, a pre-prepared solution of EDC and Ab (18 µg mL −1 ) (1:1 v/v) was mixed with an NHS solution (2:1 v/v) and rest for 30 min. This activates the -COOH groups of Abs. In the next, 10 µL of EDC/ NHS-Ab (18 µg mL) was incubated onto the modified electrode for 120 min (at 4 °C). After washing in 10 mM PBS solution (pH = 7.4), a droplet of 10 µL solution of HER-2 protein (in different concentrations) was incubated on the electrode for 3 h (at room temperature). This led to the formation of GCE-WO 3 /p-Glu-Ab-HER-2 on the electrode. The prepared electrode was carried out into an electrochemical cell in which the electrochemical measurements were proceeded using K 4 [Fe (CN) 6 ] solution as a redox agent. Both DPV and CV voltammograms were obtained in the potential range of − 0.1 to 0.5 V (0.1 V s −1 ). EIS experiments have been implemented for characterizing the sensor fabrication process. Nyquist plots consisted of two regions include a semicircle and a linear part that appeared at high frequencies low frequencies respectively. The diameter of the semicircle region (R et ) reflects the charge transfer resistance. With creasing the diameter, the resistance was increased. Figure 1 represented the DPVs, CVs, and EIS results of the consecutive preparation stages of the proposed platform. As can be seen, after deposition of the PGA/WO 3 platform, the DPV and CV current peaks were increased while the R et was decreased. This means the increased conductivity of the electrodeposited platform. As illustrated, after incubation of EDC/NHS, antibody, and target protein, the current peaks of DPV and CV were decreased and the R et was increased. This indicated that all EDC/NHS, antibody and target protein decrease the conductivity of the electrode surface. The equivalent circuit data corresponding to the Nyquist plots for electrode preparation steps were prepared and inserted in Table S1. Also, the equivalent circuit was prepared and presented in Fig. S1.
Informed consent was obtained from all the participants included in the study.
Ethics approval and consent to participate. All patients were asked to complete the informed consent.
All procedures of this study were approved by the Local Ethics Committee of Tabriz University of Medical Sciences (IR.TBZMED.VCR.REC.1400.150). All procedures were done under the declaration of Helsinki.

Results and discussion
Co-electrodeposition of WO 3 /glutamic acid. Several important tips should be considered for the cyclic voltammetry (CV) behavior of the electrochemical solutions. To this purpose, the CVs of Glu, Na 2 WO 4 , Glu/NaWO 4, and water were obtained and compared to each other. The exact exploration of the CVs can help to interpret the events on the electrode surface and prove the deposition route of the proposed nanocomposites. Glutamic acid was electropolymerized into PGA in positive potentials during which, an oxidation process occurred resulting in a cation radical (GA +°) . In the following, the formed cation radical initiates a sequence of addition reactions to the nucleophilic centers (-COO − ) of glutamic acid molecules leading to the formation of α-L-PGA 33 . The overall reaction is represented as bellow: On the other hand, according to the pieces of literature, WO 3 can be converted to its hydrate form (H x WO 3 ) and sub-stoichiometric (WO 3-y ) species in different potentials. Also, the previous studies proved that conversion of WO 3 into H x WO 3 and WO 3-y occurs on about − 0.1 and − 0.5 V vs. Ag/AgCl respectively [49][50][51][52] . The reduced forms of WO 3 provide better conductivity 52 .
(1)  57 . This mechanism proceeds as follows: Aspirating with such a mechanism, phosphate ions can be oxidized electrochemically into peroxodiphosphate in high anodic potentials (about 2 V) 58,59 . The mechanism of this route is ascribed as below: Peroxodiphosphate ions are oxidative anions that can oxidize reductive molecules. The required H 2 O 2 is produced from the following equation 60 . This reaction is accelerated by the pre-produced peroxodiphosphate anions.
Aspired by these mentioned reactions, we prepared four solutions of PBS, WO 3 /PBS, Glu/PBS, and WO 3 / Glu/PBS and applied different potential ranges on the GCE dipped into the solutions. The behavior of the solutions to the applied potential ranges discovered many interesting signs of WO 3 and p-Glu electrodeposition  www.nature.com/scientificreports/ PBS solution. These two mentioned peaks are absent in the PBS solution. In total, by using a broad potential range (− 1 to 2.5 V), both PGA and WO 3 species were deposited onto the electrode at the same time. There is an important point in this case that is, the produced reduced forms during the negative cycle can turn into their oxidized forms during the next anodic cycle. There are two tips in this view. First, these consecutive reduction-oxidation processes produce a unity film instead of a layer-by-layer film which could increase the stability of the film. Second, the oxidized products were reduced again in the next cathodic cycle. Because the concentration of oxidized forms (WO 3 and PGA + ) increased from one step to the next step, the thickness of the deposited film is not constant and increases after each step. The CVs of 10 consecutive electrodeposition cycles were illustrated in Fig. S7 A-C. As can be seen, the deposition currents were increased from one step to the next one, which represented the more electrodeposited amount of the nano-biocomposite. Also, as represented, the increase rate was smoothened from one step to the next step. This means that the effect of the number of deposition cycles on film growth is limited by the number of cycles.
Characterization. Electrochemical characterization. The effect of each modification step was investigated through CV, electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV), techniques (Fig. 2). Also, the effect of the swept potential range was studied considering the charge transferring ability alterations. All the electrochemical measurements were obtained in a PBS solution (pH = 7.4) of 5 mM With creasing the diameter, the resistance was increased. The electron transferring capability of the electrode surface and diffusion rate of redox agents were screened from the semicircle part and linear portion of EIS plots. These characteristics were individually obtained from peak heights of DPV curves. Following the modification of the electrode with WO 3 /p-Glu, the charge transferring resistance was dramatically changed. To study the effect of p-Glu and WO 3 on conductivity, the electrode was separately modified with p-Glu and WO 3 . According to the results, both WO 3 and p-Glu boosted the charge transferring ability of the surface and we noted that the effect of WO 3 was more than that of the p-Glu. As expected, the co-electrodeposition of WO 3 /p-Glu showed a slight increase in conductivity compared to the p-Glu. After incubation of EDC/ NHS-Ab, the resistance was enhanced as a result of the steric hindrance. This can be interpreted as a successful attachment protocol of Ab onto the electrode surface. To probe the effect of the potential range, the same protocol was adopted for two potential ranges (0-2.5 V and -1 to 2.5 V). The EIS results (Fig. 3B) represented that the resistance was enhanced when potential swept in the range of 0-2.5 V in comparison to the -1 to 2.5 V range. In this range, two moieties are formed including WO 3 nanostructures and p-Glu. But there is no formation of reduced forms of WO 3 which are form in − 0.1 and − 0.7 V vs Ag/AgCl. Because the reduced forms of WO 3 represent better conductivity than WO 3 , the reduction of current density can have correlated to the lack of reduced forms production. In other words, in this potential range, in the obtained WO 3 /p-Glu nanocomposite, the ratio of p-Glu is higher than WO 3 . We noted that resistance is lower than the p-Glu-GCE. This may be due to the co-participation of the tungsten along with the growth of p-Glu on the electrode surface. The associated CVs readouts were recorded for each notified step which was in line with EIS results (Fig. 3A). As a proof of principle, the best results were obtained for the -1 to 2.5 V potential sweep range (Fig. 3C,D).

Morphology and roughness characterization.
To prove the electro-formation of p-Glu and WO 3 , morphology, lattice structure, and size distribution of the WO 3 nanoparticles, scanning electron microscopy (SEM) imaging was employed. Also, the elemental analysis of the modified electrode surface was studied using Energy Dispersive X-ray spectroscopy (EDX). Figure 4 illustrated the surface modification quality of the electrode by WO 3 /p-Glu (− 1 to 2.5 V) nanoarchitectures. The SEM results presented a uniform, porous and high-quality electrodeposited nanocomposite on the electrode. For further confirmation, the dot mapping analysis was performed from the surface of the WO 3 /p-Glu modified electrode. As expected and shown in Fig. 4G-I, the W, O, and C atoms are uniformly distributed on the modified electrode surface with relatively close distance to each other. These relatively close distances between the W-O, W-C, and C-O correspond to the successful synthesis of WO 3 /p-Glu nanobiocomposite. The EDX results represented the appropriate electrodeposition of the WO 3 /p-Glu nanocomposites. The distribution quality of the electrodeposited nanocomposite was evidence by dot mapping photos. As shown in Fig. S2A and S2B, a satisfactory and homogeneously distribution manner was obtained.
Optimization of the electrosynthesis step conditions. The number of cycles affects the electrochemical performance of the biosensor from two aspects via the thickness of the electrodeposited layers. First, the WO 3 nanoparticles which cause the conductivity to be significantly boosted, and second, p-Glu which exerted a decreasing effect on charge transferring ability. On the other hand, the amount of p-Glu on the electrode plays a vital role in the amount of Abs which success to be covalently immobilized on the platform. In this regard, the different number of cycles (2, 5, 8, 12, 15, and 20) at the same scan rate (0.1 V s −1 ) and the same potential range (− 1 to 2.5 V), were investigated. The results illustrated in Fig. S3, as can be seen, the best results were obtained for 8 cycles. Further increase of cycle numbers has no obvious change on the signal outputs. By increasing the cycle number to 20, the electrochemical signals were decreased. This can be correlated to the alteration of the WO 3 /p-Glu ratio to be decreased.  Fig. 5, the signal readouts were declined by increasing HER-2 concentration. The peak heights presented good linearity with the logarithm of the concentration of HER-2 protein) (1 ng mL −1 to 1 fg mL −1 ). The limit of detection (LOD) was gained to be 1 fg/mL. Compared to the formerly reported electrochemical 17,61,62 for HER-2 determination, the proposed nanoimmunoassay possesses lower LOD and consequently better sensitivity. This high performance can be ascribed to the electrocatalytic activity and high conductivity of WO 3 nanostructures.
To evaluate the reproducibility, the relative standard deviation (RSD%) was measured for 1 fg/mL of HER-2 protein using three different electrodes. The obtained RSDs indicated good reproducibility for both concentrations (3.42%). The results were presented in Fig. S4.
The signal stability of the prepared immunosensor was assessed via 10 consecutive DPV measurements. The obtained RSD for 10 consecutive DPV readouts represented good signal stability of about 1%. This can be obtained from two important features of the designed platform: (I) the rich functional groups of p-Glu and its strong binding to the Ab molecules; (II) highly ordered and stable electrodeposited WO 3 /p-Glu nanocomposite with high durability and strength. The obtained DPV voltammograms and correlated histograms are shown in Fig. S5.
To assess the specificity of the developed electrochemical immunosensor several possible interferences (carcinoma embryonic antigen (CEA), bovine serum albumin (BSA)) and the mixture of them in real samples were examined in a 100-fold concentration of HER-2 (Fig. S6). Weak electrochemical readouts were perceived in the presence of annoying species only. Whiles, an intense change in electrochemical responses were observed by adding HER-2. These observations proved the desirable selectivity of the proposed biosensor.
To give a lucid view of the proposed strategy, it was compared with several previously reported methods. The summary of the comparison was represented in Table 1. According to the evidence, the introduced framework possessed a desirable figure of merits in comparison with the other protocols.
There are some tips about the above-mentioned methodologies. Of course, we noted that each protocol has its advantages and restrictions and this deal is not to underestimate or minimize their qualities. www.nature.com/scientificreports/ In the development of electrochemical biosensors using nanomaterials, two important tips should be notified. First, the biocompatibility and abundance of the ingredients (include nanoarchitectures) of the transducing framework are of great significance from the economical-environmental viewpoint. Some (nano) materials are high-performance but poor in biocompatibility like cadmium-based materials [68][69][70] . Some nanomaterials are of high biocompatibility but highly expensive and low abundance like platinum, gold, and silver nanomaterials One of the most important drawbacks of the Au nanomaterials is their high costs which prevent them to be employed in commercial and even, sometimes, experimental extent 71,72 . Some other (nano)materials represent desirable biocompatibility but with (very) low conductivity and then efficiency than the high conductive materials (such as Pt, Au, Ag) like several biopolymers (such as glutamic acid). On the other hand, some nanomaterials are of great interest for their conductivity and biocompatibility nut poor in functionability like WO 3 nanostructures. The second tip is the preparation of the applied (nano)materials. Electrochemical synthesis strategies are promising ways of synthesis in which there is no need for extra reduction or other reagents which are most hazardous. Just a proper potential or current are implemented with no environmental issues. Considering these facts, the combination of biopolymers with WO 3 nanostructures could be a response to the ask of new platforms with low costs and high biocompatibility. Regarding the presented experiments and the results which illustrated the proposed platform of p-Glu/WO 3 nano-biocomposite as a high-performance platform for cancer screening the suggested www.nature.com/scientificreports/ strategy could address the present problems like biocompatible and final costs. As represented in Table 1, electrochemical synthesis routes possess a cost-effective methodology compared to the other procedures. In addition, the low volume consumed reagents is of great importance from commercial and environmental standpoints. A quick view of Table 1 can be a schematic comparison of different synthesis methods from several aspects.

Clinical samples.
To evaluate the applicability of the suggested strategy, it was implemented for untreated normal and patient samples. The normal sample was spiked with HER-2 protein (1 ng/mL) before analysis for matrix effect exploration (n = 2). The recovery results represented a matrix effect of 125% which is good considering the measurement in untreated serum matrix. Also, we tried an untreated HER-2 positive serum sample which indicated the competency of the proposed framework for biological and clinical point of care utilities. All the results were depicted in Fig. 6.

Conclusions
WO 3 /p-Glu nanocomposite was synthesized through a biocompatible electrochemical process. To this end, the nanocomposite was synthesized by only dipping an electrode into the PBS solution containing Na 4 WO 4 and glutamic acid. This process was followed by an appropriate potential sweep on the electrode. Compared to the previous methods developed for electrodeposition of WO 3 nanostructures, which employed H 2 O 2 as an extra co-reactant, no H 2 O 2 was added to the precursor solution, but H 2 O 2 was produced in situ using the water oxidation-splitting route. WO 3 nanoparticles and p-Glu were prepared simultaneously in the sense that