Development of an electrochemical biosensor for the detection of mammary gland carcinoma using molybdenum enhanced poly taurine nano-biofilms confirmed pathological findings

Abstract

Background

Developing high-performance sensing frameworks for diagnosing anaplastic changes is the subject of debate. The lack of on-time diagnosis in patients with suspicious cancers can affect the prognosis and survival rate. As a correlate, the emergence of de novo strategies for developing transducing frameworks has an inevitable role in advanced biosensing. The combination of green chemistry procedures with eco-friendly and biocompatible materials is of high desirability in this context. The synthesis of new biocompatible and cost-effective nanomaterials to meet the emerging needs of rising demands appeals to new synthetic methodologies.

Methods

Here, we applied the electrochemical synthesis method to the fabrication of biocompatible and subtly governed Molybdenum trioxide/poly taurine nano-bio films to monitor human epidermal growth factor receptor-2 (HER-2) in sera from breast cancer patients. Morphological and elemental assessments were performed using a scanning electron microscope, energy-dispersive X-Ray spectroscopy, and dot mapping analyses. In addition, HER-2 immunohistochemistry (IHC) staining was performed on tissue samples, and data were compared to the values obtained by Molybdenum trioxide/poly taurine nano-bio films.

Results

We also noted our platform is eligible for feasible, rapid, and specific determination of HER-2 factor in human samples. The method had a lower limit of quantification of 0.000001 ng/mL and a linear dynamic range between 0.1 ng/mL and 0.000001 ng/mL. IHC imaging showed that the degree of anaplastic changes in breast samples (intensity of HER-2 factor) was closely associated with the intensity of signals obtained by our developed immunosensor.

Conclusions

According to the obtained desirable coordination with pathological studies, the designed biosensor has excellent capability to use as a reliable diagnostic tool in clinical laboratories.

Graphical Abstract

Introduction

Breast cancer (BC) is one of the most devastating malignancies and the second-leading mortality cause in females worldwide. Traditional methods for BC screening include magnetic resonance imaging (MRI), ultrasound, mammography, or positron emission tomography (PET scans). Unfortunately, these approaches possess several drawbacks, such as high cost, low sensitivity, invasiveness, and difficult operation with stressful conditions for patients (Kim et al. 2020; Loyez et al. 2020). Several BC biomarkers, such as nucleotides, cancer cells, proteins, and some small molecules, have been explored to support a more precise and convenient diagnosis than conventional methods. Of these biomarkers, the human epidermal growth factor receptor-2 (HER-2), a specific oncoprotein, is changed during the development and progression of BC (Nasrollahpour et al. 2021a). It was suggested that the overexpression of HER-2 in breast cancer cells stimulates proliferation rate and tumor expansion (Chen et al. 2019; Sharma et al. 2018). Currently, the biomarker assisted diagnosis of HER-2⁺ BC is based on conventional techniques like cytological analyses using fine-needle aspiration (FNA), fluorescence in situ hybridization (FISH), gene expression monitoring, immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA) procedures. Among the above-mentioned approaches, FNA and direct sampling are time-consuming and operationally invasive and can result in iatrogenic tumor implantation or metastasis (Tabasi et al. 2017). Importantly, laboratory data have indicated that the level of HER-2 in the serum of BC patients ranged from 220 pM to 1.1 nM whereas these values reached 30 pM to 220 pM in healthy people, showing the much overlaps in the systemic levels of HER-2 under physiological and pathological conditions (Ranganathan et al. 2020; Chun et al. 2013; Shen et al. 2018). Consequently, there is an urgent need to develop more accurate and low-cost methodologies with the ability for rapid and sensitive recognition in BC patients.

Biosensors have been developed as key responses to circumvent the problems associated with HER-2 screening (Karimzadeh et al. 2020; Pourakbari et al. 2019). Electrochemical biosensors are one of the most employed, highly sensitive, and easily implemented tools for detecting target molecules in biological samples (Cesewski and Johnson 2020; Wang et al. 2017; Min et al. 2021; Khalilzadeh et al. 2019; Mansouri et al. 2020). One of the most appealing interests in electrochemical biosensing strategies is using nano-biomaterials to modify electrode surface structure (Chenaghlou et al. 2021; Isildak et al. 2020). The simultaneous application of nanomaterials in electrochemical sensors can yield excellent achievements (Zhang and Chen 2019; Wongkaew et al. 2018; Gupta et al. 2021). The synthesis protocol of nanomaterials is one of the limiting factors in their applicability, quality, final costs, and environmental viewpoints (Chen et al. 2018; Kolahalam et al. 2019). Thanksgiving to colloidal procedures, electrosynthesis of nanomaterials is easily performed by dipping the working electrode in the electrochemical cell containing the precursor solution. A load of the same nanostructures on working electrodes using electrochemical methods is thought to increase detection outcomes (Wang et al. 2020; Ansari et al. 2020; Zhang et al. 2020; Nasrollahpour et al. 2021b). In this case, there are no reducing or stabilizing agents in the growth solution, which were applied in colloidal methodologies. Instead, a specified potential or current is implemented across the reaction cell to form nanoparticles. Integrating the electrosynthesis methods with the fast-growing nanotechnology field can present a unique gift to progress the synthesis chemistry for many applications (Mozafari and Parsa 2020; Ulyankina et al. 2020; Fani et al. 2020; Ma et al. 2021).

Molybdenum trioxide (MoO₃) semiconductor is highly interesting among other semiconductors because of desirable biocompatibility, high electrical conductivity, big band gape, acceptable catalytic activity, tuning plasmon, large surface area, and lower final production costs (Zhu et al. 2017; Huang et al. 2018). These superiorities facilitate the application of MoO₃ in several domains, such as electro-catalysis (Yang et al. 2020; Afsharpour and Dini 2019), energy storage device development (Wu et al. 2017; Dwivedi et al. 2018), and fabrication of chemical sensors (Wei et al. 2020; Samdani et al. 2017; Xue et al. 2019; Pandey et al. 2018). To this end, MoO₃ nanomaterials have been used in different shapes, sizes, and functionalities in biosensing strategies. One of the most prevalent interests in such an investigation is merging Mo nanostructures with other nanomaterials like platinum (Wang et al. 2016), graphene (Achadu et al. 2020), and gold (Wei et al. 2020) nanomaterials. All these combinations are proposed to boost the performance of the Mo nanostructures and cover their application gaps like poor functionality and lower conductivity compared to the noble metal nanomaterials.

In this research, we used MoO₃/poly-Tau (MoO₃/p-Tau) nanofilms as a high-performance framework for the detection of HER-2 in serum samples of BC patients. P-Tau is a conductive biopolymer that can raise the sensitivity of the biosensing framework by increasing the conductivity, enhancing the specific surface area, and effectively binding the other (nano)materials onto the electrode. In addition, taurine is considered a functional group-rich compound for nitrogen and sulfur containing groups in the structure. Considering its high biocompatibility and desirable features, combining p-Tau with other (nano)materials can be a promising tool for designing high-performance biosensors. This research applied an electrosynthesis route to fabricate the nanofilms onto the electrode for breast cancer screening. To our knowledge, this is the first report of MoO₃/p-Tau nano-films application in the bioassaying field. The highly homogeneous distribution of Mo nanoparticles into the p-Tau films was monitored using an appropriate potential range.

Experimental

Material and instruments

HER-2 antibody (Ab) and HER-2 protein were obtained from Abcam. Na₂MoO₄ powder and taurine were purchased from Merck. N-ethyl-N′-(3-(dimethylamino) propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) solutions were prepared in deionized water (Sigma-Aldrich). Phosphate buffer solution (PBS, pH = 7.4) was prepared by dissolving 200 mg KCl, 1.44 g Na₂HPO₄, 8 g NaCl, 245 mg KH₂PO₄, in the deionized water at room temperature. Electrochemical measurements were done by using a Metrohm Autolab system and Nova software. The electrochemical system was composed of a three-electrode system, including a glassy carbon electrode (GCE) as a working electrode with 3 mm in diameter, a Pt wire as the counter electrode, and an Ag/AgCl as the reference electrode. All analyses were performed at room temperature. Before experiments, an ultrasonic bath (Model: 420; Transsonic) was applied to homogenize the solutions. The pH of solutions was controlled via a pH meter (Corning, model 120). Solutions were mixed using a magnetic stirrer,. The SEM, EDX, and dot mapping records were recorded using a Tescan instrument (Model: MIRA3).

Fabrication of the electrochemical immunosensor

Immunosensor was manufactured on a pre-cleaned 3 mm diameter GCE as the working electrode. The electrosynthesis process was performed in a three-electrode system through the cyclic voltammetry technique. All the potentials were applied versus Ag/AgCl electrode. At pH = 7, the electrodeposition was switched by dipping the working electrode inside the electrochemical cell containing an aqueous solution of 5 mM Na₂MoO₄ and 0.1 M taurine. Afterward, cyclic voltammetry was carried out in the range of − 1.5 to 2.3 V with a scan rate of 0.05 V per second for 5 cycles. The modified electrode was tagged as MoO₃/p-Tau/GCE. In the following, the modified electrodes were rinsed in deionized water. Then, a mixture of EDC/NHS/Ab (2:1:1 v/v) was prepared and after a homogenizing process, left to rest for 30 min. Antibody molecules attached to the surface via amid bound between carboxylic groups of the antibody and nitrogen groups of the poly taurine moiety of the electrodeposited film. Next, a 10 µL of the mixture was drop cast on the MoO₃/p-Tau/GCE and left for 90 min. Finally, a determined amount of HER-2 protein was incubated on the Ab-EDC-NHS/MoO₃-p-Tau/GCE for 90 min. The prepared electrode was washed with PBS solution (pH = 7.4) and analyzed with an AutoLab instrument. For comparison, other electrodes, including p-Tau/GCE and MoO₃/GCE, were fabricated using a similar approach.

HER-2 IHC staining

In this study, we performed HER-2 IHC staining to find the correlation of anaplastic changes in breast tissue with HER-2 levels in the serum of BC patients. For this purpose, 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. In BC patients, histopathological examination is touted as the gold standard for consolidated diagnosis and determination of BC anaplastic changes. The tissue fragments sampled during the surgical procedure and referred to the pathological lab were used in this study without any interference with the treatment protocol of patients. Here, we monitored the HRE-2 levels in BC samples. For IHC staining, samples were fixed in 10% formalin solution, 5 µm thick slides were prepared. Samples were incubated with 3% hydrogen peroxide solution for 20–30 min to neutralize endogenous peroxidase activity. To retrieve antigens, slides were kept in citrate buffer (pH = 6.0) for 15 min. Thereafter, an antibody targeting human HER-2 was used according to the manufacturer's instructions. After PBS washes, a secondary HRP-conjugated antibody was used. Diaminobenzidine (DAB) was used as a chromogen agent. Finally, the slides were visualized and imaged using Olympus microscopy.

Results and discussion

Clarification of the electrosynthesis methodology

To evaluate the efficiency of the protocol, the developed electrochemical biosensors were monitored using two different approaches. The cyclic voltammetry (CV) technique was used in the range of − 1.5 to 2.3 V to prepare the MoO₃/p-Tau nanofilms. According to the literature, p-Tau was electropolymerizable in positive potentials about 1.8 V vs. SCE. The electropolymerization of p-Tau was initiated by an oxidation process of a taurine molecule on its NH₂ end in positive potentials. The oxidized taurine molecule linked to a sulfur atom in another taurine molecule released an H₂O molecule to form an N-S bund between the two taurine molecules. Equation 1 depicts the whole mechanism of p-Tau formation (Hasanzadeh et al. 2014). MoO₃ was electrodeposited in the negative ranges (Yao et al. 2012; Zhao et al. 2020). In this regard, we used a potential range between − 1.5 and 2.3 V (vs. Ag/AgCl) to cover both negative and positive potentials. This feature led to the simultaneous formation of homogenous MoO₃ and p-Tau as highly ordered nanofilms. According to our data (Fig. 1A, B), two important potential points were notified on the voltammogram following the electrodeposition of p-Tau. The first point correlates with the oxidation of p-Tau and enhanced electropolymerization process at 1.8 V. The peak at -0.55 V would be related to the reduction of the positive p-Tau byproducts on the electrode surface. We found that both these peaks were also detectable in the CV after co-electrodeposition of MoO₃/p-Tau. Data showed three distinct points in the CV voltammograms of MoO₃/p-Tau. First, in both CV voltammograms, there are two pairs of potentials (represented as a₁, a_2, and c₁, c₂). As can be seen, the two pairs of potentials related to the MoO₃/p-Tau surface exhibited a sharp and reversible response compared to the electrode coated with MoO₃ alone, indicating a more rapid electron transferring ability of MoO₃/p-Tau nanofilm versus MoO₃ alone. The peak at about − 0.5 V on MoO₃/p-Tau nanofilms' growth (c₄) can be correlated with the reduction of p-Tau species which were produced during the anodic potentials. The cathodic peak (c₃) in the MoO₃ voltammogram is associated with the reduction of produced O₂ during the anodic cycle (Genies et al. 1998). This peak was not detected in MoO₃/p-Tau and p-Tau voltammograms. One reason would be that the reduction of O₂ by oxidized p-Tau species disappeared from the relevant peak.

Fig. 1

Clarification of the electrosynthesis of frameworks (Taurine, MoO₃, and MoO₃/p-Tau). A The correlated CV voltammograms and B The magnified voltammograms between − 1.5 to 1 V. All the represented arrows are associated with the formation of the nano-biomaterials onto the electrode

$${\mathrm{n NH}}_{2}-{\mathrm{CH}}_{2}-{CH}_{2}-{SO}_{3}H\stackrel{Applying potential (Cyclic Voltammetry)}{\to }{\left[NH-{CH}_{2}{-CH}_{2}{-SO}_{2}-NH{-CH}_{2}{-CH}_{2}-{SO}_{2}\right] }_{n}$$

(1)

Characterization of the co-electrosynthesized MoO₃/p-Tau nanofilms

To confirm the electrodeposition and electrocatalytic activity of the electrosynthesized nanofilms, electrochemical and morphological characterizations were conducted. The CV and electrochemical impedance spectroscopy (EIS) techniques were implemented to prove the effectiveness of each component on the electrochemical signal's intensity. The experiments proceeded in 5 mM K₄[Fe(CN)₆] (Additional file 1: Fig. S1A, B). Noteworthy, the peak currents were increased after electropolymerization of p-Tau in comparison with bare GCE. This can be due to the increased surface area and electro-conductivity of p-Tau film. According to the CV data, the signal further increased with the co-electrosynthesis of MoO₃/p-Tau. For further confirmation, the voltammograms of electrodes coated with Molybdenum alone were studied. Data supported the fact that the peak heights of the MoO₃/p-Tau modified electrode were more than that of the MoO₃, indicating the higher conductivity of the MoO₃/p-Tau electrode. It is inferred that the combination of MoO₃ and p-Tau have a synergic and/ or structural improvement effect of increasing current compared to when used alone. The stepwise preparations and relevant data are presented in Fig. 2A, B). After incubation of electrodes with the EDC/NHS/antibody mixture, the signal intensities declined, which is linked to the steric hindrance of the EDC/NHS/antibody combination. EDC/NHS couple was used to activate the carboxylic groups of Abs. Using this strategy, the incubation time of Ab is decreased dramatically. Along with these changes, a dramatic decrease in the intensity of signals was notable after incubating the HER-2 protein. These effects can be related to the existence of antigen–antibody interaction and the gained steric repulsion of HER-2 protein.

Fig. 2

Electrochemical characterization of the preparation steps of the proposed nanobiosensor. All the signals were obtained in K₄[Fe(CN)₆] (5 mM)

In order to confirm the results, morphological and semiquantitative studies were performed using SEM, EDX, and dot mapping analyses (Fig. 3 and Additional file 1: S2). SEM imaging revealed that MoO₃ nanoparticles were evenly distributed inside the p-Tau matrix with a narrow particle size (about 22 nm) distribution. As can be seen from the figures visibly, the surface-to-volume ratio is the best by MoO₃/p-Tau compared to the p-Tau and MoO₃ films. The distribution quality was also confirmed by data from dot-mapping plots (Additional file 1: Fig. S2), which indicated a high-density electrode deposition of the platform o the electrode. Also, to give a clearer vision of the deposition manner of the nanofilm, we employed elemental analysis results with MoO₃/p-Tau modified electrode using EDX. According to the results (Additional file 1: Table S1), the weight percent of elements reflect the deposition quality of the proposed platform onto the electrode.

Fig. 3

The SEM images of three different modified electrodes at different magnitudes of 200, 500 and 1000 nm. A–C The electrode modified with electrosynthesized MoO₃ nanoparticles. D–F) The electrode modified with electrografted p-Tau nano-scaffolds. G–I The electrode modified with electrosynthesized MoO₃/p-Taunanofilms

Optimization of experimental conditions

Several parameters, such as the number of deposition cycles and incubation time should be considered to boost the sensing efficiency. As a correlate, the number of deposition cycles was investigated. According to previous data, scan numbers critically impact the thickness and quality of the nanofilms. In this way, five different scan numbers, including 1, 3, 5, 8, and 10 were performed. The electrochemical readouts were increased with increasing cycle number (Additional file 1: Fig. S3A, B). This can be correlated to increasing the specific surface area as the nanofilm was grown further from 1 to 5 cycles. In cycles more than 5 cycles, the response intensity was decreased, resulting from much thickness of the nanofilms and/or decreased porosity. The incubation time of the antibody on the modified electrode surface was also optimized. Considering the time required for biorecognition and the promotion of covalent interaction on the modified electrode surface, the reduction of signal intensity is a result of the steric hindrance. By increasing the incubation time, the number of antibody molecules attached to the electrode surface is thought to be increased. As shown in Additional file 1: Fig. S4A, B), the signal intensity was decreased with an increase in the incubation time from 15 to 90 min. Of note, a higher amount of time (more than 90 min) did not alter the signal readouts, which can mean the saturation of the attachment sites by Ab molecules.

Analytical performance characteristics

Quantitative recognition of the target protein was tried out under the optimized conditions by incubating six different concentrations of HER-2 protein (0.1, 0.01, 0.001, 0.0001, 0.00001, and 0.000001 ng/mL) with the designed transducer. As illustrated in Fig. 4A, B), the electrochemical response was orderly decreased upon the increased HER-2 concentration. The data represents a good linear correlation between the signal intensity and related concentrations (r² = 99.78). We found a lower limit of quantification (LLOQ) at the range of 1 fg/mL. These data demonstrated the suitability of the developed immunosensor for the analysis of HER-2 protein at the femtogram levels. The specificity of the biosensor was evaluated. In this regard, we assessed the effect of three possible interferences (CEA, CA-15-3, and BSA proteins) on the electrochemical readouts (Additional file 1: Fig. S5A, B). The one-by-one interfering effects and also the cumulative effect of them were checked. Also, the 10 and 100 folds of interferences concentrations were applied in cumulative format for better comparison. Based on the recorded results, the mentioned proteins have little effect on the signals. Additionally, the prepared bioassay was successfully checked in normal serum samples by spiking the target HER-2 protein.

Fig. 4

A HER-2 bioassaying by application of DPV between 0.1–0.000001 ng/mL, B calibration curve of minus logarithm of HER-2 concentration (ng/mL) vs average current (µA) (n = 3)

To evaluate the effect of electrode type on the recorded responses, three different electrodes were modified by the same procedure, and the electrochemical response was gained for 0.01 pg/mL of HER-2. Again, signals displayed a practicable reproducibility using our protocol (Additional file 1: Fig. S6A, B). The stability of signals was also assessed by the fabrication of electrodes under the optimized conditions and by recording ten repetitive DPV signals exposed to 1 fg/mL HER-2. Data showed an RSD of 0.51% (Additional file 1: Fig. S7A, B) and Table 1).

Table 1 Comparison of the analytical features with other biosensors for HER-2 protein

The performance of biosensors can be compared together from several domains. First, the employed (nano) materials and implemented synthetic protocols should be attended. It is highly significant that the synthesis strategies and the materials in the framework's constructions be biocompatible, environmentally friendly, eco-friendly and easy to prepare. The second option is the analytical performance, such as sensitivity, selectivity, stability, and repeatability, which should have as high as possible. As shown in Table 1 the summarized details regarding other investigations can help us develop further analyses. There are some tips that can be considered from the current experiments. First, our synthesis methodology is an electrosynthesis procedure that correlates with low reagent consumption and high preparation rate, making the protocol more biocompatible and environmentally friendly. The latter point is that we used biocompatible materials to construct the sensing platform. MoO₃ is a cheap and biocompatible compound that makes a desirable combination with p-Tau biopolymer for biosensing purposes. The obtained lower limit of quantification (LLOQ) of the present study offers a more sensitive strategy for immunosensing of HER-2 in serum levels compared to other available approaches.

Real sample analysis

A calibration curve was plotted using the standard addition method to obtain a more reliable and accurate analysis. This way, six different concentrations of HER-2, including 0.1, 0.01, 0.001, 0.0001, 0.00001, and 0.000001 ng/mL, were prepared in the independent serum samples of a healthy individual (Fig. 5A, B). In comparison to Fig. 4, similar responses were recorded according to Fig. 5 but with decreased signals. This is correlated to the matrix effect of the real untreated samples. 6 (A and B) is illustrated the obtained signal readouts for four HER-2 positive volunteer patients.

Fig. 5

Biosensing of HER-2 via standard addition methodology of determined amount of the protein (0.1–0.000001 ng/mL) in untreated serum samples. A DPV curves and B the correlated histograms (n = 3)

Pathological studies

Histopathological examination revealed the existence of HER-2⁺ cells inside the breast tissues with malignancies. The intensity of HER-2 and the number of HER-2⁺ cells were different in different samples enrolled in this study. The uncontrolled increase of HER-2 correlates with aggressive histological remodeling and poor diagnosis in BC patients. We noted numerous infiltrating carcinoma cells into the mammary gland tissue, forming aggregates. These cells were positive to a membrane tyrosine kinase, namely HER-2, showing malignancy. However, the intensity and number of HER-2⁺ varied in samples of different BC patients. These data showed that in HER-2 positive BC, ductal cells could express large levels of this factor that varies between the samples. The pathological images are presented in Fig. 6C.

Fig. 6

Real sample analysis of HER-2 A DPV voltammograms of unprocessed serum sample analysis with developed bioassay, B Corresponded histograms and C correlated phatological images (samples 1, 2, 3 and 4) (n = 3)

Conclusions

This research successfully constructed a high-performance and ultrasensitive electrochemical immunoassay for BC analysis based on a biocompatible and environmentally friendly MoO₃/p-Tau nanofilm. The nanostructure was fabricated onto the electrode through the electrosynthesis approach as an environmentally and eco-friendly synthesis route. It is suggested that p-Tau can be employed for two purposes: I) increasing the specific surface area for attachment of MoO₃ and antibody molecules; II) providing a highly stable substrate to hold MoO₃ nanoparticles from releasing during the measurements. Besides, MoO₃ was utilized to enhance the conductivity, boosting the signal intensity. The designed nano-immunoassay displayed a desirable LLOQ of 1 fg/mL with a wide dynamic range of 0.1 ng/mL to 1 fg/mL. We implemented two calibration plots (in standard solutions and healthy blank serum) to give more insights and prove the proposed nanobiosensor’s reliability. Also, to establish the applicability of the proposed framework, it was tested for the analysis of HER-2 positive real samples. Interestingly, the obtained results were confirmed by pathological studies. The results indicated an excellent ability for the biosensor in diagnosing breast cancer.