Human serum albumin (HSA), HER2 antigen (human-HER2 mouse monoclonal), and HER2 antibody (anti-HER2-rabbit IgG) were purchased from Sigma (Sigma-Aldrich, United States). Bovine Serum Albumin (BSA), K₄Fe(CN)₆, K₃Fe(CN)₆, mercaptoacetic acid (MAA), mercaptoethanol, and aminobenzoic acid were obtained from Sigma-Aldrich. HAuCl₄ 4H₂O, methacrylic acid, 1,5-diaminonaphthalene, ammonium hydroxide (25 wt%), 4-pyridine carboxaldehyde, ferrous chloride tetrahydrate, and 4,4′-oxybisbenzoic acid (H₂oba) were purchased from Merck (Darmstadt, Germany).

Electrochemical studies were carried out at room temperature using an Autolab potentiostat–galvanostat model PGSTAT30 using the NOVA software (version 1.11). A conventional three-electrode system composed of the modified GCE/Fe₃O₄@ TMU-24-AuNPs/MCA/Ab₁ as the working electrode, a platinum wire as the counter electrode, and Ag/AgCl (saturated KCl) electrode as the reference electrode was used for all measurements. The Fourier transform infrared (FT-IR) was investigated in the transmission mode by Thermo Scientific Nicolet IR100 (Madison, WI, United States). A scanning electron microscope (SEM) (Philips instrument, Model XL 30) and energy-dispersive X-ray spectroscopy (EDX) was used to evaluate the modified surface electrode morphology.

Step-by-step assembly strategies were used to construct the magnetic framework composites (MFCs). Under stirring for 24 h, 0.3 g of Fe₃O₄ was added to 50 ml of ethanol solution containing 0.29 mM of MAA. The obtained Fe₃O₄@MAA was collected by magnetic separation and washed five times with DMF. Products were dispersed in 10 ml of 45 mM Zn(NO₃)₂.6H₂O DMF solution for 30 min, followed by dispersion in 10 ml of a 4-nbpy and H₂oba DMF solution (45 mM) for 30 min. Magnets were used to collect the resulting products between each step. Both of these steps were repeated 15 times. Fe₃O₄@TMU-24 is finally washed and dried under vacuum at 80°C.

The general approach for the synthesis of Pt: CdTe d-dots was performed by the previous procedure (Najafi et al., 2018; Safari et al., 2019). At first, sodium hydrogen telluride (NaHTe) was prepared by reducing Te powder with NaBH₄ in deionized water under stirring conditions and N₂ purging. After 3 h, the freshly NaHTe nanoparticle was prepared. In another flask, 0.3 g of CdCl₂.5H₂O and 0.2 mg of Na₂PtCl₄ were dissolved in 40 ml ultrapure water and after the addition of 200 µl of TGA under stirring conditions, its pH was adjusted to 10 by adding dropwise of NaOH solution (1 M) under N₂ purging. Both free oxygen solutions were mixed and transferred into a Teflon-lined stainless steel autoclave and heated in an oven at 120°C for 3 h. At the end of the reaction, the obtained TGA coated Pt: CdTe QDs precipitate was washed with ethanol several times to remove the excess contaminants.

To the Pt: CdTe QDs conjugated with anti-HER2, 1.0 ml of Pt: CdTe QDs was activated by adding EDC (500 μl of 5 mg ml⁻¹) and NHS (500 μl of 0.25 mg ml⁻¹). The mixture was rotated for 15 min at room temperature and then added with 500 μl of anti-HER2 (50 μg ml⁻¹) and oscillated at 4°C overnight. Finally, it was stored at 4°C.

Before modification, the GCE was polished successively with (1.0 and 0.05 μm) Al₂O₃ water slurry using a polishing cloth, and it was rinsed with doubly distilled water. Then, 1 mg Fe₃O₄@TMU-24 was dispersed in 10 ml DMSO containing 2.5% (V/V) Nafion with ultra-sonication for 1 h. Then, 4 µl of the suspension was dropped on the surface of the GCE and dried at room temperature to obtain GCE/Fe₃O₄@. Then, the AuNPs were electrodeposited onto the GCE/Fe₃O₄@ by CV scanning from −0.2–1.2 V in a solution of 0.5 M H₂SO₄ containing 0.6 M of HAuCl₄ at a scan rate of 50 mV s⁻¹ for 15 cycles. The constructed electrode was immersed in a 40 mM MAA solution for 5 h to form a self-assembled monolayer. Then, the prepared electrode (GCE/Fe₃O₄@-AuNPs/MAA) was rinsed carefully with double distilled water to remove physically adsorbed MAA and MAA carboxyl groups were activated on the electrode surface using EDC/NHS. Then, the electrode was then washed with PBS and immersed in a solution of Ab₁ (4 μg ml⁻¹) for 40 min, then, was washed with PBS. Then, to block the residual active sites on the working electrode it was immersed in the 3 ml mercaptohexanol for 30 min, followed this step, the GCE/Fe₃O₄@-AuNPs/MAA was washed with PBS. The obtained immunosensor was stored at 4°C for later use (Scheme 1).

The performance of the immunosensor for sensing the HER2 was carried out by the Amperometric technique with 5.0 μl of HER2 varying concentrations after 45 min incubation at 37°C. By using 0.8 mg ml⁻¹ Ab₂-Pt: CdTe QDs (Ab₂ labeled probe) attached to the surface of GCE/Fe₃O₄@-AuNPs/MAA/Ab₁/HER2 by the specific binding of antigen and antibody, thereby catalyzing H₂O₂ to obtain the higher reducing current signals. For the determination of HER2, a constant voltage of -0.4 V, the H₂O₂ (10 μl, 5.0 mM) was stirred with a constant voltage of −0.4 V, and the amperometric signal was then collected. The CV of the biosensor was scanned in 0.1 M KCl containing 5.0 mM of [Fe(CN)₆]^3−/4−, which was used as the electrochemical probe. The Nyquist plots were taken under the frequency ranging from 10⁵ to 0.1 Hz and AC amplitude 5 mV in the solution of 0.1 M KCl containing 5.0 mM of [Fe(CN)₆]^3−/4− as the probe and then the charge transfer resistance (Rct) was tracked.

The FT-IR spectroscopy of TMU-24 is presented in Figure 1A. The peaks at 1,378 and 1,427 cm⁻¹ referring, as C=C bonds, appear in TMU-24. Peaks at 2,752 and 2,851 cm⁻¹ corresponded to the stretching vibration of C–H bonds in the benzene ring. The peak at 1,611 cm⁻¹ is attributed to the stretching vibration adsorption peaks of the C=O bonds.

The morphology of TMU-24 was investigated by SEM. As shown in Figure 1B, the particle of nanomaterials is presented XRD patterns are similar to the previously reported data that revealed syntheses of TMU-24 (Figure 1C). Based on the SEM images, the average diameter of the TMU-24 studied here is about 350 nm.

From N₂ isotherm at 77 K, TMU-24 was found to be nonporous materials with negligible uptake, which reveal an “N₂-phobic” behavior. We reasoned that this behavior could be due to the existence of different structural rearrangements depending on the MOF during sorption or when exposed to cryogenic temperatures and/or under vacuum, reducing/preventing access to the porosity. However, other factors cannot be fully excluded, such as the existence of strong interactions between N₂ and the channel walls at 77 K that hinder diffusion into the material. Interestingly, contrariwise to the adsorption of N₂, the CO₂ sorption isotherm revealed that TMU-24 is porous, and CO₂ uptake at 203 K was calculated to be 6.3 mmol g⁻¹ with the pore volume of 0.22 cm³ g⁻¹ at 760 Torr.

FT-IR spectroscopy was applied to explore the surface functionalization of the TGA capped Pt: CdTe and TGA. Figure 2A shows the FT-IR spectra of TGA-capped Pt: CdTe QDs and TGA alone. In TGA-capped Pt: CdTe QDs spectrum a broad and strong peak at 3,441 cm⁻¹ was observed assigning to the stretching vibrations of O-H while this peak is not observed in TGA. Instead, the stretching vibration of the S-H bond and C=O in TGA appeared at 2,565 cm⁻¹ and 1716 cm⁻¹, which in the spectrum of TGA-capped Pt: CdTe QDs the first peak was disappeared that may be due to the binding of metal cations with thiol group and the second peak was shifted to 1,578 cm⁻¹. These results confirmed the attachment of TGA onto the quantum dots.

The particle size and morphology of the as-prepared TGA-capped Pt: CdTe QDs were studied by the TEM method. As shown in Figure 2B the prepared QDs with nearly spherical morphology have a uniform size with a diameter of about 2 nm. The elemental composition of the TGA capped Pt: CdTe d-dots NPs was corroborated by EDX analysis (Figure 2C). The EDX indicates the presence of Pt, Cd, and Te in the prepared QDs.

The crystal structure of undoped Pt: CdTe QDs were investigated with an XRD pattern and is shown in (Figure 2D). The XRD pattern achieved for the Pt: CdTe QDs illustrates four peaks positioned at 2θ = 22.57°, 39.70°, 45.82°, and 57.54° that are corresponding to (111) (220), (311), and (422) planes, which their positions are consistent with the values of the standard diffraction patterns of the cubic structure of CdTe (JCPDS no. 03-065-1,046).

The UV–Vis absorption and fluorescence spectra of Pt: CdTe QDs were presented in Figure 3A. As seen in Figure 3A curve the absorption spectrum of Pt: CdTe QDs was observed with the excitonic at 495 nm and it is a wide spectrum The Pt: CdTe QDs fluorescence was emitted at 515 nm upon excitation at 360 nm (curve b). The emission of Pt: CdTe QDs was close to its absorption onset indicating the emission arising from the direct recombination of charge carriers between conduction and valence bands.

To confirm the binding of Pt: CdTe QDs to Ab₂, the fluorescence spectra of Pt: CdTe QDs, Ab₂, and Ab₂- Pt: CdTe QDs were also recorded and compared with the emission spectra of Pt: CdTe QDs. As Figure 3A shows, after, the attachment of Ab2 to QDs and covering the surface of QDs with Ab₂, the related fluorescent signal decreases and shows a redshift at 525 nm which can prove the interaction between Ab₂ and QDs due to their conjugation.

Similar to UV-Vis studies, fluorescence studies show the same trend. The QDs have an intense band of fluorescence at 515 nm, while the Ab₂ has almost no band at all (Figure 3B). After the attachment of Ab₂ to QDs, the related fluorescent signal decreases with a redshift to 525 nm. These studies suggest that the Ab₂-QDs are successfully formed.

The surface morphologies of the constructed immunosensor during modifications were characterized by the SEM as an excellent procedure. Figure 4 shows that the composite film of Fe₃O₄@ and Nafion is covered the surface of GCE. The size of Fe₃O₄@ with a diameter of about 50–100 nm with the ball shape in the Nafion film is distinguishable in this image. The distribution of the Au NPs with an average diameter of about 36 nm at the Fe₃O₄@ surface is illustrated in Figure 4B. This film by its nanostructure morphology can increase the surface area of the electrode, which in turn can increase a biosensor, especially for immobilization of an antibody.

The EDX images (Figure 1B) and elemental mapping (Figure 1C) indicate the presence and dispersion of different elements of Au, C, O, N, Fe, and Zn at the prepared composite of elements at the platform immunosensor (Figure 4C). The CV and EIS techniques were used to monitor the surface changes during the construction process of the HER2 sandwich assay. Figure 5 shows different CVs of bare GC (curve a), GC/Fe₃O₄@ TMU-24 (curve b), GC/Fe₃O₄@ TMU-24 –AuNPs/MAA (curve c), GC/Fe₃O₄@ TMU-24 -Au NPs/MAA/Ab₁ (curve e), GC/Fe₃O₄@ TMU-24-AuNPs/Ab₁/HER2 (curve f) electrodes. In the solution of 5.0 mM [Fe(CN)₆]^3-/4- (0.1 M KCl) and a scan rate of 50 mV s⁻¹, the bare GC electrode shows well-known reversible peaks of redox probes. After the position of Fe₃O₄@ TMU-24 on the GC electrode surface, the peak current is increased and the peak-to-peak separation (ΔE_p) is decreased, which can be contributed to the electron transfer process being facilitated by using Fe₃O₄@ TMU-24 as an excellent signal amplifying material.

Electrodeposition of AuNPs on GCE/Fe₃O₄@ TMU-24 increased the value of the redox current of the probe, which clearly shows the deposition of Au NPs in the resulted electrode. This nanoparticle improves the electrode conductivity and facilitates the kinetic electron transfer. Next, by appending the MAA on GCE/Fe₃O₄@ TMU-24-AuNPs, the semicircle diameter is also dramatically, decreased from the current value, indicating MAA as a low conductive film.

As was expected, after immobilization of Ab₁ in GCE/Fe₃O₄@ TMU-24-AuNPs/MAA/Ab₁, the current is dramatically decreased and the current decrease is continued by the addition of antigen. These observations prove that the surface GC/Fe₃O₄@ TMU-24-AuNPs/MAA was successfully modified with Ab0₁. The CV was used to monitor the surface changes during the construction process. The results of the CV are in good agreement with the results of the EIS.

TMU-24 exhibited excellent electrochemical behavior to facilitate the electron transfer and prominent redox properties by charge compensation. Furthermore, it existed a large surface area and high affinity to AuNPs in terms of strong interaction between -CN and AuNPs.

The amperometric was used to record the steps of immunosensor fabrication. As Figure 6 shows, bare GCE did not show any catalytic effect on the reduction of H₂O₂ (curve a) and GCE/Fe₃O₄@ TMU-24-AuNPs had a large current signal (curve b). While, the current signal declined after Ab₁, and HER2 were modified on the surface (curves c and d), successively. It can be attributed to proteins that block the electroactive material from coming into contact with the platform. As result, confirms the formation of the antigen-antibody complex. As a result, to produce high-sensitivity signals, the catalytic activity of Pt: CdTe QDs and CdTe QDs in the label of Ab₂ were compared. As demonstrated in Figure 6, the current response of Pt: CdTe QDs -Ab₂₀ (curve f) was greater than CdTe QDs-Ab₂ (curve e). The experiment result confirmed that.

Pt: CdTe QDs-Ab₂ possessed efficient catalytic activity towards H₂O₂. Doping Pt to CdTe QDs has led to a significant increase in the immunosensor response signal. As a result, Pt can act as catalytic labels for the reduction of H₂O₂ to amplify the signal of the electrochemical immunosensor. Therefore, when the HER2 is sandwiched by the Ab₁ and Pt: CdTe QDs -Ab₂, the current signal increased rapidly. It can be attributed to Pt: CdTe QDs -Ab₂ with high catalytic activity. Therefore, the immunosensor was fabricated successfully.

We optimized the following parameters for maximum performance of the immunosensor: incubation times of Fe₃O₄@ TMU-24-AuNPs/Ab₁ with HER2 and Ab₂- Pt: CdTe QDs/HER2 with pH. The incubation time effect of 10 ng ml⁻¹ of HER2 on the surface of GC/Fe₃O₄@ TMU-24-AuNPs/MAA/Ab₁ electrode on signal response was evaluated. As it can be seen in Supplementary Figure S1A, the immunosensor response increases with time raise and after 40 min the signal is almost unchanged, the maximum signal response was obtained then it became almost saturated. Therefore, the time of 40 min was chosen as the incubation time for determining the HER2.

The incubation time of Ab₂- Pt: CdTe QDs labeled on the surface of GCE/Fe₃O₄@ TMU-24-AuNPs/MCA/Ab₁/HER2 was evaluated with the same amperometric method (Supplementary Figure S1B) proves, the incubation time of Ab₂- Pt: CdTe QDs labeled 40 min was selected and applied for HER2 biomarker analysis.

The pH of PBS buffer has a high influence on the electrochemical behavior of the stabilized peptide. The pH optimization of PBS solution was evaluated from pH 5.5 to 9.5. The amperometric response of the immunosensor increased up to 7.5 and then decreased. The maximum signal response of the sensor was obtained at pH 7.5 in Supplementary Figure S1C, which is close to the isoelectric point of the protein. Therefore, pH 7.5 was selected for the following.

In optimum conditions, the analytical performances of immunosensors were evaluated for different concentration detection of HER2 using Pt: CdTe QDs as labels. The amperometric’s signals of the immunosensor were recorded at −0.4 V in pH 7.0 PBS. As Figures 7A,B show, the increase in incubation of HER2 at the sensor surface caused a gradual increase in amperometric signals (Figure 7A). The amperometric current was charted against of logarithm of HER2 concentration and the calibration chart had shown a linear relationship in the range of 1 pg ml⁻¹–100 ng ml⁻¹ with a regression equation of I = -32.34 log (C ng·mL⁻¹) + 260.8 (R ² = 0.9865) (Figure 7B) and an appropriate response with a detection limit of 0.175 pg ml⁻¹. The proposed immunosensor exhibits a comparable and even better performance for the determination of HER2 relative to that of the reported sensors.

A comparison between immunosensor presented in previous studies has been shown in Table 1. The proposed immunosensor has a decent function (For example, lower detection limit, and wide linear range).

The stability of the proposed immunosensor was evaluated by monitoring of signal of 10 ng ml⁻¹ of HER2 stored at 4°C in 0.1 M PBS of pH 7.0 solution. After one week no change, but 2 weeks of storage under similar conditions reduced the initial current of immunosensor by about 12%. The stability of the sensor is attributed to the presence of Fe₃O₄@ TMU-24 as a robust sublayer, which can assist the composite to keep intact the established architecture in stabilizing the probe connected to the electrode surface. The relative standard deviations (RSD) of six immunosensor were found to be 3.15% suggesting the acceptable reproducibility of the electrode. The binding specificity of the proposed immunosensor to 10 ng ml⁻¹ of HER2 was compared with those obtained by equal concentration (100 ng ml⁻¹) of HSA, hepatitis B surface antigen (HBS), carcinoembryonic antigen (CEA), and human immunoglobulin (IgG) and the results are presented in Figure 8. As shown in Figure 8, in the presence of interfering species, the current response to HER2 did not change. These results indicate that the constructed sensor is suitable for sensing HER2.

The main advantage of using the Fe₃O₄@ TMU-24-AuNPs/Ab1 is easy and quick surface renewal after each use. Thus, the repeatability of the response signal was studied for HER2 biomarker by an amperometric method in optimized conditions. The relative standard deviation (RSD) of 1.65% was obtained.

To show the use of constructed immunosensors in the analysis of real samples, accurate HER2 determination in human serum samples was measured by the standard addition method following the addition of different concentrations of HER2 into healthy human serum samples. The results obtained from human serum samples are assembled in Table 2. The accuracy and precision of the proposed immunosensor were evaluated by recovery and the relative standard deviation calculation respectively. The recovery of the amounts of HER2 detected by the proposed immunosensors was in the range of 98 to 103.7% and the relative standard deviations were obtained to less than 5% that revealing the utilization potentiality of the assay of HER2 based on the designed sensor using Pt: CdTe QDs labeled Ab₂ for clinical analysis samples.