Materials and reagents

Sodium tetra molybdate dihydrate (Na₂MoO₄.2H₂O), 3-aminopropyl triethoxysilane [APTES (C₉H₂₃NO₃Si)] were purchased from Alfa-Aesar, India. Sodium monophosphate (NaH₂PO₄), sodium diphosphate dihydrate (Na₂HPO₄·2H₂O), N-hydroxysuccinimide [NHS (C₄H₅NO₃)], sodium chloride (NaCl), potassium ferricyanide [K₃{Fe(CN)₆}], and potassium ferrocyanide [K₄{Fe(CN)₆}3H₂O] were procured from Fisher Scientific, India. Nitric acid (HNO3) was purchased from Rankem, India. The cetyl trimethylammoniumbromide,1-(3-(dimethylamino)-propyl)-3-ethylcarbodiimide hydro-chloride [EDC (C₈H₁₇N₃)] were procured from Sigma Aldrich, USA. The analytical grade chemicals were used for all the experiments without purification. Fresh phosphate buffer saline (PBS) solution using Na₂HPO₄·2H₂O (0.05 mol/L) and NaH₂PO4 (0.05 mol/L) was prepared using Milli-Q water (resistivity of 18 MΩ cm). The ELISA kit (HER-2) was purchased from Promocell GmbH, Germany. The monoclonal antibodies (anti-HER-2) and antigen (HER-2) were obtained from Biorbyt Ltd, UK. Further the biomolecules were diluted in PBS (pH 7.0) and stored at 4 °C till further use.

Fabrication of BSA/anti-HER-2/APTES/nMoO₃/ITO biosensing platform

For the fabrication of the biosensing platform, indium tin oxide (ITO)-coated glass electrode (0.5 cm × 2.5 cm) was used. The ITO electrode was hydrolyzed with a solution of NH₃:H₂O₂:H₂O in a ratio of 1:1:5 to introduce –OH functional groups on the surface to promote the formation of nMoO₃ film.^[Reference Kumar, Kumar, Tiwari, Srivastava, Srivastava, Yadav, Kumar, Tran, Dewan and Mulchandani^35, Reference Srivastava, Kumar, Ali, Solanki, Srivastava, Sumana, Saxena, Joshi and Malhotra^36] For this purpose, a dispersed colloidal solution (1 mg/mL) of functionalized nMoO₃ (APTES/1D nMoO₃) was prepared in acetonitrile. The electrophoretic deposition (EPD) technique (50 V for 2 min) was used to obtain uniform thin film on hydrolyzed ITO electrodes. The uniform film formation was further confirmed by AFM studies of APTES/1D nMoO₃/ITO electrode (Fig. S6). The AFM results indicated a uniform deposition of film onto the ITO substrate with an average roughness of ~2.93 nm. For the EPD technique, platinum was used as the anode and the ITO electrode was used as the cathode and these were separated at a distance of 1 cm.

Anti-HER-2 were immobilized onto the APTES/1D nMoO₃/ITO electrode in a ratio 2:1 via EDC-NHS coupling chemistry. In brief, 15 µL of monoclonal anti-HER-2 (50 µg/mL) was mixed with 7.5 µL of EDC (0.4 M) and 7.5 µL NHS (0.1 M) and stored at room temperature (25 °C) for 20 min to activate the –COOH groups at F _c region of anti-HER-2. Further, this mixed solution (30 µL) was drop cast uniformly onto the APTES/1DnMoO₃/ITO electrode surface and stored in a humid chamber (2 h). The interaction resulted in the formation of covalent bond between –NH₂ groups present on APTES/1D nMoO₃/ITO electrode and the activated –COOH groups of anti-HER-2. To remove the unbound biomolecules, the electrode was washed with PBS (pH 7.0). For blocking of the non-specific binding sites, 20 µL of BSA (0.1 mg/dL) was used as it did not interact with the specific binding sites. These BSA-incorporated immunoelectrodes were kept for 2 h at room temperature (25 °C) and were subsequently washed with PBS. The fabricated BSA/anti-HER-2/APTES/1D nMoO₃/ITO immunoelectrodes were stored at 4 °C until further use. Scheme S1(c) represents the various steps relating to the fabrication of a BSA/anti-HER-2/APTES/1D nMoO₃/ITO immunoelectrode.

Morphological, structural, Raman studies

The SEM image [Fig. 1(a)] obtained for nMoO₃ indicated the formation of needle-like structure.^[Reference Bleyer and Welch^11] The magnified image [inset in Fig. 1(a)] revealed the structure of nMoO₃ to be as rods. The diameter of nMoO₃ was found to lie between 60 and 80 nm with a few micrometers in length [Fig. 1(b)]. The XRD spectra [Fig. 1(c)] of the nMoO₃ obtained in the 20–60° diffraction angle (2θ) range with a scan rate of 0.2/min revealed crystallographic (hkl) planes: (001), (101), (002), (011), (110), (012), (003), (013), (020), (−114), and (023) were well indexed with JCPDS No. 47-1320 indicating the presence of the monoclinic phase of 1D nMoO₃. The (002) plane seen at 2θ = 25.83° had the highest intensity revealing that the 1D nMoO₃ crystallites were more orderly along the (002) plane. Figure 1(d) shows the Raman spectra obtained for the nMoO₃ nanorods. The high-intensity characteristic peak found at 818.2/cm was attributed to the stretching mode of the doubly connected bridge oxygen atom in Mo–O–Mo, present in the MoO₆ octahedrons. The peak obtained at 991.5/cm was due to asymmetric stretching modes of terminal oxygen in Mo=O.^[Reference Radhakrishnan, Reed, Oyama, Seman, Kondo, Domen, Ohminami and Asakura^32] The peak at 280/cm was a doublet comprising of wagging modes of the terminal oxygen atoms. The peak at 660.7/cm was attributed to the triply coordinated oxygen atoms linked to the Mo–O stretching modes of three octahedrons.^[Reference Radhakrishnan, Reed, Oyama, Seman, Kondo, Domen, Ohminami and Asakura^32] The peaks found at 333 and 375.2/cm were assigned to the bending mode of O=Mo=O and scissoring modes of O–Mo–O, respectively.

Figure 1.

SEM image (a), TEM image (b), XRD pattern (c), Raman spectra (d) of the synthesized 1D nMoO₃.

BET studies

The specific surface area and porosity of the synthesized 1D nMoO₃ were determined via BET studies. For sampling the specific surface area, the sample was degassed in a vacuum at 200 °C for 2 h and then the nitrogen adsorption and desorption isotherms were recorded. The pore size distribution and nitrogen adsorption–desorption isotherm for the 1D nMoO₃ are shown in Fig. 2. The results of the BET studies obtained 1D revealed the total pore volume of 1D nMoO₃ to be 0.029 cc/g. The specific surface area of the 1D nMoO₃ calculated by BJH method was found to be 17.19 m²/g. The pore size distribution curve indicated that the 1D nMoO₃ was dominated by mesoporous with a minor fraction of microporous [Fig. 2 inset (a)]. The mean pore diameter was found to be 6.8 nm. The obtained results revealed the highly mesoporous behavior of the synthesized 1D nMoO₃ facilitate increased loading of anti-HER-2.^[Reference Ali, Srivastava, Mondal, Chavhan, Agrawal, John, Sharma and Malhotra^13]

Figure 2.

BET studies for 1D nMoO₃ (inset a: pore size distribution).

Fourier transform infrared spectroscopy studies

Figure 3 shows Fourier transform infrared spectroscopy (FTIR) spectra obtained for (i) APTES/1D nMoO₃/ITO and (ii) anti-HER-2/APTES/1D nMoO₃/ITO electrodes, respectively. In both the spectra, bands appearing in the range 1350–1000/cm indicated the presence of C–N (amines) group onto the nMoO₃-bound APTES molecules. Bands found at 1474, 2880/cm and in the 1640–1550/cm range were due to C–H bending vibration of alkane (back bone of APTES), –C–H bond and N–H bending for primary amine.^[Reference Srivastava, Kumar, Ali, Solanki, Srivastava, Sumana, Saxena, Joshi and Malhotra^36, Reference Singh^37] The band found at 1382/cm was due to stretching vibration of C–N bond between –NH₂ group of APTES functionalized nMoO₃ and F _c region of anti-HER-2.^[Reference Pavia, Lampman, Kriz and Vyvyan^38–Reference Liu, Liu, Mernaugh and Zeng^40] The singlet band observed at 1651/cm was due to bending vibration of C=O group of amide bond (O=C–N–H). The broad band found at 3350/cm was assigned to stretching vibration of monosubstituted N–H group of amide (R–CO–NH–R) bond. The bands seen at 2720 and 2970/cm were attributed to –CH-band of alkane of the biomolecules and APTES present in the BSA/anti-HER-2/APTES/1D nMoO₃/ITO.^[Reference Chen, Wang and Wu^41, Reference Pavia^42] These FTIR results confirmed successful covalent immobilization of anti-HER-2 biomolecules onto APTES/1D nMoO₃/ITO electrode.^[Reference Liu, Liu, Mernaugh and Zeng^40]

Figure 3.

FTIR spectra of (a) APTES/1D nMoO₃, (b) anti-HER-2/APTES/1D nMoO₃/ITO electrode.

Cytotoxicity studies

The biocompatible behavior was evaluated by conducting cytotoxicity studies. In brief, the cytotoxicity of the functionalized nMoO₃ (APTES/1D nMoO₃) was determined in the concentration range 0–30 µg/mL by MTT assay on HEK 293T cells. The level of toxicity of the 1D nMoO₃ was found to vary as a function of concentration. The APTES/1D nMoO₃ did not exhibit any significant cytotoxicity on HEK 293 cell lines up to 15 µg/mL. This was adequate for the development of the biosensing platform. Figures S2(a)–S2(d) show morphology of the HEK 293 cell lines on exposure to different concentrations. The % cell viability of APTES/1D nMoO₃ with respect to control was found to be about 75% for 15 µg/mL (Fig. S3).

X-ray photoelectron spectroscopic studies

The survey scan X-ray photoelectron spectroscopic (XPS) spectra of APTES/1D nMoO₃ and anti-HER-2/APTES/1D nMoO₃ onto the ITO electrode is shown in Fig. S4. The peaks pertaining to Mo 3d, Si 2p, O 1s and N 1s were found at 232, 102, 530.5, and 399.6 eV, respectively. During the process of anti-HER-2 immobilization, the APTES/1D nMoO₃/ITO electrode was washed with PBS (pH 7.0). The peaks corresponding to Na and Cl can be seen in the survey scan spectra. The Na (KLL), O (KLL), and C (KLL) Auger transitions were observed. The P transition was attributed to anti-HER-2 molecules. It was found that the intensity of Mo (3d) and Si (2p) peaks decreased, and the N (1s) peak intensity increased after the anti-HER-2 immobilization on APTES/1D nMoO₃/ITO electrode.

The XPS core-level spectra of Mo (3d), Si (2p), N (1s), and O (1s) were deconvoluted into corresponding binding energy peaks [Figs. 4(a)–4(d)]. The two distinct states Mo (3d _5/2) and Mo (3d _3/2) separated by 3.15 eV arising due to spin-orbit splitting were visible [Fig. 4(a)]. The presence of two distinct valance states of Mo 5+ and 6+ in the deconvoluted spectra revealed the Mo valence state. The sharp doublet at Mo(3d _5/2) = 231 eV and Mo(3d _3/2) = 234 eV corresponded to Mo⁺⁵ state while the doublet at Mo(3d _5/2) = 232.02 eV and Mo(3d _3/2) = 235.76 eV pertained to Mo⁺⁶ state.^[Reference Choi and Thompson^43] The atomic percentage area of +6 states in anti-HER-2/APTES/1D nMoO₃/ITO decreased from 66.9% to 41.2%. This could be due to immobilization of antibodies onto the APTES/1D nMoO₃/ITO electrode. The N(1s) peak observed for APTES/1D nMoO₃/ITO and anti-HER-2/APTES/1D nMoO₃/ITO electrodes deconvoluted to four peaks at 395.3, 397.4, 399.1, and 401.0 Ev corresponded to the N–O–Mo, N–CH, –NH₂, and NH³⁺, respectively [Fig. 4(b)]. It may be noted that the contribution of the highest energy peak of –NH₂ (399.1 eV) in APTES/1D nMoO₃/ITO was perhaps due to –N–C=O (398.9) in anti-HER-2/APTES/1D nMoO₃/ITO.^[Reference Choi and Thompson^43–Reference Song, Hildebrand and Schmuki^45] This was assigned to the formation of amide bond (–N–C=O) between the carboxyl terminal of anti-HER-2 and the free –NH₂ groups of APTES molecules. The deconvolution of Si (2p) yielded four peaks at 101.7, 102.5, 103.3, and 104.4 eV corresponding to Si in Si–O, SiO₂, Si–O–Mo, Si–O–C, respectively [Fig. 4(c)],^[Reference Baltrusaitis, Mendoza-Sanchez, Fernandez, Veenstra, Dukstiene, Roberts and Fairley^46] confirmed the functionalization of 1D nMoO₃ with the APTES molecules. The O (1s) deconvolution was carried out in the range 528–534 eV [Fig. 4(d)]. The three deconvoluted peaks were ascribed to the oxygen linked to the O–Si–, O–Si–R wherein R was attributed to the presence of CH₂/NH₂ groups in APTES and the O–Mo.^[Reference Martin, Schulz, Bumgardner and Walters^44] After immobilization of anti-HER-2, an additional peak found at 530.5 eV was due to oxygen atom of the amide bond in O=C–N–.^[Reference Ali, Srivastava, Mondal, Chavhan, Agrawal, John, Sharma and Malhotra^13, Reference Kumar, Sharma, Maji and Malhotra^47] These results confirmed decrease in N–O–Mo and O–Mo bonding in N 1s and O 1s core-level spectra after the anti-HER-2 immobilization [Figs. 4(b) and 4(d)], respectively.

Figure 4.

Deconvoluted core-level XPS spectra of (a) Mo (3d), (b) N (1s), (c) Si (2p), and (d) O (1s) and of APTES/1D nMoO₃/ITO and anti-HER-2/APTES/1D nMoO₃/ITO.

Electrochemical studies

The effect of pH (6.0–8.0) on BSA/anti-HER-2/APTES/1D nMoO₃/ITO immunoelectrode was investigated using DPV in PBS buffer (50 mM, 0.9% NaCl) containing 5 mM of [(Fe (CN)₆)]^3−/4− ions. The maximum current response was recorded at pH 7.0 [Fig. 5(a)]. This could perhaps be due to the fact that at pH 7.0, biomolecules (e.g., antibodies, antigens, enzymes, DNA, mRNA, etc.) existed in their original form and had the highest bio-molecular activity. However, in the acidic or basic medium, the biomolecules perhaps got denatured due to the interaction of H⁺ or OH⁻ ions in the amino acid sequence of the antibodies.^[Reference Solanki, Singh, Rupavali, Tiwari and Malhotra^25] In further electrochemical studies, PBS (pH 7.0) were used.

Figure 5.

pH study of (a) BSA/anti-HER-2/APTES/1D nMoO₃/ITO with respect to pH (ranging from 6.0 to 8.0) in PBS (50 mM, 0.9% NaCl) containing 5 mM of [Fe (CN) ₆]^−3/4− (b) DPV studies at each step of electrode fabrication. Scan rate studies of (c) APTES/1D nMoO₃/ITO and BSA/anti-HER-2/APTES/1D nMoO₃/ITO (d) with square root of scan rate versus current (inset i) and peak potential versus square of scan rate (inset ii).

To investigate the electrochemical behavior of ITO, APTES/1D nMoO₃/ITO, anti-HER-2/APTES/1D nMoO₃/ITO, and BSA/anti-HER-2/APTES/1D nMoO₃/ITO electrodes, the DPV studies were conducted in the potential range −0.2 to 0.6 V [Fig. 5(b)]. The peak current obtained for the APTES/1D nMoO₃/ITO (0.31 mA) was found to be higher as compared to that of ITO (0.20 mA) due to high electrocatalytic efficiency and large surface area of the nMoO₃ nanorods leading to more efficient electron transfer between the electrolyte and the electrode surface.^[Reference Malik, Srivastava, Verma, Kumar, Kumar and Singh^48] The value of current decreased to 0.24 and 0.23 mA after immobilization of anti-HER-2 antibodies. This decrease was attributed to the insulating nature of the anti-HER-2 and BSA that acted as a barrier to free electron movement to the immunoelectrode.^[Reference Malik, Srivastava, Verma, Kumar, Kumar and Singh^48]

Interfacial kinetics studies on the APTES/1D nMoO₃/ITO and BSA/anti-HER-2/APTES/1D nMoO₃/ITO electrodes were carried out as a function of scan rate (40–140 mV/s) via CV. The magnitude of peak currents obtained for both the electrodes exhibited a linear relationship with square root of scan rate [inset (i) of Figs. 5(c) and 5(d)] indicating that the electrochemical reaction was a diffusion-controlled process and followed Eqs. (1)–(4).

(1) $$\eqalign{&I_{{\rm pa}({\rm APTES}/{\rm 1D}\,{\rm nMoO3}/{\rm ITO})} \cr &= - [12.7 {\rm \mu}{\rm A}({\rm s}/{\rm mV}) \times (\hbox{scan rate}\,[{\rm mV}/{\rm s}])^{1/2}] \cr & \quad\hskip 1pt - {10.3}\,{\rm \mu}{\rm A},\;R^{2} = 0.996,\;{\rm SD} = 24.8 \times 10^{ - 4},} $$

(2) $$\eqalign{I_{{\rm pc}({\rm APTES}/{\rm 1D\,nMoO3}/{\rm ITO}))}R^2 = 0.996,\;{\rm SD} = 34.5 \times 10^ - 4,} $$

(3) $$\eqalign{& I_{{\rm pc}({\rm BSA}/{\rm anti} - {\rm HER} - {2}/{\rm APTES}/{\rm 1D\,nMoO3}/{\rm ITO} )} \cr & = [14.9\,{\rm \mu} {\rm A}({\rm s}/{\rm mV}) \times (\hbox{scan rate}\,[{\rm mV}/{\rm s}])^{1/2}] \cr &\quad \, + 15.3\,{\rm \mu} {\rm A},\;R^{2} = 0.997,\;{\rm SD} = 13.809 \times 10^{ - {4}},} $$

(4) $$\eqalign{& I_{{\rm pa}({\rm BSA}/{\rm anti} - {\rm HER} - 2/{\rm APTES}/{\rm 1D}\,{\rm nMoO3}/{\rm ITO})}R^2 \cr& = 0.997,\;{\rm SD} = 20.9 \times 10^{ - 4}.} $$

With increasing scan rate, the oxidation and the reduction peaks shifted linearly toward more positive and negative potential, respectively. The linear variation was found between the magnitude of difference peak potentials calculated as (ΔE _p = E _pc–E _pa, where E _pa is anodic peak potential and E _pc is cathodic peak potential) and function of the square root of scan rate [inset (ii) of Figs. 5(c) and 5(d)]. The observed linear variation indicated efficient and more facile electron transfer kinetics between the medium to electrode and followed Eqs. (5) and (6).

(5) $$\eqalign{& \Delta E_{{\rm pa}({\rm APTES}/{\rm 1D\,nMoO3}/{\rm ITO} )} \cr & = [0.41\,{\rm V}({\rm s}/{\rm mV}) \times (\hbox{scan rate}[{\rm mV}/{\rm s}])^{1/2}] \cr &\quad\hskip 1pt + 0.11\,{\rm V},\;R^{2} = 0.995,\;{\rm SD} = 2.77 \times 10^{ - 4},} $$

(6) $$\eqalign{& \Delta E_{{\rm pc}({\rm BSA}/{\rm anti} - {\rm HER} - {2}/{\rm APTES}/{\rm 1D\,nMoO3}/{\rm ITO})} \cr & = [0.30\,{\rm V}({\rm s}/{\rm mV}) \times (\hbox{scan rate}[{\rm mV}/{\rm s}])^{1/2}] \cr & \quad\hskip 1pt+ 0.079\,{\rm V},\;R^{2} = 0.997,\;{\rm SD} = 4.77 \times 10^{ - 4},} $$

where R is the correlation coefficient and SD is the standard deviation. The diffusion coefficient (D) for the BSA/anti-HER-2/APTES/1D nMoO₃/ITO immunoelectrode was calculated to be 5.9 × 10⁻⁵ cm²/s by using Randle Sevick equation that is based on the assumption that mass transport occurred only by diffusion:

(7) $$I_{\rm p} = (\hbox{2}.\hbox{69} \times \hbox{1}0^{\rm 5}){\rm} n^{{\rm 3}/{\rm 2}}\hbox{A}\hbox{D}^{{\rm 1}/{\rm 2}}C\upsilon ^{{\rm 1}/{\rm 2}},$$

where I _p is the peak current obtained for the immunoelectrode, n is the number of electrons transferred (1), A is the active surface area of the immunoelectrode (0.25 cm²), D is the diffusion coefficient, C is the concentration of redox species (5 × 10⁻³ mol/cm⁻²) and υ is the scan rate (50 mV/s). The surface concentration (γ) of the BSA/anti-HER-2/APTES/1D nMoO₃/ITO immunoelectrode was calculated to be 1.049 × 10⁻⁷ mol/cm² using Brown–Anson Eq. (8)

(8) $$I_{\rm p} = n^{\rm 2}F^{\rm 2}\gamma A\upsilon (\hbox{4RT})^{ - {\rm 1}},$$

where I _p is the peak current of immunoelectrode, n is the number of electrons transferred (1), F is the Faraday constant (96485 C/mol), γ is the surface concentration of the absorbed electro-active species, A is the surface area of the electrode (0.25 cm²), υ is the scan rate (V/s), R is the gas constant (8.314 J/mol/K) and T is the temperature (25 °C).

Incubation time studies

To determine the time required for interaction between the epitope of HER-2 antigen (2.5 ng/mL) and paratope of the immobilized anti-HER-2 onto BSA/anti-HER-2/APTES/1D nMoO₃/ITO immunoelectrode, incubation time studies were performed via differential pulse voltammetry (DPV) every 5 min up to 30 min (Fig. S5).^[Reference Srivastava, Ali, Solanki, Chavhan, Pandey, Mulchandani, Srivastava and Malhotra^49] It was observed that the peak current response decreased gradually from 0 to 20 min after which it became nearly constant. This result showed that 20 min were required for complete binding of HER-2 onto BSA/APTES/1D nMoO₃/ITO immunoelectrode.

Electrochemical detection of the cancer biomarker

The electrochemical response studies of the BSA/anti-HER-2/APTES/1D nMoO₃/ITO immunoelectrode were conducted as a function of HER-2 concentration (0–110 ng/mL) in PBS (pH 7.0) containing 5 mM [Fe (CN)₆]^3−/4− via DPV technique in the potential range −0.2 to +0.6 V [Figs. 6(a) and 6(b)]. Each measurement was repeated three times for a given concentration of HER-2 (n = 3). It was observed that the anodic peak current was found to decrease proportionally with increasing HER-2 concentration. The decrease in the response current was attributed to the formation of insulating antigen–antibody complex molecules resulting due to the interaction of the specific binding sites of the HER-2 with anti-HER-2 at the transducer surface. This perhaps obstructed the electron transfer of Fe(CN)₆]^3−/4− from the bulk solution to the electrode resulting in reduced current.^[Reference Srivastava, Kumar, Ali, Solanki, Srivastava, Sumana, Saxena, Joshi and Malhotra^36] The calibration curve [Fig. 6(c)] obtained by plotting change in the magnitude of the anodic peak current of BSA/anti-HER-2/APTES/1D nMoO₃/ITO immunoelectrode as a function of HER-2 concentration in the detection range 2.5–110 ng/mL followed Eq. (9):

(9) $$\eqalign{I_{\rm p} =& \,[0.\hbox{9}0\hbox{4}\,{\rm \mu} /\hbox{Ang/mLc}\hbox{m}^{\rm 2} \times {\rm} (\hbox{conc}.\hbox{of HER - 2 in ng/mL}) \cr & + 0.0\hbox{954}\,{\rm \mu A}],\;R^{\rm 2} = 0.\hbox{997},\;\hbox{SD} = \hbox{1}.\hbox{83} \times \hbox{1}0^{ - {\rm 7}}.} $$

Figure 6.

Electrochemical response of the (a) BSA/anti-HER-2/APTES/1D nMoO₃/ITO immunoelectrode as a function of HER-2 concentration using DPV, (b) the magnified view of oxidation peak current, (c) calibration curve plot between magnitude of peak current and concentration of HER-2 (2.5–110 ng/mL) and (d) control experiment of APTES/1D nMoO₃ against HER-2.

The lower detection limit calculated using the standard equation Eq. (10) was obtained as 2.47 ng/mL

(10) $$\hbox{Lower detection limit} = \hbox{3}\sigma /m{\rm,} $$

where σ is the standard deviation (SD) of the calibration graph and m is the sensitivity. The linear detection range was obtained as 2.5–110 ng/mL (S/N = 3) and the sensitivity was calculated from the slope/surface area (0.25 cm²), which was found to be as 0.904 µAng/mL/cm².^[Reference Tabasi, Noorbakhsh and Sharifi^50]

To determine the effect of morphology onto the efficiency of biosensor, the electrochemical sensing studies of spherical nanoparticles of spherical MoO₃ were simultaneously carried out against HER-2 biomarker. Figure S7 is the SEM image of the as-synthesized spherical nanoparticles. The linear detection range was obtained as 5–60 ng/mL with sensitivity 0.52 µ/Ang/mL/cm². The poor sensitivity and narrow detection range of the spherical nanoparticle with respect to 1D nMoO₃ immunoelectrode can be ascribed to the fact that the exposed surface area available for binding of antibodies is far less as compared with that of the MoO₃ nanorods.^[Reference Barua, Yoo, Kolhar, Wakankar, Gokarn and Mitragotri^23] The higher sensitivity and the wide linear range (2.5–110 ng/mL) of the 1D MoO₃-based immunoelectrode (with respect to those reported in Table 1 and spherical MoO₃) can be attributed to the high electro activity of 1D nMoO₃.^[Reference Chen, Cheah, Madhavi and Lou^24]

Table I.

Characteristics of the BSA/anti-HER2/APTES/1D MoO₃/ITO immunosensor along with those reported in the literature.

The association constant (K _a) is known to determine the affinity of antigen toward antibodies. The higher the K _a value, the stronger is the affinity of HER-2 toward BSA/anti-HER-2/APTES/1D nMoO₃/ITO bio electrode. The affinity between the HER-2 and anti-HER-2 obtained using Hanes–Wolf plot (the plot between substrate concentration verses substrate concentration/current) (Fig. S8)^[Reference Jagadeesan, Kumar and Sumana^51, Reference Tiwari, Gupta, Bagbi, Sarkar and Solanki^52] was estimated to be 4.91 × 10¹² mol/L indicating strong affinity.

The electrochemical response of bare APTES/1D nMoO₃/ITO electrode toward different concentrations (2.5–110 ng/mL) of HER-2 antigen was determined [Fig. 6(d)] in order to evaluate the current response of the developed sensor. No significant change was observed in the peak current indicating that the APTES/1D nMoO₃/ITO electrode did not generate any false electrochemical response. This control experiment revealed that the sensor response was entirely due to the immunoreactions between anti-HER-2 and HER-2 biomolecules. Regeneration studies of the fabricated immunosensor were carried out using the same electrolyte after immersing the BSA/anti-HER-2/APTES/1D nMoO₃/ITO in 0.2 M solution of glycine-HCl buffer (pH 2.4 for 2 min). It was found that there was a gradual decrease in peak current with the increased regeneration times (Fig. S9). This could be due to the fact that at low pH, the protein binding sites repelled each other and tended to fold making the molecules move further apart.^[Reference Goode, Rushworth and Millner^14, Reference Andersson, Areskoug and Hardenborg^53] The results demonstrated that the proposed immunosensor could be regenerated and used for at least five times.

Selectivity studies

To determine the selectivity of the BSA/anti-HER-2/APTES/1D nMoO₃/ITO immunoelectrode toward various biomolecules present in serum samples of breast cancer patients, interferent studies were conducted via DPV technique (Fig. S10). The serum samples of breast cancer patients are known to contain glucose [7 mg/mL], cardiac troponin [cTn-I, >0.6 ng/mL], CYFRA-21-1 [>3.5 ng/mL],^[Reference Kumar, Kumar, Tiwari, Srivastava, Srivastava, Yadav, Kumar, Tran, Dewan and Mulchandani^35] CEA [>3 ng/mL], cTn-I [>0.19 ng/mL],^[Reference Kumar, Sen, Kumar, Augustine, Yadav, Mishra and Malhotra^54] etc. Firstly, we measured the electrochemical response of BSA/anti-HER-2/APTES/1D nMoO₃/ITO immunoelectrode toward the HER-2 (2.5 ng/mL). Thereafter we added cTn-I, glucose, and CYFRA-21-1 and ET-1 one by one in the same solution containing HER-2 and measured the electrochemical response. The measured peak current exhibited no significant change. The results obtained indicated that 1D nMoO₃ based immunoelectrode was highly selective toward the HER-2 biomarker only. The selectivity coefficient was calculated using Eq. (11):

(11) $$\hbox{SC} = \displaystyle{{I_{\rm c} + i} \over {I_{\rm c}}},$$

where I _c+i is the current response of bioelectrode for interference and I _c is the current obtained in absence, of each interferent, respectively. The SC value obtained for each interferent was ~1.

Shelf life studies

The shelf life of the BSA/anti-HER-2/APTES/nMoO₃/ITO immunoelectrode was determined at a regular interval of 2 days up to 40 days (Fig. S11). The fabricated biosensor was stored at 4 °C when not in use. The measured current response exhibited 95% of the initial electrochemical current response up to 30 days after which the current gradually decreased. The sensing characteristics of the fabricated immunoelectrode with the reported literature are summarized in Table 1.^[Reference Marques, Viswanathan, Nouws, Delerue-Matos and González-García^55, Reference Gruhl, Rapp and Länge^56]

Patient's serum samples analysis

Quantification of HER-2 in serum samples of breast cancer patients was accomplished via double sandwich ELISA. After determination of the HER-2 concentration, five serum samples of breast cancer patients were utilized to test the accuracy of data obtained using fabricated BSA/anti-HER-2/APTES/1D nMoO₃/ITO platform. The reasonable correlation with an acceptable percentage relative standard deviation was obtained between (a) HER-2 concentration in serum samples determined by ELISA and (b) standard concentration of HER-2 (Table S2).