Optical Evidence for the Assembly of Sensors Based on Reduced Graphene Oxide and Polydiphenylamine for the Detection of Epidermal Growth Factor Receptor

: Using Raman scattering and FTIR spectroscopy, new optical evidence for the assembly of sensors based on reduced graphene oxide (RGO) and polydiphenylamine (PDPA) for the electrochemical detection of the epidermal growth factor receptor (EGFR) are reported. The assembly process of the RGO sheets electrochemical functionalized with PDPA involves the chemical adsorption of 1,4-phenylene diisothiocyanate (PDITC), followed by an incubation with protein G in phosphate buffer (PB) solution and after that the interaction with EGFR antibodies solution. Taking into account the changes reported by Raman scattering and FTIR spectroscopy, a chemical mechanism of the assembling process for this sensor is proposed. The preliminary testing of the electrochemical activity of the sensors based on RGO and PDPA was reported by cyclic voltammetry. the assembly of immunosensors for EGFR detection, the information concerning the optical evidence of the stages of assembling these platforms is missing. In order to overcome this limitation, in this work, we report several optical studies carried out using Raman scattering and FTIR spectroscopy concerning the assembly of RGO sheets electrochemically functionalized with PDPA in order to be used in the future for the electrochemical detection of EGFR. In this work, a short characterization of these platforms is shown by cyclic voltammetry. Our results open up new perspectives for highly reproducible platforms for clinical screening of cancer tumors.


Introduction
The epidermal growth factor receptor (EGFR) is a biomarker often used for many tumors in various diseases such as breast cancer, gliomas, laryngeal cancers, carcinoma, and so on [1,2]. Different methods were developed for the detection of EGFR, the most used being immunohistochemistry [3,4], enzyme-linked immunosorbent assay (ELISA), [5] and Western blotting [6]. The main platforms used until now for the EGFR detection were lab-on-chip sensors [7], biochips-based on microfluids [8], Au nanoparticles which show surface plasmon resonance [9] and label-free electrochemical immunosensors [10]. In comparison with this progress, a new platform based on reduced graphene oxide (RGO) electrochemical functionalized with polydiphenylamine (PDPA) is proposed to be used for the EGFR detection in this work. These platforms are considered more attractive in comparison with the Au nanoparticles or Au plate, as a consequence of the fact that it is no longer necessary to interact with cysteamine in order to generate new amine-type bonds that would later allow interaction with 1,4-phenylene diisothiocyanate (PDITC). An example which supports this is the case of the RGO sheets electrochemically functionalized with poly(5-amino-1-naphtol) [11]. Despite the greater progress made concerning the assembly of immunosensors for EGFR detection, the information concerning the optical evidence of the stages of assembling these platforms is missing. In order to overcome this limitation, in this work, we report several optical studies carried out using Raman scattering and FTIR spectroscopy concerning the assembly of RGO sheets electrochemically functionalized with PDPA in order to be used in the future for the electrochemical detection of EGFR. In this work, a short characterization of these platforms is shown by cyclic voltammetry. Our results open up new perspectives for highly reproducible platforms for clinical screening of cancer tumors. Figure 1a shows the Raman spectrum of the SPCE-RGO/PDPA platform, which is characterized by two intense bands with the maximum at 1592 and 1350 cm −1 that are accompanied of other two Raman lines of low intensity at 1176, 1133, and 996 cm −1 .

Optical Evidences by Raman Scattering and FTIR Spectroscopy Studies Concerning the Assembling of the Sensorial Platforms for EGFR Detection
Coatings 2021, 11, x FOR PEER REVIEW 3 of 13 Figure 1a shows the Raman spectrum of the SPCE-RGO/PDPA platform, which is characterized by two intense bands with the maximum at 1592 and 1350 cm −1 that are accompanied of other two Raman lines of low intensity at 1176, 1133, and 996 cm −1 . As shown in our previous article [13], the RGO Raman spectrum at an excitation wavelength of 514 nm is characterized by two lines at 1349 and 1573 cm −1 , these being assigned to the graphitic lattice defects and E2g in-plane phonon in the Brillouin zone G point [14]. According to our previous studies, the main Raman lines of PDPA in a doped state were reported to be localized at 1176, 1342, 1367, 1492, 1585, and 1613 cm −1 , these being assigned to the vibrational modes C-H bending in the benzene ring, C-N in the N,N'-diphenyl benzidine radical cation, C=N stretching, C-C stretching in the quinoid ring + C-C stretching in the benzene ring, and C-C stretching in benzene ring + C-H bending in benzene ring, respectively [15,16]. The Raman line at 1592 cm −1 in Figure 1a, confirms the presence of PDPA on the RGO sheet's surface. The Raman line at 996 cm −1 (Figure 1a) belongs to the vibrational modes of W=O in the H4SiW12O40 [17]. The up-shift of the Raman line assigned to the C-H bending vibrational mode in the benzene rings of the polymer from 1176 to 1189 cm −1 can be explained if we accept that a covalent functionalization process of the RGO sheets with conjugated polymer takes place, when the steric hindrance effects are induced in the PDPA macromolecular chain. A puzzling fact is the presence of the Raman line at 1133 cm −1 (Figure 1a), which is located not far from the Raman line from 1150 cm −1 assigned to the vibrational mode of C-H in-plane bending of the polymers having triphenylamine as repeating units [18]. The Raman spectrum of PDITC, at the excitation wavelength of 514 nm, shows lines with peaks at 1157, 1257, 1583, and 1603 cm −1 , attributed to the vibrational modes of the C-S bending, C-H in the benzene ring + C-C stretching + C-N stretching, C=C+C-C stretching in the benzene ring, and C-C stretching + C-H bending in the benzene ring, respectively [19][20][21][22][23]. The interaction of the SPCE-RGO/PDPA with the PDITC solutions with increasing concentration leads to the following changes in the Raman spectra of Figure 1  As shown in our previous article [13], the RGO Raman spectrum at an excitation wavelength of 514 nm is characterized by two lines at 1349 and 1573 cm −1 , these being assigned to the graphitic lattice defects and E 2g in-plane phonon in the Brillouin zone G point [14]. According to our previous studies, the main Raman lines of PDPA in a doped state were reported to be localized at 1176, 1342, 1367, 1492, 1585, and 1613 cm −1 , these being assigned to the vibrational modes C-H bending in the benzene ring, C-N in the N,N'-diphenyl benzidine radical cation, C=N stretching, C-C stretching in the quinoid ring + C-C stretching in the benzene ring, and C-C stretching in benzene ring + C-H bending in benzene ring, respectively [15,16]. The Raman line at 1592 cm −1 in Figure  1a, confirms the presence of PDPA on the RGO sheet's surface. The Raman line at 996 cm −1 (Figure 1a) belongs to the vibrational modes of W=O in the H 4 SiW 12 O 40 [17]. The up-shift of the Raman line assigned to the C-H bending vibrational mode in the benzene rings of the polymer from 1176 to 1189 cm −1 can be explained if we accept that a covalent functionalization process of the RGO sheets with conjugated polymer takes place, when the steric hindrance effects are induced in the PDPA macromolecular chain. A puzzling fact is the presence of the Raman line at 1133 cm −1 (Figure 1a), which is located not far from the Raman line from 1150 cm −1 assigned to the vibrational mode of C-H in-plane bending of the polymers having triphenylamine as repeating units [18]. The Raman spectrum of PDITC, at the excitation wavelength of 514 nm, shows lines with peaks at 1157, 1257, 1583, and 1603 cm −1 , attributed to the vibrational modes of the C-S bending, C-H in the benzene ring + C-C stretching + C-N stretching, C=C+C-C stretching in the benzene ring, and C-C stretching + C-H bending in the benzene ring, respectively [19][20][21][22][23]. The interaction of the SPCE-RGO/PDPA with the PDITC solutions with increasing concentration leads to the following changes in the Raman spectra of Figure (Figure 2f), confirms a chemical adsorption of a part the EGFR antibodies and EGFR antigen onto the SPCE-RGO/PDPA-PDITC-G platform surface. This can be explained by an incomplete deactivation of the thiocyanate groups due to steric effects induced by the presence of protein G on the surface. The remaining thiocyanate groups can anchor to the surface the anti-EGFR antibodies that get non-covalently attached to protein G, and similarly the EGFR antigen that gets caught by the anti-EGFR antibodies.

Optical Evidences by Raman Scattering and FTIR Spectroscopy Studies Concerning the Assembling of the Sensorial Platforms for EGFR Detection
Additional information is obtained by FTIR spectroscopy, as shown in Figure 4. The main IR bands of the SPCE-RGO/PDPA platform peak at 694, 750, 779, 881, 916, 970, 1014, 1164, 1251, 1315, 1493, 1593, and 1651 cm −1 , being assigned to the following vibrational modes: inter-ring deformation, ring deformation, W-Oc-W (octahedral edge-sharing), C-H in-plane bending of the quinoid ring (Q), W-Ob-W (octahedral corner-sharing), W-Od (terminal), Si-Oa, C-H bending in the benzene ring (B) + quinoid ring (Q), radical cation structure, Caromatic-N stretching, C-C stretching + C-H bending, C-C stretching, and -NH + =Q=Q=NH + -, respectively [19,[25][26][27]. The interaction of the SPCE-RGO/PDPA platform with PDITC, protein G, EGFR antibodies, and EGFR antigen induces in Figure 4 the following changes: (i) a decrease in the absorbance of the IR bands at 694 and 750 cm −1 ; (ii) an up-shift of IR band from 1164 to 1184 cm −1 ; (iii) a gradual increase in the absorbance of the IR bands at 1251, 1315, 1593, and 1651 cm −1 ; and (iv) the appearance of two IR bands with maxima at 1699 and 1780 cm −1 , both assigned to the C=O vibrational mode whose absorbance gradually increases as the platform interacts with protein G, EGFR antibodies, and EGFR antigen.  (Figure 2f), confirms a chemical adsorption of a part the EGFR antibodies and EGFR antigen onto the SPCE-RGO/PDPA-PDITC-G platform surface. This can be explained by an incomplete deactivation of the thiocyanate groups due to steric effects induced by the presence of protein G on the surface. The remaining thiocyanate groups can anchor to the surface the anti-EGFR antibodies that get non-covalently attached to protein G, and similarly the EGFR antigen that gets caught by the anti-EGFR antibodies.
Additional information is obtained by FTIR spectroscopy, as shown in Figure 4. The main IR bands of the SPCE-RGO/PDPA platform peak at 694, 750, 779, 881, 916, 970, 1014, 1164, 1251, 1315, 1493, 1593, and 1651 cm −1 , being assigned to the following vibrational modes: inter-ring deformation, ring deformation, W-O c -W (octahedral edge-sharing), C-H in-plane bending of the quinoid ring (Q), W-O b -W (octahedral corner-sharing), W-O d (terminal), Si-O a , C-H bending in the benzene ring (B) + quinoid ring (Q), radical cation structure, C aromatic -N stretching, C-C stretching + C-H bending, C-C stretching, and -NH + =Q=Q=NH + -, respectively [19,[25][26][27]. The interaction of the SPCE-RGO/PDPA platform with PDITC, protein G, EGFR antibodies, and EGFR antigen induces in Figure 4 the following changes: (i) a decrease in the absorbance of the IR bands at 694 and 750 cm −1 ; (ii) an up-shift of IR band from 1164 to 1184 cm −1 ; (iii) a gradual increase in the absorbance of the IR bands at 1251, 1315, 1593, and 1651 cm −1 ; and (iv) the appearance of two IR bands with maxima at 1699 and 1780 cm −1 , both assigned to the C=O vibrational mode whose absorbance gradually increases as the platform interacts with protein G, EGFR antibodies, and EGFR antigen.
According to Figure 5, the IR spectrum of protein G is dominated by two IR bands at 1518 and 1634 cm −1 , which were assigned to the vibrational modes C-C + C-H and C=C, respectively [28]. Other IR bands of low absorbance are remarked in Figure 5a, at 1084, 1234, and 1393 cm −1 that were attributed to the vibrational modes of bonds CH, COH and COO, respectively [28]. In the case of the EGFR antibodies, the IR bands localized at 1038-1109 and 1649 cm −1 (Figure 5b) were assigned to the vibrational mode C-O + C-H and C=O [28]. All these vibrational changes can be explained by taking into account the chemical mechanism of the assembling of these platforms shown in Scheme 1.  According to Figure 5, the IR spectrum of protein G is d 1518 and 1634 cm −1 , which were assigned to the vibrational respectively [28]. Other IR bands of low absorbance are rem 1234, and 1393 cm −1 that were attributed to the vibrational m COO, respectively [28]. In the case of the EGFR antibodies, th 1109 and 1649 cm −1 (Figure 5b) were assigned to the vibrat C=O [28]. All these vibrational changes can be explained by t ical mechanism of the assembling of these platforms shown   According to Figure 5, the IR spectrum of protein G is dominated by two IR bands 1518 and 1634 cm −1 , which were assigned to the vibrational modes C-C + C-H and C= respectively [28]. Other IR bands of low absorbance are remarked in Figure 5a, at 10 1234, and 1393 cm −1 that were attributed to the vibrational modes of bonds CH, COH a COO, respectively [28]. In the case of the EGFR antibodies, the IR bands localized at 103 1109 and 1649 cm −1 (Figure 5b) were assigned to the vibrational mode C-O + C-H a C=O [28]. All these vibrational changes can be explained by taking into account the che ical mechanism of the assembling of these platforms shown in Scheme 1.    Table 1.    6 ] solution, depending on the scan rate. The main changes in the potential of the anodic and cathodic peaks as well as their current densities are summarized in Table 1.    In the case of Au electrode, the ratio between current densities of the anodic and cathodic peaks is different from one. In the case of the electrodes SPCE-RGO, SPCE-RGO/PDPA-PDITC-G-anti-EGFR/EGFR, the values of the ratio between current densities of the anodic and cathodic peaks is~1. Regardless of the electrode type, i.e., Au, SPCE-RGO, and SPCE-RGO/PDPA-PDITC-G-anti-EGFR/EGFR, the potential of separation of the anodic and cathodic peaks has a difference of 56.5/n, where n corresponds to the number of electrodes involved in the electrochemical process. These results indicate that at the electrode-electrolyte interface, an irreversible process occurs.

The Electrochemical Properties of the Platforms SPCE-RGO/PDPA-PDITC-G-EGFR Antibodies-EGFR
This process must to be understood by the electrostatic interaction between the positively charged amine entities of the SPCE-RGO/PDPA-PDITC-G-EGFR antibodies/EGFR platform and negative charges of [Fe(CN) 6 ] 3− / 4− .

Conclusions
In this work, new optical evidence of the assembly process of sensors based on RGO sheets functionalized with PDPA in a doped state are reported by Raman scattering and FTIR spectroscopy. Our results allow us to conclude that: