Highly Sensitive Electrochemical Biosensor Using Folic Acid-Modified Reduced Graphene Oxide for the Detection of Cancer Biomarker

The detection of cancer biomarkers in the early stages could prevent cancer-related deaths significantly. Nanomaterials combined with biomolecules are extensively used in drug delivery, imaging, and sensing applications by targeting the overexpressed cancer proteins such as folate receptors (FRs) to control the disease by providing earlier treatments. In this investigation, biocompatible reduced graphene oxide (rGO) nanosheets combined with folic acid (FA)-a vitamin with high bioaffinity to FRs-is utilized to develop an electrochemical sensor for cancer detection. To mimic the cancer cell environment, FR-β protein is used to evaluate the response of the rGO-FA sensor. The formation of the rGO-FA nanocomposite was confirmed through various characterization techniques. A glassy carbon (GC) electrode was then modified with the obtained rGO-FA and analyzed via differential pulse voltammetry (DPV) for its specific detection towards FRs. Using the DPV technique, the rGO-FA-modified electrode exhibited a limit of detection (LOD) of 1.69 pM, determined in a linear concentration range from 6 to 100 pM. This excellent electrochemical performance towards FRs detection could provide a significant contribution towards future cancer diagnosis. Moreover, the rGO-FA sensing platform also showed excellent specificity and reliability when tested against similar interfering biomolecules. This rGO-FA sensor offers a great promise to the future medical industry through its highly sensitive detection towards FRs in a fast, reliable, and economical way.


Introduction
Folic acid (FA) or pteroylmonoglutamic acid is an important B-group vitamin involved in various metabolic pathways regulating foetal development, genetic material synthesis, and ageing [1,2]. Being an essential nutrient, it is involved in the vital 'one-carbon transfer reaction' occurring in most of the body's metabolic pathways [3][4][5]. However, mammalian cells are incapable of producing FA, and of avoiding the nutritional deficiency, exogenous intake of this nutrient to cells is necessary. According to the Food and Drug Administration (FDA), daily FA intake should be in the range of 100-1000 µg [6]. Compared to normal cells, the proliferating cells exhibit enhanced FA consumption due to their significant contribution to biosynthetic pathways. This strongly supports the link of FA to cancer [7]. Due to its structure and pH sensitivity, the intake of this nutrient to cells is highly limited. In normal cells, the uptake of FA occurs via two major mechanisms, which involve cellular

Scope of the Study
Despite the effective localization of tumours in vivo or in vitro for targeted drug/gene/ imaging agent delivery, few studies have reported the electrochemical detection of cancer cells using FA. However, in our study for the sensor development, we utilized a biocompatible rGO nanocomposite green synthesized by Ganoderma lucidum (G.l.) extract. Herein, utilizing FA targeting property combined with the excellent electrocatalytic potential of the rGO nanosheets is explored as the electrochemical sensing platform for the real-time analysis of FR for cancer diagnosis application. This rGO-FA electrochemical sensor could be utilized to detect increased FRs in the body fluids as early detection for cancer progression. Thus, it can be used to monitor and control the progression of diseases from their advanced forms. It is a fast, reliable, and economical means of detecting cancer easily. FR is utilized in this model study to mimic the cancer cell environment. The concentration of FR can be expressed in molar units since in cancer cells, FR is usually found in picomolar. For instance, a study by Doucette et al. [67] explains that the FR levels in the case of JAR cells were found to be in the range of 4-26 pmol/mg protein, and in the case of Caco-2 cells, the FR levels were found to be in the range of 0.2-1.5 pmol/mg protein. Similarly, for MA−104 cells, the FR levels were found to be in the range of 0.3-17 pmol/mg protein. Considering these FR ranges, the limit of detection in the current study is 1.69 pM, significant and comparable with previous findings.

Synthesis of rGO
Initially, GO was synthesized from graphite flakes by modified Hummer's method [68]. rGO was prepared using G.l. extract following our previously reported protocol [59]. Briefly, an equivalent amount of GO (0.1 mg/mL) and G.l. extract was stirred at 60 • C for up to 12 h, followed by multiple washing with distilled water.

Synthesis of rGO-FA
FA was dissolved in DMSO and transferred to an aqueous solution of rGO at pH 7 (1:10 ratio). FA and rGO were allowed to react by stirring for 12 h at 37 • C. Black boxes were used during the reaction to prevent any possible light exposure. After the reaction, the solution was centrifuged at 10,000 rpm for 5 min to remove free FA, which was not bound to rGO. The produced rGO-FA was then freeze-dried.

Characterization Techniques
The UV-Vis absorption spectra of rGO, FA, and rGO-FA nanocomposite were investigated using aqueous solutions of the samples in a Lambda 35 Spectrophotometer (Perkin Elmer, Waltham, MA, USA). The morphological studies using dried samples were carried out using field emission scanning electron microscopy Quanta SEM 400 instrument (Quanta 400 FEI, OR, USA), high-resolution transmission electron microscopy (HRTEM, Philip model JOEL, Tokyo, Japan), and atomic force microscopy Agilent Technologies AFM System (Agilent Technologies, Santa Clara, CA, USA) using ultra-sharp tip (non-contact high resonance frequency, nanosensor probe). The crystalline properties of the dried samples were examined using X'Pert Pro diffractometer (XRD, PANalytical, Almelo, the Netherlands), with CuKα radiation and a step size of 0.001 • (2θ). The presence of specific chemical bonds and functional groups were examined from the characteristic vibrations and corresponding peaks in the range of 4000-400 cm −1 using a Fourier transform infrared (FTIR) spectrometer (Spectrum RX1, Perkin Elmer, TX, USA) using the dried samples mixed with KBr spectroscopic grade Potassium bromide (KBr) as the window material to make pellets to analyze the IR-based vibrations. Elemental composition was determined using X-ray photoelectron spectroscopy (XPS, Kratos Axis Ultra, Shimadzu, Japan) using Al Kα monochromatic radiation at 1486.6 eV with a step size of 0.1 eV and 20 eV pass energy.

Electrochemical Measurements
The electrochemical studies were performed using a PAR-VersaSTAT-3 electrochemical workstation (Princeton Research, TN, USA). The studies were conducted on a threeelectrode electrochemical cell system at room temperature (RT). The glassy carbon (GC) electrode was used as a working electrode; platinum and silver/silver chloride (Ag/AgCl) electrodes were used as counter and reference electrodes, respectively. Before modification, the GC electrode was cleaned by polishing with 0.03-micron alumina slurry and distilled water, followed by continuous potential cycling between +1 and −1 V in 0.1 M H 2 SO 4 . The

Results and Discussion
UV-Vis spectroscopic analysis of rGO, FA, and rGO-FA resulted in characteristic absorption peaks, as shown in Figure 1. The peak at 260 nm in rGO is a red-shifted peak from the characteristic peak of GO at 230-240 nm. In FA, the UV-Vis spectrum varied depending on the system's pH [69]. In our investigation, FA at pH 7 exhibited a sharp peak at 280 nm. rGO-FA exhibited a slight blue shift with a sharp peak positioned at 276 nm corresponding to FA deposited on rGO nanosheets. Similarly, the small hump appearing around 300-350 nm could be the red-shifted peak of rGO. Peak shift in nanomaterials is associated with changes in the size of the material. Here, similar to polymerization, the conjugation of FA molecules on the surface of rGO resulted in the change of size and the slight shift in the absorption peaks [70,71]. The FA is directly attached to the rGO sheets by π-π interaction.
Nanomaterials 2021, 11, x FOR PEER REVIEW 5 of 17 tion, the GC electrode was cleaned by polishing with 0.03-micron alumina slurry and distilled water, followed by continuous potential cycling between +1 and −1 V in 0.

Results and Discussion
UV-Vis spectroscopic analysis of rGO, FA, and rGO-FA resulted in characteristic absorption peaks, as shown in Figure 1. The peak at 260 nm in rGO is a red-shifted peak from the characteristic peak of GO at 230-240 nm. In FA, the UV-Vis spectrum varied depending on the system's pH [69]. In our investigation, FA at pH 7 exhibited a sharp peak at 280 nm. rGO-FA exhibited a slight blue shift with a sharp peak positioned at 276 nm corresponding to FA deposited on rGO nanosheets. Similarly, the small hump appearing around 300-350 nm could be the red-shifted peak of rGO. Peak shift in nanomaterials is associated with changes in the size of the material. Here, similar to polymerization, the conjugation of FA molecules on the surface of rGO resulted in the change of size and the slight shift in the absorption peaks [70,71]. The FA is directly attached to the rGO sheets by π-π interaction. In Figure 2, the water-mediated synthesis approach of rGO, FA, and rGO-FA exhibited peaks corresponding to the -OH stretching vibrations at 3400 cm −1 . rGO and rGO-FA showed peaks at 1400 cm −1 which could be due to the C=C stretching of aromatic groups. Similarly, FA and rGO-FA exhibited peaks at 1640 cm −1 corresponding to the C=O of carboxylic acid. The presence of peaks in rGO and rGO-FA at 1100 cm −1 is due to the C-O stretching vibration of carboxylic groups [59]. The multiple characteristic peaks of FA from 1600 to 1500 cm −1 correspond to different functional groups, such as C=O, C=N, C=C, and NH bending [72], and other observed peaks, as well as the corresponding functional In Figure 2, the water-mediated synthesis approach of rGO, FA, and rGO-FA exhibited peaks corresponding to the -OH stretching vibrations at 3400 cm −1 . rGO and rGO-FA showed peaks at 1400 cm −1 which could be due to the C=C stretching of aromatic groups. Similarly, FA and rGO-FA exhibited peaks at 1640 cm −1 corresponding to the C=O of carboxylic acid. The presence of peaks in rGO and rGO-FA at 1100 cm −1 is due to the C-O stretching vibration of carboxylic groups [59]. The multiple characteristic peaks of FA from 1600 to 1500 cm −1 correspond to different functional groups, such as C=O, C=N, C=C, and NH bending [72], and other observed peaks, as well as the corresponding functional groups, are listed in Table S1. In rGO-FA, the presence of similar peaks with slight red shifting confirms the conjugation of FA on rGO's surface.
groups, are listed in Table S1. In rGO-FA, the presence of similar peaks with slight red shifting confirms the conjugation of FA on rGO's surface. The XRD analysis of rGO showed a characteristic peak shown in Figure 3 at an angle of 22° with (002) crystal plane. XRD analysis of FA exhibited multiple diffraction peaks at 2θ values of 11°, 13°, 19°, 23°, 27°, 29°, 31°, 35°, 40°, and 45°, in agreement with the FA standard JCPDS files 42-1963 and 29-1716 [73,74]. In rGO-FA, similar FA peaks were observed with a slight shift in the 2θ position. Additionally, rGO-FA showed a broad peak at 21° corresponding to the (002) crystal plane of rGO.   Table S1. In rGO-FA, the presence of similar peaks with slight red shifting confirms the conjugation of FA on rGO's surface. The XRD analysis of rGO showed a characteristic peak shown in Figure 3 at an angle of 22° with (002) crystal plane. XRD analysis of FA exhibited multiple diffraction peaks at 2θ values of 11°, 13°, 19°, 23°, 27°, 29°, 31°, 35°, 40°, and 45°, in agreement with the FA standard JCPDS files 42-1963 and 29-1716 [73,74]. In rGO-FA, similar FA peaks were observed with a slight shift in the 2θ position. Additionally, rGO-FA showed a broad peak at 21° corresponding to the (002) crystal plane of rGO.  SEM images provided a 2D morphology analysis of the rGO, FA, and rGO-FA nanocomposite ( Figure 4). rGO appeared crumbled with the stacking of few layers, showing the effective conversion of GO to rGO during the reduction. The images of FA exhibit flake-like morphology with signs of agglomeration. The SEM images of rGO-FA show homogenous dispersion of FA on the surface of rGO. In the magnified image of rGO-FA, the transparent layers of rGO could be observed, which shows signs of interruption to the prior stacking due to the effective incorporation of FA in between the rGO layers. This confirms the successful loading of FA on rGO layers. A detailed morphological analysis was carried out by HRTEM ( Figure 5), where the rGO appeared wrinkled with signs of stacking of few layers, agreeing to the SEM images of rGO. In rGO-FA, transparent rGO sheets could be observed with FA uniformly distributed on the rGO sheets. FA appears as flower-like structures on the surface of the rGO sheets that facilitate and maintain the few-layer structure of rGO by preventing the rest of the exfoliated layers. A detailed morphological analysis was carried out by HRTEM ( Figure 5), where the rGO appeared wrinkled with signs of stacking of few layers, agreeing to the SEM images of rGO. In rGO-FA, transparent rGO sheets could be observed with FA uniformly distributed on the rGO sheets. FA appears as flower-like structures on the surface of the rGO sheets that facilitate and maintain the few-layer structure of rGO by preventing the rest of the exfoliated layers.  A detailed morphological analysis was carried out by HRTEM ( Figure 5), where the rGO appeared wrinkled with signs of stacking of few layers, agreeing to the SEM images of rGO. In rGO-FA, transparent rGO sheets could be observed with FA uniformly distributed on the rGO sheets. FA appears as flower-like structures on the surface of the rGO sheets that facilitate and maintain the few-layer structure of rGO by preventing the rest of the exfoliated layers. To analyze the 3D profile/thickness of the rGO-FA nanocomposite, AFM analyses were carried out. rGO-FA showed a height profile of <5 nm and based on our earlier report, the thickness of rGO produced was <2 nm [59], which supports the effective loading of FA on the surface of rGO ( Figure S1). This finding is also in agreement with the other morphological characterization.
EDAX analysis confirms the elemental composition of rGO, FA, and rGO-FA. As shown in Table 1 The XPS spectra of the rGO-FA could be used to identify the elemental composition of rGO-FA after FA binding ( Figure S2). The wide scan analysis revealed the atomic % values of C1s, O1s, and N1s as 67.36, 18.65, and 13.99, respectively. The existence of C-C, C-O, C=O, and COO bonds reveals either rGO or FA. Moreover, other bonds were also observed corresponding to N interaction with O and C, specifically NC=O bonds positioned at 401 eV and N1s peak at 399 eV corresponding to C=N. These N bonds could be contributed to by the conjugation of FA and rGO [75].
The electrochemical properties of rGO-FA were evaluated by cyclic voltammetry (CV) using the three-electrode system with glassy carbon (GC) as working electrode, Ag/AgCl as reference and platinum as the counter electrode. A redox couple [Fe(CN) 6  A stepwise modification was observed in the CV analysis of the modified electrodes compared to the bare GC, as shown in Figure 6. Changes in the peak-to-peak separation and associated anodic and cathodic current responses represented the charge transfer barriers in the electron transfer kinetics of [Fe(CN) 6 ] 3−/4− . The decrease in the redox couple's peak currents could be due to the insulating behaviour of the biomolecules [76]. Compared to the bare GC and rGO, FA's non-covalent functionalization on the rGO showed a decrease in the current in [Fe(CN) 6 ] 3−/4− . FR's introduction to the system promotes effective interaction of FA with FR, and the current flow was significantly reduced. A decrease in the current corresponds to the effective interaction of FA and FR. Upon adding FR, an insulating layer was formed on the surface of rGO-FA/GC, causing a blockage to the interfacial electron transfer and thus resistance to electron flow [77].
The electrochemical impedance spectroscopy (EIS) analysis is a sensitive method in detecting the interfacial changes in the impedance of electrodes with response to the addition of biomolecules or living cells such as macrophages, endothelial cells, fibroblasts, bacterial cells, or cancer cells. Upon cells' addition, an insulating effect is developed on the surface of these cells [78]. The EIS curve has two portions: a semicircle and a linear section. The semi-circular curve at a higher frequency corresponds to the electron transfer process, and the semicircle diameter is calculated as the electron transfer resistance, R et . The linear portion at low frequencies denotes the diffusion process [76]. EIS spectra of different GC modified electrodes in Figure 7 show the resistance developed on the electrode-electrolyte interface during the charge transfer. The bare GC exhibits the least resistance to charge transfer, and rGO-FA shows the highest resistance in charge transfer. This demonstrates the effective π-π interaction of rGO and FA, which prevents electron flow. Finally, upon the addition of FR to the rGO-FA-modified electrode, the bio-recognition of FR takes place at the modified electrode, which further prevented the charge transfer at the electrodeelectrolyte interface. The electrochemical impedance spectroscopy (EIS) analysis is a sensitive method in detecting the interfacial changes in the impedance of electrodes with response to the addition of biomolecules or living cells such as macrophages, endothelial cells, fibroblasts, bacterial cells, or cancer cells. Upon cells' addition, an insulating effect is developed on the surface of these cells [78]. The EIS curve has two portions: a semicircle and a linear section. The semi-circular curve at a higher frequency corresponds to the electron transfer process, and the semicircle diameter is calculated as the electron transfer resistance, Ret. The linear portion at low frequencies denotes the diffusion process [76]. EIS spectra of different GC modified electrodes in Figure 7 show the resistance developed on the electrode-electrolyte interface during the charge transfer. The bare GC exhibits the least resistance to charge transfer, and rGO-FA shows the highest resistance in charge transfer. This demonstrates the effective π-π interaction of rGO and FA, which prevents electron flow. Finally, upon the addition of FR to the rGO-FA-modified electrode, the bio-recognition of FR takes place at the modified electrode, which further prevented the charge transfer at the electrode-electrolyte interface. Due to the complexities of redox reactions at the biomolecular level, CV analysis for detecting FR using the modified rGO-FA/GC gave negligible current responses. Though EIS was widely used for live cell-based sensor analysis, previous studies reported an extreme change in the resistance by extending the analysis duration [76]. This could be due to the cells' possible detachment, caused by prolonged electric field associated cell death. Thus, in the current study, the detection mode of analysis was switched to differential pulse voltammetry (DPV) instead of a CV to obtain a faster and reliable response. Interestingly, DPV analysis was very sensitive towards the detection of FR. As shown in Figure  8, the highly sensitive detection of FR was conducted via DPV by introducing FR-β at regular intervals of 60 s. The addition of FR showed a significant reduction in the peak current response due to the FA-FR interaction.
The DPV analysis in the concentration range of 6−100 pM (Figure 8) resulted in a linear relationship between peak current density (j) and log FR concentration with a correlation coefficient, R 2 = 0.9101. The calibration plot of peak current vs concentration of FR from 6 to 100 pM resulted in a linear equation with I (mA) = 4.638−0.037 [FR] (pM) with a detection limit of 1.69 pM (at an S/N ratio of 3), with milliseconds response time and sensitivity of 0.037 µA pM −1 cm 2 , which is highly comparable to other earlier reported detection systems ( Table 2).  Due to the complexities of redox reactions at the biomolecular level, CV analysis for detecting FR using the modified rGO-FA/GC gave negligible current responses. Though EIS was widely used for live cell-based sensor analysis, previous studies reported an extreme change in the resistance by extending the analysis duration [76]. This could be due to the cells' possible detachment, caused by prolonged electric field associated cell death. Thus, in the current study, the detection mode of analysis was switched to differential pulse voltammetry (DPV) instead of a CV to obtain a faster and reliable response. Interestingly, DPV analysis was very sensitive towards the detection of FR. As shown in Figure 8, the highly sensitive detection of FR was conducted via DPV by introducing FR-β at regular intervals of 60 s. The addition of FR showed a significant reduction in the peak current response due to the FA-FR interaction.
The DPV analysis in the concentration range of 6−100 pM (Figure 8) resulted in a linear relationship between peak current density (j) and log FR concentration with a correlation coefficient, R 2 = 0.9101. The calibration plot of peak current vs concentration of FR from 6 to 100 pM resulted in a linear equation with I (mA) = 4.638−0.037 [FR] (pM) with a detection limit of 1.69 pM (at an S/N ratio of 3), with milliseconds response time and sensitivity of 0.037 µA pM −1 cm 2 , which is highly comparable to other earlier reported detection systems (Table 2). Interaction of similar interfering biomolecules is a critical concern in sensor development. Under normal conditions, human serum is free of FR. However, during cancer progression or macrophage associated inflammations, the overexpression of FR, especially FR-β, occurs in the serum. Blood serum involves three major serum proteins (SP): albumins, globulins, and fibrinogen. Among these, human serum albumin (HSA) is the most abundant protein in plasma, existing as <50% of the total SP [91]. Thus, the introduction of HSA could be used as a prominent interference analysis. Upon introducing SP/HSA (100 pM) to the rGO-FA/GC sensor, SP's interference did not exhibit any significant difference in the current response ( Figure 9) compared with the detection signals of FR. This finding facilitates using rGO-FA for the selective and reliable detection of FR even in the presence of interfering biomolecules, and the three repeated experiments ensured reproducibility.  CNTs@PDA-FA GC HL-60 cells (5 × 10 3 -5 × 10 5 cells/mL) 5 × 10 2 cells EIS [81] MPA/(Fc-PEI/SWNT) Au HeLa cells (10-10 6 cells/mL) 10 cells DPV [62] PNT-FA G HeLa cells (250-5 × 10 3 cells/ mL) 250 cells CV [82] PNT-FA G FR (8-13 nM) 8 nM CV [82] Au/MUA-FA Au HeLa cells (6-10 5 cells/mL) 6 cells EIS [83] Au-FA BDD HeLa cells (10-10 5 cells/ mL) 10 cells EIS [84] FA-AuNPs Au Hela cells (1.3 × 10 5 ) Not indicated CV [85] FA-GSH-GNPs -HeLa cells (10-10 5 cells/mL) 100 cells Absorbance [86] FA/PEI/CMC-G GC HL-60 cells (500-5 × 10 6 cells/ mL) 500 cells EIS [76] FA-MHDA-HT-Fc Au beads HeLa cells (10-10 6 cells/mL) 10 cells DPV [87] rGO Conventionally, protein/antibody/DNA conjugated gold electrodes were utilized for sensitive detection approaches to attain similar LoD values, which are expensive and need special handling [88][89][90]. However, in the current report, the developed rGO-FA-modified GC is a novel system, which is economically feasible, sensitive, and allows rapid detection. Reproducibility of the rGO-FA-modified GC was ensured by three repeated experiments.
Interaction of similar interfering biomolecules is a critical concern in sensor development. Under normal conditions, human serum is free of FR. However, during cancer progression or macrophage associated inflammations, the overexpression of FR, especially FR-β, occurs in the serum. Blood serum involves three major serum proteins (SP): albumins, globulins, and fibrinogen. Among these, human serum albumin (HSA) is the most abundant protein in plasma, existing as <50% of the total SP [91]. Thus, the introduction of HSA could be used as a prominent interference analysis. Upon introducing SP/HSA (100 pM) to the rGO-FA/GC sensor, SP's interference did not exhibit any significant difference in the current response ( Figure 9) compared with the detection signals of FR. This finding facilitates using rGO-FA for the selective and reliable detection of FR even in the presence of interfering biomolecules, and the three repeated experiments ensured reproducibility.

Conclusions
Recognizing FA's vital role as a potential biomarker for detecting FR, which is associated with cancer progression and immune response associated inflammations, a nanocomposite was developed by the conjugation of FA with rGO for the sensitive detection of FR. rGO-FA was well characterized using SEM, TEM, AFM, XRD, XPS, EDAX, and FTIR techniques. The electrocatalytic properties of rGO-FA were analyzed utilizing a three-electrode electrochemical cell with an rGO-FA-modified GC electrode via a DPV technique. Upon adding FR, rGO-FA-modified GC showed significant current responses in a linear concentration range from 6 to 100 pM, with a detection limit of 1.69 pM. SP was introduced to the system to analyze the effect of interfering molecules, resulting in no significant current responses. The specificity and reproducibility of the electrode were analyzed repeatedly. Apart from usual sensing studies, replacing gold or platinum working electrode with a GC as the working electrode proves this system's economic feasibility. Compared to the typical expensive FR detection systems that involve gene/protein/antibodies for the detection of biomolecules, the current rGO-FA electrode offers an economical, fast, and sensitive sensing to detect FR biomarkers. The current novel rGO-FA nanocomposite for FR sensing enunciates a great promise for future cancer detection systems.

Conclusions
Recognizing FA's vital role as a potential biomarker for detecting FR, which is associated with cancer progression and immune response associated inflammations, a nanocomposite was developed by the conjugation of FA with rGO for the sensitive detection of FR. rGO-FA was well characterized using SEM, TEM, AFM, XRD, XPS, EDAX, and FTIR techniques. The electrocatalytic properties of rGO-FA were analyzed utilizing a three-electrode electrochemical cell with an rGO-FA-modified GC electrode via a DPV technique. Upon adding FR, rGO-FA-modified GC showed significant current responses in a linear concentration range from 6 to 100 pM, with a detection limit of 1.69 pM. SP was introduced to the system to analyze the effect of interfering molecules, resulting in no significant current responses. The specificity and reproducibility of the electrode were analyzed repeatedly. Apart from usual sensing studies, replacing gold or platinum working electrode with a GC as the working electrode proves this system's economic feasibility. Compared to the typical expensive FR detection systems that involve gene/protein/antibodies for the detection of biomolecules, the current rGO-FA electrode offers an economical, fast, and sensitive sensing to detect FR biomarkers. The current novel rGO-FA nanocomposite for FR sensing enunciates a great promise for future cancer detection systems.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/nano11051272/s1, Figure S1: AFM image of rGO-FA and the height profile at cross-section with average thickness as 5 nm, Figure S2: XPS analysis of rGO-FA, displaying various bonds supporting the conjugation of rGO and FA and elemental analysis as an atomic percentage. Table S1: FTIR peaks of FA and its corresponding functional groups.