Carbon fiber based electrochemical sensor for sweat cortisol measurement

This study examines the use of a conductive carbon fiber to construct a flexible biosensing platform for monitoring biomarkers in sweat. Cortisol was chosen as a model analyte. Functionalization of the conductive carbon yarn (CCY) with ellipsoidal Fe2O3 has been performed to immobilize the antibodies specific to cortisol. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) chemistry has been used to immobilize the antibodies onto the Fe2O3 modified CCY. Crystallinity, structure, morphology, flexibility, surface area, and elemental analysis were studied using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (FE-SEM/EDS) and Brunauer–Emmett–Teller (BET) analysis. Mechanical properties of the fiber such as tensile strength, young’s modulus have also been investigated. Under optimal parameters, the fabric sensor exhibited a good linearity (r2 = 0.998) for wide a linear range from 1 fg to 1 μg with a detection limit of 0.005 fg/mL for the sensitive detection of cortisol. Repeatability, reliability, reproducibility, and anti-interference properties of the current sensor have been investigated. Detection of cortisol levels in human sweat samples has also been investigated and the results were validated with commercial chemiluminescence immunoassay (CLIA) method.

Bleached and scoured CCY with the density and diameter of 0.35 g/cm 3 and ~350 μm was purchased from Vinpro Tech, Hyderabad, India. The purchased CCY was ultrasonicated and dried at ambient temperature under vacuum for 24 h to remove any impurities prior to characterization and material deposition.

Synthesis of ellipsoidal Fe 2 O 3 nanostructures integration with CCY (Fe 2 O 3 /CCY). Ellipsoidal
Fe 2 O 3 was integrated with CCY using the simple hydrothermal method, wherein stoichiometric amounts of pluronic f127 were added to an aqueous solution of FeCl 3 .6H 2 O with continuing magnetic stirring. Following this, the obtained homogeneous solution was transferred into a Teflon vial (65 mL). CCY was immersed in this solution, placed in a sealed autoclave and heated at 180 °C for twelve hours. The resulting yarn was allowed to cool down to room temperature naturally, the yarn was collected and washed with alcohol, double distilled water for four times to remove remaining impurities. Then the product was dried at 80 °C overnight. The working electrode was kept at ~5 cm in length and 4 cm length fiber was soaked into the electrolyte solution.
A standard process, reported previously, has been utilized to immobilize Anti-C mab covalently onto Fe 2 O 3 /CCY electrode 41 . The immobilization of Anti-C mab with Fe 2 O 3 /CCY electrode was achieved via electrostatic interaction using EDC as the coupling agent and NHS as the activator due to the high isoelectric point (IEP) of Fe 2 O 3 (8.5) and antibody (4.5) using physical adsorption. To fabricate the electrochemical immunosensor electrode, 70 μL of 2 μg/mL, Anti-C mab solution was mixed with the Fe 2 O 3 /CCY in 5 mL PBS (10 mM, pH 7.0) solution containing 0.4 M NHS and 0.4 M EDC and incubated for 2 h in a humid chamber. The fabricated Anti-C mab /Fe 2 O 3 /CCY electrode was washed with PBS (10 mM, pH 7.0) to remove any loosely bound molecules. Following this the sensor electrode was immersed in 50 μL of BSA (10 μg/mL) in PBS (10 mM, pH 7.0) and incubated for 30 mins for blocking nonspecific binding sites on the Anti-C mab /Fe 2 O 3 /CCY electrode surface. As fabricated BSA/Anti-C mab / Fe 2 O 3 /CCY electrode was further washed using PBS (10 mM, pH 7.0) and stored at 4 °C. Figure 1a,b shows the schematic of Fe 2 O 3 /CCY preparation by hydrothermal method and subsequent sensor fabrication.
Characterization. Structural features of Fe 2 O 3 nanostructures were investigated using a PANalytical (X'Pert-Pro) diffractometer (using Cu Kα radiation at a wavelength of 1.5406 Å) and the XRD patterns were recorded in the diffraction angle (2 Theta) ranging from 20° to 70°. The functional groups of the nanocomposite materials were identified by Bruker Tensor 27 Fourier transform infrared spectrometer (FT-IR). Raman spectra were recorded at using a Horiba Jobin-LabRam-HR system at 514 nm excitation focused through a 100x microscope objective for a total spot size of 1 μm. Excitation power was held constant at 150 μW for all samples. The morphologies were observed by field emission scanning electron microscopy (FESEM) on FEI Quanta-250 FEG microscope. The compositions of the materials were confirmed using energy dispersive X-ray spectrometry (EDS) by the FESEM attachment. The high-resolution transmission electron microscopy (HRTEM) images were recorded on a JEM-2100FS instrument (JEOL) operating at 200 kV to further confirm the morphology of the samples. Surface area measurements were carried out using Brunauer-Emmett-Teller (BET) Quantachrome Nova 1200e (USA) instrument with N 2 as the analysis gas. The mechanical properties of the pristine and modified CCY were examined using the universal testing machine (UTM) (Zwick Roell).

Electrochemical characterizations.
Electrochemical measurements were carried out to analyze the electro active behavior of the electrodes. The electro activity of CCY, Fe 2 O 3 /CCY, Anti-C mab /Fe 2 O 3 /CCY and BSA/ Anti-C mab /Fe 2 O 3 /CCY electrodes was studied in a 20 mL of PBS (pH 7.0) containing 5 mM Fe(CN) 6 3−/4− . The conventional three-electrode electrochemical workstation (BioLogic SP-50) was used for all the experiment. In the cell, the BSA/Anti-C mab /Fe 2 O 3 /CCY was used as the working electrode. A platinum wire and Ag/AgCl was used as the counter and reference electrodes, respectively. All the electrochemical measurements were made thrice and the average is used for analysis. Sweat collections for analysis. Sweat samples were collected immediately after 30-45 minutes of vigorous exercise were the age group of 27-35 with proper permission. The exercise entailed either running on treadmill or rowing on an ergometer for a minimum of 10 minutes. Sweat samples were collected in the evening to examine whether sweat cortisol concentrations approximated the well-established theoretical value. For sweat collection, a cotton swab was rubbed over the scalp hair and neck, allowed to saturate and placed it in a 5 mL Eppendorf tube 42 . The collected samples were centrifuged for 5 minutes and 1 mL of the supernatant was pipetted out in and stored at −20 °C to maintain its biological characteristics. These samples were further used to detect cortisol using electrochemical immunosensor and for comparison with commercial cortisol assay. Ethical approval for this study was granted by the Department Ethics Committee of Bharathiar University (BU). All experiments were performed in accordance with relevant guidelines and regulations and all experimental protocols were approved by the Department Ethics Committee, BU. Informed consent was obtained from all subjects.

Results and Discussion
Structure and morphology of the CCY and α-Fe 2 O 3 . The crystal structures and the phases of samples were investigated by XRD measurements and the resulting XRD diffraction patterns are displayed in Fig. 2A 45 .
In order to investigate the interaction between Fe 2 O 3 and CCY, the FT-IR spectrum has been performed. Figure 2B shows the chemical information and major functional groups existing in the pure CCY and  47,48 . In addition, weak peak around 1413 cm −1 roots in C-O deformation vibration, implying the existence of intramolecular hydrogen bonding between CCY and Fe-OH, which is beneficial in keeping the stability of material structure. A remarkable decrease in the absorption of C=O, O-H (deformation vibration) and the C-O groups were observed, proving that most of the oxygen containing groups were removed 31 . From the FT-IR results, we corroborate that the Fe 2 O 3 are covered to the CCY surface uniformly.
Raman spectroscopy was used to further confirm the structural characteristics of nanocomposite electrode materials. Raman spectra of the pure CCY and Fe 2 O 3 /CCY are taken in the range of 100-3500 cm −1 and shown in Fig. 2C. The D-band located at 1355 cm −1 and the G-band at 1584 cm −1 are the characteristic Raman shifts of carbon, which can be observed in both pure CCY and Fe 2 O 3 /CCY. The G band assigned to the first order scattering of the E 2 g phonon of CCY represent the in plane bond stretching vibration of sp 2 -bonded carbon atoms in a 2D hexagonal lattice. The D band is associated with the breathing mode of K-point phonons of A 1 g symmetry with vibration of carbon atoms with angling bonds in plane terminations of disordered carbon yarn. In addition to this, the appearance of two weak peaks at 2693 and 2944 cm −1 can be assigned to the 2D band, which originates from the second-order Raman scattering process 49 . As we can see from the spectra of Fe 2 O 3 /CCY, the Fe 2 O 3 sample exhibited the bands at 210, 274 388 and 610 cm −1 indicating the presence of Fe 2 O 3 (hematite) phase with the D 6 3d crystal space group 50,51 . As expected, the spectra of Fe 2 O 3 /CCY sample also exhibited peaks due corresponds to carbon. This result implies a tight integration of Fe 2 O 3 nanostructures on CCY and supporting the XRD results very well.
The morphology of the CCY and Fe 2 O 3 /CCY were elucidated by FESEM and the corresponding images are shown in Fig. 3A. As seen in Fig. 3A(a), the morphology of pure CCY is consists of smaller fibers and reveals that there are no impurities on the smooth surface. The inset figure shows the clearer version of the smooth fiber. High-magnification FESEM images shown in Fig. 3(b-d) provide clear information about the ellipsoidal Fe 2 O 3 nanostructures. As depicted the carbon yarn surfaces are uniformly covered by ellipsoidal Fe 2 O 3 nanostructures and it reveals that the material consists of uniform size with the diameter of 300-350 nm, while the length is ~800-850 nm. The large number of Fe 2 O 3 nanoparticles were uniformly anchored onto the carbon yarn surface via self-assembly due to the differences in surface charges resulting in strong electrostatic interactions, which is expected to improve the electrochemical properties of Fe 2 O 3 resulting in enhanced sensing performance. Figure 3A(e,f) show images of the integrated ellipsoidal Fe 2 O 3 /CCY electrode which can be freely rolled up with tweezers. It can be clearly observed that the electrodes exhibit excellent flexibility, which makes them possible for application in flexible and wearable devices.
The successful synthesis of Fe 2 O 3 ellipsoid on CCY and its chemical composition was further confirmed by EDS mapping analysis. As shown in Fig. 3B, iron and oxygen are two elements coated on CCY apart from the carbon that relates to the substrate. Moreover, elemental mapping technique shown in Fig. 3C further established the presence of Fe 2 O 3 on yarn. These images verify a homogeneous coating of the yarn with Fe 2 O 3 nanoparticles.
To extensively study the morphology of the nanostructures, we have examined the HRTEM imaging and selected area electron diffraction (SAED) patterns for the synthesized Fe 2 O 3 . Figure 4(a,b) depicts the typical HRTEM images of Fe 2 O 3 nanoparticles and indicates the ellipsoidal morphology with a homogeneously well dispersed structure which is consistent with the FESEM micrograph. The ellipsoidal Fe 2 O 3 nanostructure is beneficial for electrode materials due to the large surface area. In Fig. 4c   for electrochemical sensor to evaluate its cortisol sensing performance. CV was used to study the electro activity of the functionalized electrode to understand the electrochemical behavior of the electrode. Figure 5 shows the CV studies of the bare CCY, Fe 2 O 3 /CCY, Anti-C mab /Fe 2 O 3 /CCY, BSA/Anti-C mab /Fe 2 O 3 /CCY immunoelectrode in PBS (10 mM, pH 7.0). The bare CCY exhibited oxidation and reduction current magnitude in the range of ~10 −6 A, which is a typical characteristic of bare CCY (as inset). The oxidation current response increases to 120.7 μA for Fe 2 O 3 /CCY electrode and it only shows the redox peak of Fe 2 O 3 which , the anodic oxidation peak corresponds to the oxidation of Fe 2+ to Fe 3+ and the cathodic peak corresponds to the reduction of Fe 3+ to Fe 2+ . The magnitude Effect of pH and scan rate. To investigate the optimal pH, the activity of BSA/Anti-C mab /Fe 2 O 3 /CCY immunoelectrode was investigated in the pH range of 6.0 to 8.0 (Fig. 6). It was observed that the oxidation and   Fig. 6). However, it was observed the most stable oxidation and reduction peak area and current response is high for pH 7.0. Thus, pH 7.0 was selected as the working electrolyte pH. Also, pH 7.0 mimics biological conditions and thus chosen as the supporting electrolyte pH for all successive experiments. The electrochemical behaviour of the BSA/Anti-C mab /Fe 2 O 3 /CCY immunoelectrode was also studied using CV as a function of scan rates from 5-100 mV/s in PBS (10 mM, pH 7.0) which is shown in Fig. 7 and the change in anodic and cathodic current response with scan rate is plotted in Fig. 7 as insert. It is observed that the magnitude of the current is linearly dependent on the scan rates and the equations are given in Eqs 1 and 2.  The well-defined redox peak suggests that the diffusion of electron is surface controlled. The separation of peaks suggests that the process is not perfectly reversible but the stable redox peak current and position during the repeated scans at a particular scan rate suggests that the immunoelectrode exhibits a quasi-reversible process 7 . It is observed that the prepared immunoelectrode showed characteristic redox peak for Fe 2 O 3 in PBS and exhibit stability up to 100 mV/s with slight peak -to -peak separation (ΔE p ) of 122 mV, which evidenced the two electron transfer process. Additionally, the anodic peak to cathodic peak current ratio of (I pc /I pa ) of prepared BSA/ Anti-C mab /Fe 2 O 3 /CCY immunoelectrode is 0.822 52 . The obtained low working potential helps to avoid possible interference from the biological samples. Further, the fabricated BSA/Anti-C mab /Fe 2 O 3 /CCY immunoelectrode can be used as a mediator free electrochemical biosensor. It was found that at a scan rate of 50 mV/s, the electrode exhibit stability and equal oxidation and reduction peak area and current values. Thus, all further CV studies were carried out at a scan rate of 50 mV/s.

Cortisol response studies of BSA/Anti-C mab /Fe 2 O 3 /CCY immunoelectrode. The electrochemical
response of BSA/Anti-C mab /Fe 2 O 3 /CCY immunoelectrode has been studied using CV technique using PBS (pH 7.0, 10 mM) at scan rate of 50 mV/s as a function of cortisol concentration ranging from 1 fg to 1 μg in three electrode system. Different BSA/Anti-C mab /Fe 2 O 3 /CCY immunoelectrode prepared in the same batch were found to exhibit similar current values with maximum variation of ±3%. The magnitude of the electrochemical current response decreases as a function of increasing cortisol concentration. The formation of insulating immune complex between Anti-C mab and cortisol that hinders electron transport is attributed to decreased current response. This insulating behavior of cortisol binding was investigated and shown in Fig. 8A. As shown in Fig. 8B, the calibration curve between the current response and logarithm of cortisol concentration has been plotted in the range   (Table 1). Therefore, the demonstrated method exhibited a good analytical performance for cortisol detection and could be used as a wearable platform for the detection of cortisol in real samples.  Performance of BSA/Anti-C mab /Fe 2 O 3 /CCY immunoelectrode by DPV. Also, the electrochemical response of BSA/Anti-C mab /Fe 2 O 3 /CCY immunoelectrode has been studied by DPV under similar condition used for CV. The DPV is sensitive analytical technique to study the electrochemical changes during biological reaction on the surface when the analyte concentration is very low 53 . Figure 9 revealed the peak current of the   BSA/Anti-C mab /Fe 2 O 3 /CCY immunoelectrode decreasing when increasing the cortisol concentration. This confirms the effective formation of an immune-complex between antigen and antibody and the hindrance in electron transfer to the electrode due to the insulating behavior of cortisol. It is clear from Fig. 9A that linear curve attained between the logarithmic concentration of cortisol and decrement of current revealed good linear range from 1 fg -1 μg. The linear regression equation was obtained as I (μA) = 2.133 + 83.665 log C cor (g/mL) with a correlation coefficient (R 2 ) of 0.9977. The detection limit was estimated to be about 0.003 fg/mL. The results from three successive experiments (n = 3) for the concentration on different immunoelectrode are indicted by the error bars. As the DPV method is highly sensitive compared to CV in trace level analyte detection, the lower detection of limit was calculated from DPV outcomes as 0.003 fg. The high sensitivity of the BSA/Anti-C mab /Fe 2 O 3 /CCY immunoelectrode might resulted from high specific surface area and electrochemically accessible active surface area. The high specific surface area of Fe 2 O 3 /CCY (146.02 m 2 /g) is calculated using BET analysis for ellipsoid morphological Fe 2 O 3 which is higher than the other morphologies given in the previous reports. Also, the electrochemically accessible active surface area (A e ) were calculated using the standard Randle-Sevcik equation. The calculated A e values (bare CCY is 0.0700 cm 2 and Fe 2 O 3 /CCY is 0.0802 cm 2 ) also ensured the high sensitivity of the prepared electrode 54 .
Interference studies. It is also important to evaluate how specifically and precisely the proposed sensing platform can detect cortisol in the presence of various interfering samples. The selectivity of the BSA/Anti-C mab / Fe 2 O 3 /CCY immunoelectrode towards cortisol (100 fg/mL) have been tested with interference especially cortisol analogous, for progesterone (100 fg/mL), cortisone (100 fg/mL), BSA (100 fg/mL) and cholesterol (100 fg/mL) using CV technique in PBS (10 mM, pH 7.0). As shown in Fig. 10, CV spectra exhibited a clear distinction of cortisol over progesterone, cortisone, BSA and cholesterol and decrement of electrochemical current response upto 8% to cortisol analogous, it can be concluded that the flexible and mediator free immunosensor electrode is highly selective towards cortisol estimation.

Reproducibility and stability of the immunosensor.
To examine the repeatability of the BSA/ Anti-C mab /Fe 2 O 3 /CCY immunoelectrode, five separate electrodes were prepared and analyzed by CV technique. The average relative standard deviation (RSD) of immunosensor was found to be 3.48% for five measurements of 100 fg/mL of cortisol. Additionally, the immunosensor was stored at 4 °C for 30 days and it was used to detect cortisol samples. The BSA/Anti-C mab /Fe 2 O 3 /CCY immunosensor still retained 95.28% of its response. The excellent stability of the immunosensor attributed to the strong interaction to the cortisol. The slight decrement in response might be due to the long-term deactivation of the immobilized biomolecules. Therefore, these results specified the fabricated immunosensor has agreeable reproducibility and stability.
Determination of cortisol in sweat samples. We further examined the practicability of prepared immunosensor through analyzing real sweat samples and the results are given in Table 2. Herein, CV method was used to detect the cortisol level in sweat. The RSD of the proposed immunosensor from 3.403% to 4.064% and the recovery rates of the samples ranged between 99.62% and 104.21%. The significant recovery percentage of cortisol in various sweat samples were determined. The outcome values were validated using commercially available CLIA sensing method as shown in Fig. 11. The sweat cortisol readings were validated using commercial chemiluminescence immunoassay (CLIA) kit purchased from Abbott Diagnostics (IL, USA). This is a competitive CLIA which uses polyclonal anticortisol antibodies (Supplementary Materials ESI: S2). A good correlation between the electrochemical measurements and CLIA results was observed. The results of both techniques are summarized and tabulated (Table 2).

Conclusions
A highly sensitive and conductive yarn based flexible electrochemical sensor has been developed for the cortisol detection in sweat. EDC/NHS chemistry has been used to immobilize specific cortisol antibodies on the electrode surface. The immunosensor electrode successfully exhibits a sensing range of 1 fg -1g, detection limit of 0.005 fg/ mL with the regression coefficient of 0.9987. The immunoelectrode indicated excellent selectivity to cortisol when compared to cortisol analogous such as cortisone and progesterone and cholesterol. Cortisol detection recovery was 99.62 to 104.21% in human sweat samples. Response time of the immunosensor is 120 sec and the sensing results are correlated well with chemiluminescence immunoassay. The proposed conductive yarn based system has great potential for commercialization as this platform could be readily integrated with fabrics and garments.