Impedimetric Detection of Femtomolar Levels of Interleukin 6, Interleukin 8, and Tumor Necrosis Factor Alpha Based on Thermally Modified Nanotubular Titanium Dioxide Arrays

An inexpensive, easy to prepare, and label-free electrochemical impedance spectroscopy-based biosensor has been developed for the selective detection of human interleukin 6 (IL-6), interleukin 8 (CXCL8, IL-8), and tumor necrosis factor (TNFα)—potential inflammatory cancer biomarkers. We describe a, so far, newly developed and unexplored method to immobilize antibodies onto a titanium dioxide nanotube (TNT) array by physical adsorption. Immobilization of anti-IL-6, anti-IL-8, and anti-TNFα on TNT and the detection of human IL-6, IL-8, and TNFα were examined using electrochemical impedance spectroscopy (EIS). The impedimetric immunosensor demonstrates good selectivity and high sensitivity against human biomarker analytes and can detect IL-6, IL-8, and TNFα at concentrations as low as 5 pg/mL, equivalent to the standard concentration of these proteins in human blood. The calibration curves evidenced that elaborated biosensors are sensitive to three cytokines within 5 ÷ 2500 pg/mL in the 0.01 M phosphate-buffered saline solution (pH 7.4).


Introduction
Chronic inflammatory reactions have received much attention in recent years due to wrong effector mechanisms or immune response [1]. Both may be harmful or cause life-threatening diseases such as cancer, septic shock, aortic valve stenosis, Alzheimer's, heart attack, kidney failure, and surgical complication. Proinflammatory cytokines first appear in the area of damaged tissue (i.e., cancer cell division) and play an important role in transmitting signals into leukocytes through endothelium activation [2]. Several publications [3][4][5] have appeared in recent years documenting the increased expression of interleukin 8 (CXCL8, IL-8), interleukin 6 (IL-6), tumor necrosis factor (TNFα) in cancer cells, infiltrating neutrophils, endothelial cells and tumor-associated macrophages.
IL-8 affects gene expression (via regulation of numerous transcription factor activities), modulates the cellular proteome, and affects cell cytoskeleton organization. IL-8 signaling increases the proliferation and survival of endothelial cells, promotes angiogenic responses, and potentiates endothelial cell migration and the infiltration of neutrophils in the affected area [5]. This confirms the significant role of IL-8 within the inflammatory microenvironment. Accordingly, IL-8 expression correlates with the course of septic shock, angiogenesis, and metastasis of tumors.
Several works [4] recently showed the increased expression of IL-6 and/or its receptors in bone metabolism, hematopoiesis, pathogenesis, inflammatory responses, and cancer progression. explain the physisorption mechanism of the antibody on the TNT layer and substantially supplement the existing literature reports. A newly elaborated method to immobilize antibodies onto TNT by physical adsorption has allowed us to detect IL-6, IL-8, and TNFα protein at 5 pg/mL (192,625, and 295 fM, respectively) in phosphate buffered saline (PBS). The obtained limit of detection is lower than in the standard ELISA kit, matching the physiological level, whereas the higher concentration of cytokines includes the levels corresponding to inflammation states. The total time of determining the concentration of IL-6, IL-8, and TNFα was reduced from 6 to 2 h.
All electrochemical measurements-i.e., anodization of titanium foil, open circuit potential (OCP), and electrochemical impedance spectroscopy (EIS) measurements-were carried out with the AutoLab PGSTAT 302N potentiostat/galvanostat (Eco Chemie, Utrecht, The Netherlands) equipped with a voltage multiplier (30 V) using the standard three-electrode configuration with a titanium electrode and Ti/TNT as the working electrode, the standard Ag/AgCl − silver chloride electrode (E Ag/AgCl = 0.222 V) as the reference electrode, and a platinum foil as the auxiliary electrode at 25 ± 1 • C.

TiO 2 Nanotubes Fabrication
The titanium foil was sonicated in acetone, ethanol, and distilled water and dried in nitrogen. The formation of nanotube oxide layers was performed by electrochemical anodizing in various ethylene glycol solution concentrations with 0.65% wt. NH 4 F. The formation process consists of two stages: the first potentiodynamic and the second potentiostatic. In the beginning, the foil was polarized up to 17 V with a scan rate of 0.5 V/s and then was kept at that potential in the same electrolyte for a further 3750 s. Field emission scanning electron microscopy (FESEM, JEOL JSM-7600F, Tokyo, Japan) and energy-dispersive X-ray spectroscopy (EDS, INCA, Oxford Instruments, Oxford, UK) was used to investigate surface morphology and chemical composition.

Thermal Modification of TNT
The obtained TNT layers were annealed in argon at 550 • C for 2 h with the heating rate of 6 • C/min to the desired annealing temperature. Field emission scanning electron microscopy (FESEM, JEOL JSM-7600F, Tokyo, Japan) and energy-dispersive X-ray spectroscopy (EDS, INCA, Oxford Instruments, Oxford, UK) were used to investigate surface morphology and chemical composition.

Evaluation of the Open Circuit Potential (OCP) and Contact Angle of TNT
The OPC measurements were performed using Ti/TNT samples before and after thermal modification immersed in PBS solution (0.01 M, 50 mL, pH 7.4) for 1800 s.
Dynamic contact angle measurements were performed using distilled water at room temperature using a PG-3 Goniometer equipped with a microscope, a camera, and an integrated micro-pump, which deliver accurate droplets in 0.5 µL steps. Antibodies and IL-6, IL-8, TNFα Detection IL-6, IL-8, TNFα were immobilized on the biosensor platform using the simple drop coating method at room temperature. Annealed in argon, Ti/TNT samples were incubated with 10 µL of IL-6, IL-8, TNFα antibody solutions for different times and then treated with 10 µL of IL-6, IL-8, TNFα antigens solutions of various concentrations ranging from 0 to 2500 pg/mL. After the immobilization process, each sample was washed with a buffer solution (3 × 100 µL) to remove non-specifically bound biomolecules. Spectrophotometric measurements were performed using a Hitachi UV-2310II Spectrophotometer at a wavelength of 450 nm.

Functionalization of TNT Layers with
The response of impedimetric immunosensor was recorded using Electrochemical Impedance Spectroscopy (EIS) method. Impedance analysis was performed over a frequency range from 0.1 Hz to 100 kHz (10 points/decade), at zero DC potential, with an AC amplitude of 10 mV and an integration time of 10 s. The effects of electrolyte pH on the response of elaborated biosensors were studied using IL-6, IL-8, and TNFα antigens with a concentration of 300 pg/mL.

Characteristics of Titanium Dioxide Nanotubes
As shown in Figure 1, the TNT formed in the anodizing process, according to the applied parameters, results in a uniform 1000 ± 100 nm thick layer of vertically aligned nanotubes ( Figure 1a) with a diameter of about 50 ± 5 nm. SEM pictures show homogenous arrays of opened from the top, closed at the bottom, and vertically oriented nanotubes completely covering the titanium foil. No damage was observed on the TNT layer after annealing (Figure 1b). Obtained SEM images confirm that the thermal modification does not influence TNT layer homogeneity and morphology-its height and diameter were not changed. The height and diameter of TNT were chosen based on previous electrochemical analysis, indicating the most favorable sensing and biosensing properties [31]. This structure is characterized by OCP values close to zero, one of the smallest impedance module, and a high Ipa/Ipc ratio for potassium ferricyanide, which confirms good conductivity and good adsorption properties of this structure [29]. Moreover, titanium nanotubes annealed at a temperature of 550 • C in an argon atmosphere have more anatase structures that promote biological element adsorption [32]. However, previous experiments do not attempt antibody immobilization on the TNT surface.
The EDS microanalysis results for samples formed in ethylene glycol and thermally modified in argon are presented in Table 1. According to the formation conditions, the results showed the difference in concentrations of elements in the TiO 2 nanotubes layer. After thermal treatment, fluoride is not identified, and obtained titanium oxide is non-stoichiometric. Fluoride uptake caused an increase in oxygen content in TNT annealed in argon (33.9%). Annealing in argon could change the ingredients of the oxide layer leading to the formation of oxygen vacancies. The possible explanation is reducing titanium ions from Ti 4+ to Ti 3+ ions and the formation of oxygen vacancies [32][33][34][35]. Table 1. Elemental investigation of titania nanotubes (50 nm diameter, 1000 nm thickness) before and after thermal modification (annealing in argon, in temperature 550 • C for 2 h).

(wt.) Non-Annealed Annealed in Argon
The Open Circuit Potential (OCP) curves ( Figure 2) show stable potential recorded values during the 1800 s. Small current oscillations seen in OCP curves for TNT formed in ethylene glycol electrolyte confirm ongoing oxidation and metastable nature of nanotubular oxide layers [29]. Annealing in argon causes the increase in the OCP value (E OCP = 55.1 ± 4.2 mV) for Ti/TNT layers compared to the OCP value for non-annealed samples (E OCP = −220.1 ± 12.1 mV). The contact angle measurements (WCA) carried out on the annealed samples for doubly ionized (DI) water confirmed that all obtained surface layers are hydrophilic. The value of the contact angle presented in Figure 2 for the non-annealed TiO 2 nanotubes, which amounts to 20 • ± 2 • after annealing in argon, drops to 80 • ± 5 • . Anodic titania oxides exhibit semiconductive properties attributed to defects: oxygen vacancies, Ti 3+ interstitials, and solvent components and impurities. TNT's conductive properties may be improved by annealing in controlled atmospheres, which causes the semiconductor material to obtain sufficiently higher electronic and ionic conductivity and improve wettability [33]. More positive values of the open circuit potential are favorable for the protein immobilization on the TNT platform, according to the amino acid residues (e.g., HRP) and human antibodies exhibiting negative charges in PBS (0.01 M, 50 mL, pH 7.4). The negatively charged protein is easily attached to the biosensor's positive matrix by direct physisorption, resulting from weak electrostatic or van der Waals interaction [54]. The material characterized by WCA values near 90 • can affect hydrophobicity and hydrophilicity, which is favorable for the immobilization process. Protein adsorption is stronger on the hydrophobic surface, but hydrophilic material avoids the protein decomposition [55].
The surface topography, electrical, and adsorption properties of titanium nanotubes (TNT) are important factors for the successful immobilization of biological substances. However, chemical modification of a TNT platform using currently available methods (i.e., silanization) often results in the effective immobilization process via covalent binding. Still, this makes the manufacturing of biosensor time-consuming and costly.
Recently, direct immobilization on the TNT surface was confirmed for the chemical compounds that exist as a cation in aqueous solutions [56]. Physisorption by the electrostatic forces of human antibodies onto the TNT surface, taking into account the values of the isoelectric point of human antibodies, requires the surface charge's positive value. Most biological substances have an isoelectric point lower than 7.4, which results in anions formed in human plasma and serum. Therefore, the immobilization of enzymes and proteins on the surface of TNT requires the presence of mediators [57,58]. Physisorption by the electrostatic forces of human antibodies onto the TNT surface, taking into account the values of the isoelectric point of human antibodies, requires the surface charge's positive value. The surface treatment was performed before the immobilization process to change TNT's surface charge from negative to positive.

IL-6, IL-8, TNFα Antibodies Adsorption
Cyclic voltammetry (CV) studies of TNT and Ab/TNT electrodes in the PBS solution without any external mediator are shown in Figure 3. It is important to note that the bare TNT platform does not show any redox peak, which is related to the fact that TNT does not have any redox couple for the electron transfer from the reaction site to the electrode. Flat cyclic voltammograms confirm the platform's stability in the electrolyte and the lack of redox reaction on TNT's surface. TNT annealed in argon indicates higher current values that prove TNT's high conductivity and provides a fast electron transfer path.
It is clearly seen that the current response serially decreases (Ab IL-6, IL-8, TNFα) when the electrode is modified with antibodies, as seen in Figure 3. This might be attributed to the insulating Ab protein layers on the electrode, which would hinder the electron transfers. This is related to the fact that the macromolecules of Ab IL-6, IL-8, TNFα restrict the flow of charge carriers. The results indicated that the immunosensing substrate had formed successfully and could be used in the proposed immunoassay.
After understanding the surface charge of TNT, IL-6, IL-8, TNFα antibodies, adsorption was studied. The direct immobilization of IL-6, IL-8, and TNFα antibodies on the TNT surface was performed by the drop technique for 60 min. The shortest time and the immobilization efficiency (% of the antibodies attached to the platform surface) were evaluated based on the spectrophotometric analysis of the slopes on every stage of the immobilization process.
According to ELISA tests, immobilization time was defined as 60 min for each antibody, 120 min for TNFα antigens, and 150 min for IL-6 and IL-8 antigens. The average value of the immobilization efficiency of IL-6, IL-8, TNFα immobilization onto TNT's surface was 87.5% (Figure 4a), and for IL-6, IL-8, TNFα antigens was 83.1%. This proves the high physical affinity of IL-6, IL-8, TNFα to the prepared platform covered with TNT and confirms TNT's suitability for effective protein immobilization and biosensing. For each cytokine, the shortest antibody immobilization time was defined as 30 min and the shortest antigen immobilization time as 90, 120, and 60 min for IL-6, IL-8, and TNFα, respectively. This period was considered sufficient for the effective binding of biomolecules to the TNT matrix based on the performed measurements.

Label-Free Detection of IL-6, IL-8, TNFα using Electrochemical Impedance Spectroscopy
The impedimetric response of the TNT layer (diameter of 50 ± 5 nm, the height of 1000 ± 100 nm) annealed in argon, immobilized with IL-6, IL-8 and TNFα antibody on the varying concentration of IL-6, IL-8, and TNFα antigens, were analyzed using EIS measurements carried out in the frequency range 10 5 ÷ 0.10 Hz in PBS (0.01 M, pH 7.4) solution (Appendix A). An example of Nyquist and Bode plots for the determination of IL-6, IL-8, and TNFα are shown in Figure A1 (Appendix A), including the statistical analysis of performed measurements (Table A1), whereas the calibration curves are presented in Figure 5. The calibration curves ( Figure 6) show a linear variation in |Z| from 5458 ± 110 Ω to 9771 ± 195 Ω, from 5831 ± 115 Ω to 21,706 ± 430 Ω, from 5517 ± 110 Ω to 18,834 ± 375 Ω over a wide range of IL-6, IL-8 and TNFα concentration (5-2500 pg/mL), respectively. The straight line is fitted to the data by linear regression analysis defined by the equations with a regression coefficient (R2), shown in each graph. The regression coefficient values are close to 1, indicating good linearity of the calibration curve. Obtained EIS spectra for TNT/antibody/antigen for the assumed range of concentrations of IL-6, IL-8, TNFα were fitted to the equivalent circuit using Nova 1.8 software. The circuit characterized by the best fit (based on the results of mean square errors ×2 of matching the equivalent circuit using the nonlinear least-squares method) was the one whose graphical form is shown in Figure 6, where Rs-electrolyte resistance; R1-resistance of the porous/nanotubular layer; R2-resistance of the ionic interface between the double layer and the solution; R3-resistance of the biological layer; C1-capacitance of the porous/nanotubular layer; CPE1-double-layer capacitance; CPE2-capacitance of the biological layer.
The obtained results confirm the correlation between the determined cytokine concentration and the parameters of the replacement system ( Table 2). As the concentration of human IL-6 increases the electrolyte resistance (Rs) increases, the resistance of the porous layer (R1) increases, and the capacity of the biological layer (CPE2) increases. As the concentration of human IL-8 increases the resistance of the porous layer (R1) increases, the capacity of the porous layer (CPE1) decreases, and surface heterogeneity and roughness (α1) increase. With the increase in the concentration of human TNFα the resistance of the electrolyte (Rs) increases, the resistance of the double layer (R2) increases, and surface heterogeneity and roughness (α1) increase.

Effect of pH of Electrolyte on IL-6, IL-8, TNFα Biosensor Response
The effect of pH on the impedance biosensor response was investigated for the IL-6, IL-8, and TNFα antigens at 300 pg/mL concentrations. Measurements were made in the range from 105 to 0.  The curves illustrating the influence of pH electrolyte on the impedimetric biosensor response have a similar course for determining cytokines. In the range of pH 2 ÷ 7.4 for IL-6, IL-8, and TNFα, it was observed that increasing the pH electrolyte results in an increase in the impedance modulus |Z| measured in the frequency of 0.1 Hz. For each of the determined cytokines, the maximum biosensor response was recorded in an electrolyte at pH values of 7.4, which is analogous to human plasma pH. The increase in biosensor response observed in the pH ranges from 6.8 to 7.4 is consistent with literature reports concerning the stability of the antigen-antibody complex and cytokine activity [59,60].
Reverberi and Ward's research showed that, in the pH range of 6.8-7.4, with increasing pH value, the stability of the antigen-antibody bond increases, and biological substance activity increases. Above the pH value of 7.4, a decrease in the value of the impedance modulus was observed, which may be due to the increase in the number of Na + ions from the PBS alkalizing reagent. This factor has a destabilizing effect on the conformational structure of proteins and prevents antigen-antibody complex formation. The nature of the dependence of the impedance biosensor response for the detection of IL-6, IL-8, and TNFα concentrations on the pH of the analyte confirms both the sensitivity of the biosensor to a change in analyte pH and the broad spectrum of activity of the analyzed substances and their ability to form antigen-antibody complexes. Considering the potential use of a biosensor to measure cytokine concentration in human plasma, it is advantageous to achieve a maximal biosensor response for an analyte pH corresponding to human plasma.

Target
Composition of Sensitive Film Detection Method Limit of Detection Linear Range Ref.

Conclusions
The development of an efficient electrochemical immunosensor to detect IL-6, IL-8, and TNFα using TNT has been successfully demonstrated. The thermal modification of TNT (diameter: 50 ± 5 nm, height: 1000 ± 100 nm) in argon atmosphere results in a change in the value of open circuit potential from negative to positive, increasing the resistive character and decreasing the nonhomogeneity. The successful immobilization of the antibody of IL-6, IL-8, and TNFα onto the TNT surface via physisorption was carried out for 30 min, which is a much shorter time than currently available analytical methods.
The high sensitivity of the prepared substrates to pH changes was observed in impedimetric tests, and the highest impedance modulus values were measured in the analyte at pH 7.4. The obtained results indicate that the tested cytokine antibodies and antigens are not inactivated in a wide pH range, which ensures the universality of the biosensors being developed.
Calibration curves of impedance biosensors for each of the determined cytokines showed that the detection limits for IL-8, IL-6, TNFα are 192, 625, and 295 fM, respectively, which correspond to physiological norms, where the detection range covers clinically important concentrations from 5 to 2500 pg/mL.

Conflicts of Interest:
The authors declare no conflict of interest.

Appendix A
The impedimetric response of TNT layer (diameter of 50 ± 5 nm, the height of 1000 ± 100 nm) annealed in argon, immobilized with IL-6, IL-8 and TNFα antibody on the varying concentration of IL-6, IL-8, and TNFα antigens were analyzed using EIS measurements carried out in frequency range 10 5 ÷0.10 Hz in PBS (0.01 M, pH 7,4) solution. An example of Nyquist and Bode plots for the determination of IL-6, IL-8, and TNFα were shown in Figure A1. Each measurement was performed with a minimum of 3-times repeatability. To calculated the values of standard deviation (SD) relative standard deviation (RSD) for electrochemical parameter (EIS) the equations were used accordingly (1), (2) where Xi -each of the values of the data for 3 samples, X -the mean of Xi, n -the number of data points equal to 3. The results of statistical analysis were shown in Table A1.
The phase shift angle values recorded at the lowest frequency (0.1 Hz) presented in Bode's graphs ( Figure A1) range from 50 • to 90 • for each of the determined cytokines (Table A1). Correlations between its values (measured at a frequency of 0.1 Hz) and the determined IL-8 antigen concentrations were observed ( Figure A1B). With the increase in the concentration of the determined cytokine, the value of the phase angle decreases. Along with the increase in the concentrations of antigens: IL-8 and TNFα, the phase angle values for the frequency of 0.1 Hz decrease, which proves that the heterogeneity of the TNT surface decreases due to combining the antigen with antibodies immobilized on the surface of TNT. Forming antigen-antibody complexes, filling the pores, reduce its porosity.
Data illustrating changes in the phase angle (Table A1) for high concentrations of the determined cytokines IL-6, IL-8 and TNFα (concentrations greater than 250 pg/mL for Il-6, 600 pg/mL for IL-8, and 1000 pg/mL for TNFα) indicate the existence of two-time constants, which proves the presence of two layers on the electrode surface: the first is the layer of immobilized antibody, the second is the layer of antibody-antigen complexes. Greater heterogeneity (porosity) the surface layer for lower concentrations may result from incomplete antibody layer coverage with antigens. As antigens increase and more antibody-antigen complexes form, it decreases the value of the phase angle determines the layer's non-uniformity, and its value measured at high frequency (10 5 Hz) is also lowered, corresponding to the resistance of the electrolyte penetrating the porous electrode surface. The served increasing values of the phase angle for the time constant in the frequency range from 10 to 50 Hz well illustrate its dependence on the increasing concentration of the antigen. It is also confirmed by the correlation between the phase angle values and different concentrations of IL-6 in the studied range.  (Table A1). The lowest value of the impedance modulus (~3 × 10 3 Ω) was recorded for the TNT layer with a diameter of 50 nm, height 1000 nm, annealed in argon, and not functionalized. For each of the determined cytokines, an increase in the impedance modulus's value was observed with the increase in their concentration. This means that as the TNT layer is coated with antibodies and then with increasing antigens concentrations, which results in the formation of antigen-antibody complexes, the impedance modulus of the test layer increases. This tendency corresponds to the characteristics of biological systems covering the electrodes [58].
Nyquist curves of TNT with IL-6, IL-8, and TNFα ( Figure A1C) antibodies immobilized on its surface, similarly to before immobilization, have the shape of large semi-circles -characteristic of thin oxide layers. With the increase in the concentration of IL-6, IL-8, TNFα, the angle of the curves and the radius of the circles decreased, which indicates a decrease in the charge exchange resistance in the presence of an increasing number of antigen-antibody bonds. Their presence on the biosensor electrode's surface may stimulate the exchange of electrons, causing an increase in the electrical conductivity of the layer, thus reducing its resistance. Table A1. Results of electrochemical impedance spectroscopy obtained for the varying concentration of IL-6, IL-8, TNFα immunoelectrode measured in 0.01 M PBS solution, where: SD -standard deviation, RSD-relative standard deviation.