Improvement in Sensing Performance of H2O2 Biosensor Electrodes through Modification of Anatase TiO2 Nanorods and Pretreatment of Electrochemical Reduction

Electrochemical biosensors are essential health monitors that aid in the detection and diagnosis of diseases. In this research, anatase titanium dioxide (TiO2) nanorods (TNR-A) were synthesized on a titanium (Ti) substrate in three stages, namely, hydrothermal, alkali, and heat treatments, and utilized as a modified electrode (TNR-A/Ti). The pretreatment of electrochemical reduction was adopted to increase the Ti3+ amount in TiO2 nanorod surfaces of the electrodes, thus improving electron transfer. The electrodes were characterized by X-ray diffractometry (XRD), fieldemission scanning electron microscopy (FESEM), and X-ray photoelectron spectroscopy (XPS). The electrodes after pretreatment (Ti3+-TNR-A/Ti) showed a better electrochemical response to hydrogen peroxide (H2O2), indicating that pretreatment improves the performance of electrodes. The assembled biosensor electrode (Nafion/HRP/Ti3+-TNR-A/Ti) exhibited a sensitivity of 6096.4 μA∙mM−1∙cm−2, a detection limit of 0.008 μM, a linearity of 0.04–700 μM, and an apparent Michaelis–Menten constant K M of 0.0027 μM, which are higher than those in previous research studies on TiO2 or similarly nanostructured modified biosensor electrodes. This research could provide a potential competent method that can be used to modify electrodes for high-performance amperometric biosensors.


Introduction
Electrochemical biosensors are one of the essential health monitors used to help detect and diagnose diseases, among which hydrogen peroxide (H 2 O 2 ) biosensors are deemed important. (1)(2)(3)(4) This is because H 2 O 2 is the main component of reactive oxygen species (ROS) in living organisms, and increasing levels of H 2 O 2 in cells could indicate oxidative stress and cellular damage. (5) This damage could result in many diseases such as cancer, neurodegenerative disorders, diabetes, and atherosclerosis. (6) Thus, H 2 O 2 biosensors with high accuracy, high sensitivity, good selectivity, and good anti-interference ability play vital roles in detecting and monitoring the H 2 O 2 concentration under these conditions.
To increase sensing capacity, enzymes are utilized. The enzyme adsorption on electrodes and direct electron transfer between them have been recognized as key factors that affect biosensor performance. (7) Horseradish peroxidase (HRP) is one such enzyme, which belongs to the superfamily of peroxidases and heme-containing glycoproteins. (8) Therefore, HRP is always used to determine H 2 O 2 . (9) If a mediator with nanostructures is built on the electrodes, it could increase the surface area and facilitate more enzyme immobilization to improve biosensor performance. (9,10) Nanostructured TiO 2 has a strong protein adsorption ability and great biocompatibility. (11) Moreover, TiO 2 with an isoelectric point of 8.9 is very appropriate for HRP enzyme immobilization. (12) In our previous work, TiO 2 nanodot film and rutile TiO 2 nanorods were reported as a mediator to construct a H 2 O 2 biosensor electrode and showed good performance. (13,14) However, nanostructured anatase TiO 2 was reported to well retain the biological activity of the enzyme and it can be used to load an increased enzyme concentration. (15) The electrical conductance of TiO 2 is closely related to the Ti 3+ amount. If there are more Ti 3+ ions in the nanostructured TiO 2 surface, it should favour electron transfer within the electrode. An electrochemical reduction or self-doing approach was reported to be effective for producing Ti 3+ ions on a TiO 2 surface and demonstrated to have the capability of improving the performance of TiO 2 -nanotube-based biosensor electrodes. (16,17) The main problem with H 2 O 2 biosensors is their dependence on the dissolved oxygen concentration. This problem can be overcome by utilizing 'mediators' that transfer the electrons directly to the electrode bypassing the reduction of the oxygen cosubstrate. (13) The advantage of these biosensors is that they react rapidly with the reduced form of the enzymes. Moreover, they are sufficiently soluble, in both oxidized and reduced forms, to be able to rapidly diffuse between the electrode surface and the active site of the enzyme. (13,18) The purpose of this work is to develop a highly sensitive electrode by intensifying direct electron transfer between the electrode substrate and the functional enzyme through TiO 2 nanorod modification.
In this work, anatase TiO 2 nanorods on Ti substrates were prepared via three stages, hydrothermal, alkali, and heat treatments, following a pretreatment of electrochemical reduction. The modified electrodes were then characterized and their electrochemical behaviour and performance were measured. The effect of pretreatment on the performance of the electrodes was also evaluated.

Materials and reagents
Titanium (Ti) foils with a purity of 99.99% and a thickness of 0.1 mm were used as a conductive material for the construction of anatase TiO 2 nanorod biosensor electrodes. The structure with dimensions of 1 × 2 cm 2 (length × width) was then cleaned in a mixture of ethanol, distilled water, and acetone at a ratio of 1:1:1. 250 U•mg −1 HRP (Aladdin Chemistry Co., Ltd.) was used. Nafion (5 wt%) was purchased from Sigma-Aldrich. All the chemicals used in the experiments that were of analytical reagent grade were purchased from Sinopharm Chemical Reagent Co., Ltd. The 0.1 M phosphate buffer solution (PBS) used in the experiments was prepared with the constituents of NaH 2 PO 4 (AR, >99.0), Na 2 HPO 4 (AR, >99.0), NaCl (AR, >99.5), and H 2 O. The HCl (37 wt%) and NaOH (AR, >96.0) used to adjust the pH of the PBS were prepared with various pH values. The PBS solution was deoxygenated by bubbling pure N 2 gas for 30 min, prior to use.

Electrode construction and modification
Anatase TiO 2 nanorods were obtained through three steps. The first step was a hydrothermal process described as follows: The hydrothermal solution was prepared by mixing 0.45 g of picric acid (AR, >99.8%), 15 mL of ethanol, 60 mL of H 2 O, 40 mL of HCl, and 220 mL of titanium tetrabutoxide (TBOT, CP, >98%). After stirring the chemicals sufficiently, the Ti substrate and mixture were placed in a Teflon vessel of a hydrothermal autoclave and then into an oven at 160 °C for 4 h. After the hydrothermal treatment, the autoclave vessel was taken out and allowed to cool to room temperature. Rutile TiO 2 nanorod films were obtained by rinsing with deionized water and ethanol. In the second step, the rutile TiO 2 nanorod films were hydrothermally alkalitreated in a 100 mL Teflon vessel with a solution of hydroxides (KOH/NaOH = 1:1) at 200 °C for 2 h, then washed with deionized water and soaked in 0.1 M HCl for 4 h. In the third step, the films with acidic treatment were heat-treated at 500 °C for 2 h, after which anatase TiO 2 nanorod films on Ti substrates (TNR-A/Ti) were obtained. According to the literature, (16) the electrochemical reduction method was performed by applying −1.5 V to the electrodes in 1 M (NH 4 ) 2 SO 4 (AR, >99.0%) to obtain a Ti 3+ -TNR-A/Ti electrode. Field-emission scanning electron microscopy (FESEM) (Hitachi, S-4800) was performed to observe the morphology of the anatase TiO 2 nanorods, and X-ray diffractometry (XRD; PANalytical, X'pert PRO) was carried out to analyse the phase and crystal of TiO 2 nanostructure films. Also, X-ray photoelectron spectroscopy (XPS, ESCALAB 25OXi) was performed to analyse the role of pretreatment in changing the surficial Ti valence for the electrochemical reduction pretreatment. The anatase TiO 2 nanorod biosensor electrodes were assembled via the same procedure using the same enzyme and protective membrane, and the steps of biosensor electrode construction were started by sealing the electrodes with epoxy resin except for an area of 2.5 × 2.5 mm 2 , which was left as the measuring area. The physical adsorption method was performed to immobilize the HRP enzyme on the electrode surface, where 10 µL of HRP solution was dropped on the electrode surface and left to dry at room temperature. The HRP solution was prepared by dissolving HRP in 0.01 M PBS (pH = 7.4 (13,19) ) in order to obtain a 0.01 g mL −1 solution. Routinely, Nafion has been used as a biosensor and as an immobilisation matrix for the enzyme, which helps in maintaining the stability of the biosensor. (20) Hence, as the final step, 4 µL of a 0.5 wt% solution of Nafion was dropped onto the biosensor surface to protect the enzyme and make the biosensor biocompatible, and then was left to dry at room temperature (Fig. 1). The Nafion/HRP/Ti 3+ -TNR-A/Ti and Nafion/HRP/TNR-A/Ti biosensor electrodes were washed after storing at 4 °C for 1 d. (9) When not in use, the modified anatase TiO 2 nanorod biosensor electrodes were stored at 4 °C.

Measurement of performance characteristics of the biosensor electrodes
An electrochemical workstation device (CHI 660D, from Shanghai Instrument Co., Ltd.) was used to characterize the modified biosensor electrodes. The Pt foil and saturated calomel electrodes (SCEs) served as the counter and reference electrodes, respectively. The modified biosensor electrodes were used as the working electrode. The cyclic voltammetry technique was used to test the modified electrodes in 20 mL of PBS (PBS, 0.1 M at 25 °C and purged with pure nitrogen for 30 min in order to remove oxygen) with an optimum pH. Through this technique, we can investigate and study the electrochemical behaviour of the modified electrodes and the effect of electrocatalytic activity towards the redox reaction of H 2 O 2 . The scan rate effect on the electrochemical behaviour of the electrodes was also studied by cyclic voltammetry. The amperometric technique was carried out on the biosensors in order to study the performance characteristics of the modified biosensor electrodes, such as sensitivity, limit of detection (LOD), linearity, selectivity, and stability, by applying an optimum voltage. The optimum applied voltage was selected via the amperometric technique by applying a voltage in the range between −0.3 and −0.9 V; the voltage with high current response was selected as the optimum applied voltage. The optimum pH of the PBS was selected depending on the best performance of the modified electrodes.

Characterization of anatase TiO 2 nanorod modified electrodes
After alkali, acidic, and heat treatments of hydrothermally grown rutile TiO 2 nanorods, the XRD pattern (Fig. 2) of the nanorods on Ti substrate fits well with the anatase TiO 2 phase (JCPDS No. 21-1272). The formation of the anatase TiO 2 phase starts with breaking in the Ti-O-Ti building units in rutile TiO 2 to form Ti-O-Na (Ti-O-K). After the Na + and K + ions are exchanged with H + ions in acid wash, Ti-OH bonds are formed during alkali treatment. The resulting hydrogen titanate is finally transformed to anatase TiO 2 after heat treatment at 500 °C (Fig. 3).

Effect of electrochemical reduction on the electrodes
After the electrochemical reduction treatment was performed, the Ti 3+ contribution in the XPS spectra ( Fig. 4) increased, and the calculated Ti 3+ molar percentage on the surface also increased from 19 to 40.8%. This indicated that the electrochemical reduction treatment is really an effective surface reduction procedure.

Electrochemical behaviours
When a biosensor electrode was assembled, its cycle voltammograms (CVs) [ Fig. 5(a)] showed that the electrochemical activity of the electrode (Nafion/HRP/Ti 3+ -TNR-A/Ti) enhanced after the pretreatment, and the biosensor electrode had a higher oxidation peak current when 50 µM H 2 O 2 was added, as observed in the CV curve [ Fig. 5(b)].
The CVs [ Fig. 6(a)] of the Nafion/HRP/Ti 3+ -TNR-A/Ti electrode at different scan rates showed that the current response increased proportionally with the scan rate, and that the reduction current peak (I pc ) was linear as a function of the scan rate [ Fig. 6(b)], implying that the behaviour followed a surface-controlled process with direct electron transfer. (21,22) Hence, the improvement in the electrochemical response of the biosensor electrode could be attributed to enhancing direct electron transfer within the electrode by electrode electrochemical reduction pretreatment.

Sensing performance of the biosensor electrodes
After the optimization of the applied voltage [ Fig. 7(a)] and the pH of the PBS [ Fig. 7(b)] for the electrode with the highest response towards 20 µM H 2 O 2 , the amperometric response of the Nafion/ HRP/Ti 3+ -TNR-A/Ti electrode for the successive addition of H 2 O 2 was measured under the applied voltage of −0.55 V and 0.1 M PBS with pH 6.5.
The amperometric technique was used to test the sensing performance of the biosensor electrodes (Fig. 8) and the sensitivity of Nafion/HRP/Ti 3+ -TNR-A/Ti with 6096.4 µA•mM −1 •cm −2 (curve i). The limit of detection (LOD) was found to be 0.008 µM, the linearity ranged from 0.04 to 700 µM with the correlation coefficient R = 0.999 (n = 28) in the inset, and the required time to reach 95% of the steady-state current was less than 3 s. Compared with Fig. 8 curve ii, the electrode with pretreatment had a 1.15-fold increase in the sensitivity. Table 1 shows a comparison of the TiO 2 nanorod electrode before and after the electrochemical reduction pretreatment and other previously reported electrodes.    This indicates that both the present nanostructure and the pretreatment provide an effective way of enhancing the sensing performance. Moreover, the apparent Michaelis-Menten constant K app M could reflect the enzyme affinity. (27) The K app M of the electrodes before and after the electrochemical reduction pretreatment was calculated to be 0.029 and 0.027 mM, respectively. The results show that the value decreases after the reduction pretreatment, indicating that the enzyme achieves a higher catalytic efficiency at a low H 2 O 2 concentration owing to the higher affinity of the enzyme with the mediator.

Selectivity and stability of Nafion/HRP/Ti 3+ -TNR-A/Ti biosensor electrode
Selectivity is very important for the biosensor electrode with anti-interference towards other species. The amperometric technique was used to study the selectivity. The electrode was examined in the presence of 10 µM H 2 O 2 , where the current response of the electrode was clearly rapid and strong. In contrast, when each 100 µM of uric acid (UA), ascorbic acid (AA), and glucose were successively injected into the PBS, no notable current response was observed, as indicated in Fig. 9. However, when 10 µM H 2 O 2 was again injected into the same supporting electrolyte solution, an immediate current response was observed (Fig. 9). The obtained results suggest that the biosensor electrode had good anti-interference and a high selectivity. Thus, it could be used for the determination of H 2 O 2 from real species.
Owing to its stability, the amperometric technique was also used. About 96% of its initial current response to H 2 O 2 still remained after the electrode was stored for 12 d at 4 °C and 93% of it remained after the electrode was stored for 28 d (Fig. 10). This indicates that the Nafion/HRP/Ti 3+ -TNR-A/Ti electrode behaved as a biosensor in an acceptable manner for applications.

Conclusions
Anatase TiO 2 nanorods for modifying the electrode surface and electrochemical reduction pretreatment for increasing the amount of Ti 3+ on the surfaces of the nanorods were adopted to improve the sensing performance of the biosensor electrodes. The nanostructure gave a high  sensitivity and the pretreatment resulted in further improvement in sensing performance. The Nafion/HRP/Ti 3+ -TNR-A/Ti electrode showed a sensitivity as high as 6096.4 µA•mM −1 •cm −2 . The nanostructure could increase the affinity of the enzyme and the pretreatment could increase the direct electron transfer within the electrodes. The present approach is a facile and effective manner to improve the amperometric biosensor performance.