3D Hydrogen Titanate Nanotubes on Ti Foil: A Carrier for Enzymatic Glucose Biosensor

Glucose oxidase (GOx) based biosensors are commercialized and marketed for the high selectivity of GOx. Incorporation nanomaterials with GOx can increase the sensitivity performance. In this work, an enzyme glucose biosensor based on nanotubes was fabricated. By using Ti foil as a carrier, hydrogen titanate nanotubes (HTNTs), which present fine 3D structure with vast pores, were fabricated in-situ by the hydrothermal treatment. The multilayer nanotubes are open-ended with a diameter of 10 nm. Then glucose oxidase (GOx) was loaded on the nanotubes by cross-linking to form an electrode of the amperometric glucose biosensor (GOx/HTNTs/Ti electrode). The fabricated GOx/HTNTs/Ti electrode had a linear response to 1–10 mM glucose, and the response time was 1.5 s. The sensitivity of the biosensor was 1.541 μA·mM−1·cm−2, and the detection limit (S/N = 3) was 59 μM. Obtained results indicate that the in-situ fabrication and unique 3D structure of GOx/HTNTs/Ti electrode are beneficial for its sensitivity.


Introduction
Diabetes is widely recognized as one of the leading causes of death and disability [1]. Closely monitoring the blood glucose level of patients is necessary for preventing and reducing diabetes. Minimally invasive interventional blood glucose detection is the most promising and potential method. It is mainly based on electrochemical methods. For an electrochemical glucose sensor, the bioreceptor is the core part and the performance is closely related to it.
It is well known that enzymes are highly efficient and specific biocatalytic molecules [2]. Enzyme biosensors, using an enzyme as the molecular recognition element, have the advantages of high selectivity, high sensitivity and a fast response. The enzyme immobilization technique involves binding the target enzymes at a certain place through physical or chemical ways [3]. The immobilization technique limits the free flow of enzyme molecules. Immobilized enzymes have the following advantages: (i) improved thermal and operational stability compared with free enzymes, (ii) improved convenience of separating reaction products from mixtures and (iii) easy recovery by removing the enzyme from the reaction mixture, thus reducing the cost [4]. The development of materials science Figure 1 shows the XRD pattern of the fabricated HTNTs/Ti. The superior peaks were observed mainly as diffraction planes of Ti (JCPDS No. 44-1294). As the samples were synthesized on top of Ti foils, the titanium peaks were very high and sharp. Apparently, titanate hydrate could be observed. As shown in the inserted graph of Figure 1, there was a visible diffractions peak located at 2θ = 41.0 • , which was attributed to the (6 0 -3) diffraction of H 2 Ti 3 O 7 (JCPDS No. 47-0561). It seemed that HTNTs was successfully fabricated on the Ti foil after the hydrothermal treatment.
Sensors 2020, 20, 1024 3 of 12 An optimized cross-linking technique [29] was carried out to immobilize the GOx onto the HTNTs/Ti electrode. 25 mg BSA and 100 μL glutaraldehyde were dissolved in 1 mL PBS and mixed evenly. Then 100 uL mixed solution was deposited onto the surface of the HTNTs/Ti and dried at room temperature. 12.5 mg GOx was dissolved in 500 μL of PBS and mixed evenly. Then 100 uL of the mixed solution was deposited on the HTNTs/Ti electrode surface, and dried in the refrigerator at 4 °C overnight to obtain the GOx/HTNTs/Ti electrode.

Characterization Techniques
X-ray powder diffraction (XRD) pattern was obtained by using a RigakuD/MAX-2500 X-ray diffractometer with a working voltage and current at 40 kV and 40 mA, respectively. Morphologies of samples were characterized by scanning electron microscope (SEM, JEOL JSM-7500F) at 10.00 kV and transmission electron microscopy (TEM, FEI T20) at 200 kV. The chemical composition and oxidation state of elements on the surface of samples were identified by X-ray photoelectron spectroscopy (XPS; Kratos Axis Ultra DLD), and the binding energy was calibrated by taking C 1s peak at 284.6 eV as reference. Electrochemical experiments were performed with an electrochemical workstation (Zahner Zennium, Germany). A three-electrode system was used in this process, which employed the GOx/HTNTs/Ti electrode as the working electrode, a platinum sheet as the counter electrode, and an Ag/AgCl electrode as the reference. Cyclic voltammetric (CV) and amperometric response (I-t) were used to study the electrochemical performance of the electrode. Figure 1 shows the XRD pattern of the fabricated HTNTs/Ti. The superior peaks were observed mainly as diffraction planes of Ti (JCPDS No. 44-1294). As the samples were synthesized on top of Ti foils, the titanium peaks were very high and sharp. Apparently, titanate hydrate could be observed. As shown in the inserted graph of Figure 1, there was a visible diffractions peak located at 2θ = 41.0°, which was attributed to the (6 0 -3) diffraction of H2Ti3O7 (JCPDS No. 47-0561). It seemed that HTNTs was successfully fabricated on the Ti foil after the hydrothermal treatment. The surface morphology of Ti foil after the hydrothermal treatment was investigated by SEM, as shown in Figure 2. It can be observed that the surface of Ti foil was fully covered by 1D nanomaterials. The 1D materials intertwined together, and formed fine 3D structure with vast pores. The length of the 1D nanomaterials was more than hundreds of nanometers. Compared with the The surface morphology of Ti foil after the hydrothermal treatment was investigated by SEM, as shown in Figure 2. It can be observed that the surface of Ti foil was fully covered by 1D nanomaterials. The 1D materials intertwined together, and formed fine 3D structure with vast pores. The length of the 1D nanomaterials was more than hundreds of nanometers. Compared with the array structure made by the anodic oxidation method [30], HTNTs/Ti provided a larger surface area and stronger adsorption capacity. That could provide more effective immobilization for GOx.

Surface Morphology
Sensors 2020, 20, 1024 4 of 12 array structure made by the anodic oxidation method [30], HTNTs/Ti provided a larger surface area and stronger adsorption capacity. That could provide more effective immobilization for GOx. The detailed structure of the 1D materials fabricated on the Ti foils was further observed by TEM. For this, some of the HTNTs were scraped off from the surface of the Ti foil and then dispersed in alcohol. Figure 3 shows the TEM images of the 1D nanomaterials. It can be clearly seen that the sample shows a 1D and tubular structure. The multilayer nanotubes were open-ended with an outer diameter of about 10 nm and an inner diameter of about 5 nm. So, the structure of the Ti foil surface was a network made of HTNTs of high quality. The detailed structure of the 1D materials fabricated on the Ti foils was further observed by TEM. For this, some of the HTNTs were scraped off from the surface of the Ti foil and then dispersed in alcohol. Figure 3 shows the TEM images of the 1D nanomaterials. It can be clearly seen that the sample shows a 1D and tubular structure. The multilayer nanotubes were open-ended with an outer diameter of about 10 nm and an inner diameter of about 5 nm. So, the structure of the Ti foil surface was a network made of HTNTs of high quality.
By analyzing the XPS spectra of HTNTs, the chemical state of each element in the tubular catalyst was obtained. Figure 4a is a full spectrum of HTNTs, in which the peaks of Ti2p, O1s and C1s appeared in the diagram. The carbon resulted from the residual organic precursors and the XPS itself. The binding energy of Ti 2p 1/2 and Ti 2p 3/2 were 464.4 eV and 458.7 eV, indicating the existence of Ti 4+ . Figure 4c shows the high resolution XPS spectrum of O1s with the relative atom ratio of 55.76%. The peak of the binding energy at the position of 529.93 eV could be assigned to the crystal lattice oxygen connected by the Ti-O bond, appeared with the proportion of 77.30%. The peak of the binding energy at the position of 531.33 eV was assigned to the surface hydroxyl oxygen, with the proportion of 22.70%.  By analyzing the XPS spectra of HTNTs, the chemical state of each element in the tubular catalyst was obtained. Figure 4a is a full spectrum of HTNTs, in which the peaks of Ti2p, O1s and C1s appeared in the diagram. The carbon resulted from the residual organic precursors and the XPS itself. The binding energy of Ti 2p1/2 and Ti 2p3/2 were 464.4 eV and 458.7 eV, indicating the existence of Ti 4+ . Figure 4c shows the high resolution XPS spectrum of O1s with the relative atom ratio of 55.76%. The peak of the binding energy at the position of 529.93 eV could be assigned to the crystal lattice oxygen connected by the Ti-O bond, appeared with the proportion of 77.30%. The peak of the binding energy at the position of 531.33 eV was assigned to the surface hydroxyl oxygen, with the proportion of 22.70%.

Electrochemical Characterization
The electrochemical behavior of the GOx/HTNTs/Ti electrode is shown in Figure 5 by cyclic voltammetry. For the GOx/HTNTs/Ti electrode, cycling in PBS exhibited redox peaks at 0.1 V, which should be attributed to the reduction of Ti(IV) to Ti(III). A significant enhancement in the anodic peak current with a peak potential at about 0.55 V (vs. Ag/AgCl) can be observed after glucose was added. That should be attributed to the electro-oxidation of glucose to gluconolactone by GOx. The reaction mechanism is as follows.
The result indicates that the GOx was immobilized on the HTNTs and the fabricated enzyme electrode revealed good detection performance for glucose. The electrochemical response of the GOx/HTNTs/Ti electrode towards glucose oxidation was investigated at different scan rates ( Figure 6). The GOx/HTNTs/Ti electrode was scanned from 5 to 200 mV·s −1 . The anodic currents were found to be proportional to the square root of the scan rate (inset of Figure 6). A good linearity between the square root of scan rate and peak current was obtained within the range of 5-200 mV·s −1 (correlation coefficient R 2 = 0.9914), which indicates that the electro-oxidation of glucose was indeed diffusion-controlled when its concentration was 5 mM.

Electrochemical Characterization
The electrochemical behavior of the GOx/HTNTs/Ti electrode is shown in Figure 5 by cyclic voltammetry. For the GOx/HTNTs/Ti electrode, cycling in PBS exhibited redox peaks at 0.1 V, which The result indicates that the GOx was immobilized on the HTNTs and the fabricated enzyme electrode revealed good detection performance for glucose. The electrochemical response of the GOx/HTNTs/Ti electrode towards glucose oxidation was investigated at different scan rates ( Figure  6). The GOx/HTNTs/Ti electrode was scanned from 5 to 200 mV•s −1 . The anodic currents were found to be proportional to the square root of the scan rate (inset of Figure 6). A good linearity between the square root of scan rate and peak current was obtained within the range of 5-200 mV•s −1 (correlation coefficient R 2 = 0.9914), which indicates that the electro-oxidation of glucose was indeed diffusioncontrolled when its concentration was 5 mM.  The result indicates that the GOx was immobilized on the HTNTs and the fabricated enzyme electrode revealed good detection performance for glucose. The electrochemical response of the GOx/HTNTs/Ti electrode towards glucose oxidation was investigated at different scan rates ( Figure  6). The GOx/HTNTs/Ti electrode was scanned from 5 to 200 mV•s −1 . The anodic currents were found to be proportional to the square root of the scan rate (inset of Figure 6). A good linearity between the square root of scan rate and peak current was obtained within the range of 5-200 mV•s −1 (correlation coefficient R 2 = 0.9914), which indicates that the electro-oxidation of glucose was indeed diffusioncontrolled when its concentration was 5 mM.   Typical CV curves obtained at the GOx/TNTs/Ti electrode in the glucose solution (1-10 mM) are shown in Figure 7. With the increasing of glucose concentration, the oxidation peak currents gradually increased at +0.55 V. Inserted graph shows the corresponding linear calibration curve of the GOx/HTNTs/Ti electrode. The linear range was 1-10 mM (correlation coefficient R 2 = 0.9938). It seemed that more complex reactions occurred due to the change of glucose concentration. When the scanning rate was fixed, the current value was influenced by the diffusion ability of glucose, the reaction rate of GOx and glucose, the amount of GOx on the electrode, etc. As a result, the log relationship between the peak current and glucose concentration was formed.
the GOx/HTNTs/Ti electrode. The linear range was 1-10 mM (correlation coefficient R 2 = 0.9938). It seemed that more complex reactions occurred due to the change of glucose concentration. When the scanning rate was fixed, the current value was influenced by the diffusion ability of glucose, the reaction rate of GOx and glucose, the amount of GOx on the electrode, etc. As a result, the log relationship between the peak current and glucose concentration was formed. The typical amperometric responses at the GOx/HTNTs/Ti electrode for each successive addition of 0.1 M glucose are shown in Figure 8. It can be seen that the current of the electrode rose rapidly, and then stabilized rapidly after the glucose solution was added. When the concentration of glucose changed, it showed a sensitive response, and the overall current change showed a step-bystep growth. The as-prepared electrode could achieve a well-defined and step-rise current within 1.5 s (inset of Figure 8B). The results indicate that glucose was oxidized rapidly on the electrode surface. The released electrons were transferred to one end of the electron collector rapidly, and then converted into a response current. The mechanism is a glucose oxidase direct electron transfer process [31]. The typical amperometric responses at the GOx/HTNTs/Ti electrode for each successive addition of 0.1 M glucose are shown in Figure 8. It can be seen that the current of the electrode rose rapidly, and then stabilized rapidly after the glucose solution was added. When the concentration of glucose changed, it showed a sensitive response, and the overall current change showed a step-by-step growth. The as-prepared electrode could achieve a well-defined and step-rise current within 1.5 s (inset of Figure 8B). The results indicate that glucose was oxidized rapidly on the electrode surface. The released electrons were transferred to one end of the electron collector rapidly, and then converted into a response current. The mechanism is a glucose oxidase direct electron transfer process [31].  Table 1 summarizes the comparison of the analytical performance of various glucose biosensors. From the table, it can be seen that GOx/HTNTs/Ti sensor had a relatively wider linear range, which is a benefit for the construction of minimally invasive interventional glycemic apparatus. Compared with the response time of similar electrodes, the response time of GOx/HTNTs/Ti electrode was shorter, which should be attributed to the unique structure of the HTNTs/Ti electrode. As shown in the above SEM and TEM images, the surface of HTNTs/Ti electrode was covered by nanotubes. The 3D structure with vast pores results in more effective immobilization of GOx, more active sites for  Table 1 summarizes the comparison of the analytical performance of various glucose biosensors. From the table, it can be seen that GOx/HTNTs/Ti sensor had a relatively wider linear range, which is a benefit for the construction of minimally invasive interventional glycemic apparatus. Compared with the response time of similar electrodes, the response time of GOx/HTNTs/Ti electrode was shorter, which should be attributed to the unique structure of the HTNTs/Ti electrode. As shown in the above SEM and TEM images, the surface of HTNTs/Ti electrode was covered by nanotubes. The 3D structure with vast pores results in more effective immobilization of GOx, more active sites for glucose and a higher reaction rate. Moreover, the HTNTs were in-situ fabricated on the surface, and the conductivity of Ti foil was good. Accompanied by the 1D structure of HTNTs, the electronic energy could be quickly transmitted to the external circuit. The corresponding calibration curve is shown in the insert graph A of Figure 8. It can be seen that the biosensor shows a sensitive response to glucose in the concentration range of 1-10 mM. The amperometric response to glucose at 0.55 V exhibited a linear relationship with a glucose concentration in this range. Compared with the linear range reported in the literature [36], the linear range of GOx/HTNTs/Ti electrode was relatively wide. The linear regression equation is I/uA = 1.541Cg/mM + 8.333 (correlation coefficient R 2 = 0.9965), where I is the current and Cg is the glucose concentration. The corresponding sensitivity of the glucose biosensor was 1.541 µA·mM −1 ·cm −2 . The detection limit (LOD) of the sensor can be obtained from the formula LOD = 3 sb/S (where, sb = standard deviation of blank signal and S = sensitivity), and the calculated value was 59 µM.
However, GOx will lose its activity when pH < 2 or pH > 8, and can be irrevocably damaged at temperatures over 40 • C [37]. So, the recycling of the enzyme carrier is necessary. The self-cleaning properties of HTNTs/Ti electrode were consequently investigated. Under the illumination of a 500 Xe lamp for several seconds, the current responses of the GOx/HTNTs/Ti electrode decreased to zero. After proper irradiation, the enzyme and fouling substances on the HTNTs/Ti electrode could be entirely decomposed into CO 2 and H 2 O to achieve self-cleaning. So, the recycling of the HTNTs/Ti electrode should be convenient.

Conclusions
In summary, the GOx/HTNTs/Ti electrode with good sensitivity performance for glucose was successfully fabricated. In this electrode, HTNTs/Ti, with a 3D structure and vast pores, was fabricated in-situ by a simple hydrothermal process, and firstly served as an enzyme carrier as well as an electron transporter to monitor glucose. The unique 3D structure resulted in effective immobilization of GOx. The in-situ fabrication and 1D structure of HTNTs could accelerate electrons transport between the biomolecules and electrode. Consequently, the response time of the GOx/HTNTs/Ti electrode was short. Moreover, the HTNTs/Ti electrode exhibited self-cleaning properties, which facilitated its recycling. Other kinds of enzymatic biosensors based on HTNTs/Ti carrier are expected.