Platinum and Iridium Oxide Co-modified TiO2 Nanotubes Array Based Photoelectrochemical Sensors for Glutathione

Oriented TiO2 nanotubes, which are fabricated by anodic oxidation method, are prospective in photoelectrochemical analysis and sensors. In this work, Pt and IrO2 co-modified TiO2 nanotubes array was prepared via a two-step deposition process involving the photoreductive deposition of Pt and chemical deposition of IrO2 on the oriented TiO2 nanotubes. Due to the improved separation of photo-generated electrons and holes, Pt-IrO2 co-modified TiO2 nanotubes presented significantly higher PEC activity than pure TiO2 nanotubes or mono-modified TiO2 nanotubes. The PEC sensitivity of Pt-IrO2 co-modified TiO2 nanotubes for glutathione was also monitored and good sensitivity was observed.


Introduction
Photoelectrochemical (PEC) detection is a rapidly developing technique, due to its characteristics of high sensitivity, fast response speed, and simple instrumentation [1][2][3][4][5][6], and attracts a lot of research attention for detection of biomolecules, such as NADH, glucose, and glutathione, which are closely related to many serious diseases [7]. In PEC biosensors, photocurrent is produced by the physical and chemical interactions between biomolecules; photoactive species are identified as detection signal. The relationship between the biomolecule concentrations and photocurrent provides the foundation for PEC sensors. This promising analytical technology relies intensively on photoelectrodes. Hence, the selection of a proper photoelectrode is critical.
Diverse nanomaterials, such as Au [8], ZnO [9,10], CdS [11,12], TiO 2 [13,14], and porphyrin [15,16], have been exploited as electrodes. Among them, TiO 2 nanomaterials have been widely investigated due to their high PEC activity, stable performance, good biological compatibility, and obvious surface effect [17][18][19]. TiO 2 nanotube arrays can be grown directly on Ti substrates (TiO 2 NTs/Ti) by anodic oxidation, which are good candidates for PEC electrodes. For such electrodes, Ti substrates have superior performance of electronic conduction, stability, and exhibit excellent compatibility, which enable them to be excellent implantable devices [20]. In addition, the unique one-dimensional However, the application of TiO2 in PEC detection is limited by its wide band gap and fast recombination of photogenerated charges, which can lower the photoenergy conversion efficiency and sensitivity of PEC sensors. A typical method to solve these problems is modification the TiO2 photoelectrode with noble metals [22][23][24] or metal oxides [25,26]. The presence of noble metal results in the formation of a Schottky barrier at the metal-semiconductor interface, which can reduce the recombination of photogenerated charges and promote the separation of photogenerated charge carriers [27][28][29]. For instance, He's group prepared Pt particles modified TiO2 film, and the photocurrent of 3 wt% Pt modified TiO2 film under UV irradiation was 1.5 times higher than that of bare TiO2 film [30]. In contrast to Pt, IrO2 can intercept photogenerated holes from semiconductors and mediate the hole transfer process [31,32]. The synergistic effect of Pt and IrO2 has been reported by Yuan's group [33]. Pt functioned as an electron collector while IrO2 functioned as a hole capture, which may have led to significant charge separation and increased photocatalytic activity. Glutathione (γ-glutamyl-cysteinyl-glycine, GSH), an important tripeptide, plays a major role in many biological functions such as gene expression regulation, cell protection, immune regulation, enzyme activity, and metabolic regulation, etc. [34,35]. Cellular concentration of GSH is related to a variety of human diseases [34,36], and the detection of GSH is urgent because of its importance in physiological circumstances. In this paper, Pt and IrO2 nanoparticles were loaded on TiO2NTs/Ti electrodes to form a Pt-IrO2/TiO2NTs/Ti electrode as shown in Scheme 1, leading to a novel PEC biosensing platform, which was then applied for the biodetection of GSH. This biosensor showed good sensing performance of GSH with a rapid response. The functions of Pt and IrO2 in this system were also explored.

Reagent and Apparatus
All chemical reagents were analytical grade and used without further purification. Reduced glutathione (GSH), PBS (0.1 M phosphate buffer, pH = 7.4 at 25 °C), H2IrCl6·xH2O and Ti foil were purchased from Sigma (Merck Life Science (Shanghai) Co., Ltd. Shanghai, China). H2PtCl6·4H2O (Keruisi Chemical Reagent Co., Ltd. Tianjin, China) was used as platinum precursor. All aqueous solutions were prepared with 18 MΩ ultra purified water.
The chemical state of the elements on the photoelectrode was determined by Axis Ultra DLD multi-technique X-ray photoelectron spectrometer (Kratos Analytical Ltd., Manchester, UK). The phase structure of the catalysts was characterized by Rigaku D/Max-2500 X-ray diffractometer with Cu Kα radiation (Rigaku, Tokyo, Japan). Transmission electron microscopy (TEM) was performed using a Talos F200X G2 transmission electron microscope (FEI, Waltham, MA, USA). Scanning electron microscopy (SEM) was performed using a JSM-7500F scanning electron microscope (JEOL, Scheme 1. Schematic representation of preparation process of Pt-IrO 2 /TiO 2 NTs/Ti electrode.
The chemical state of the elements on the photoelectrode was determined by Axis Ultra DLD multi-technique X-ray photoelectron spectrometer (Kratos Analytical Ltd., Manchester, UK). The phase structure of the catalysts was characterized by Rigaku D/Max-2500 X-ray diffractometer with Cu Kα radiation (Rigaku, Tokyo, Japan). Transmission electron microscopy (TEM) was performed using a Talos F200X G2 transmission electron microscope (FEI, Waltham, MA, USA). Scanning electron microscopy (SEM) was performed using a JSM-7500F scanning electron microscope (JEOL, Tokyo, Japan).

Preparation of TiO 2 NTs/Ti
According to the reported anodic oxidation method [37,38], TiO 2 nanotubes arrays were prepared on Ti foil (30 mm × 10 mm × 0.127 mm). First, Ti foils were cleaned by sonicating in acetone, alcohol, and deionized water for 15 min, respectively. Then they were etched in mixture liquor HF: HNO 3 : H 2 O = 1:4:5 for 30 s to remove the oxide layer. The prepared Ti foils were anodized by anodic inoxidation at 30 V for 6 h in an electrolytic solution (0.3 wt% NH 4 F +2 vol% H 2 O in ethylene glycol) under magnetic stirring at room temperature. The samples were sonicated for 15 min in ethanol and then dried in air. Finally, the as-prepared samples were calcinated at 400 • C for 2 h in a muffle furnace to obtain TiO 2 NTs/Ti.

Preparation of Pt-IrO 2 /TiO 2 NTs/Ti
Pt-IrO 2 /TiO 2 NTs/Ti was prepared via a two-step deposition process involving the photoreductive deposition of Pt and chemical deposition of IrO 2 on TiO 2 NTs/Ti. First, TiO 2 NTs/Ti was dipped into a 0.05 mM H 2 PtCl 6 solution (ethanol and deionized water with volume ratio 1:1) under the irradiation of 300 W UV light for 3 h to get the Pt modified TiO 2 nanotube array electrode (Pt/TiO 2 NTs/Ti). Then, the Pt/TiO 2 NTs/Ti was dipped into a 0.5 mM H 2 IrCl 6 aqueous solution for 10 min, and dried in the oven at 150 • C for 10 min. After repeating the immersion process 5 times under the same conditions, the as-obtained sample was further annealed at 400 • C for 2 h to obtain Pt-IrO 2 /TiO 2 NTs/Ti. For comparation, IrO 2 modified TiO 2 NTs (IrO 2 /TiO 2 NTs/Ti) were prepared by using TiO 2 NTs/Ti as support.

Photoelectrochemical Measurement System
The photoelectrochemical experiments were performed with a CHI 604D electrochemical analyzer (CH Instruments, USA) using a three-electrode system. In the system, Ag/AgCl in 3 M KCl, platinum wire, and prepared electrodes were used as the reference electrode, counter electrode, and working electrode, respectively. An LED light source (M365L2: 365 nm, 90 mW) was fixed 30 cm above the working electrode and modulated by Transistor-Transistor Logic (TTL) out from a SR830 lock-in amplifier. PEC detection was carried out in PBS (pH = 7.4) containing different concentrations of GSH at room temperature. The switching on and off of the light source was controlled manually.

Microstructure Analysis
The XRD patterns of Ti foil, bare TiO 2 NTs, and the modified TiO 2 NTs/Ti are shown in Figure 1.  Finally, the as-prepared samples were calcinated at 400 °C for 2 h in a muffle furnace to obtain TiO2NTs/Ti.

Preparation of Pt-IrO2/TiO2NTs/Ti
Pt-IrO2/TiO2NTs/Ti was prepared via a two-step deposition process involving the photoreductive deposition of Pt and chemical deposition of IrO2 on TiO2NTs/Ti. First, TiO2NTs/Ti was dipped into a 0.05 mM H2PtCl6 solution (ethanol and deionized water with volume ratio 1:1) under the irradiation of 300 W UV light for 3 h to get the Pt modified TiO2 nanotube array electrode (Pt/TiO2NTs/Ti). Then, the Pt/TiO2NTs/Ti was dipped into a 0.5 mM H2IrCl6 aqueous solution for 10 min, and dried in the oven at 150 °C for 10 min. After repeating the immersion process 5 times under the same conditions, the as-obtained sample was further annealed at 400 °C for 2 h to obtain Pt-IrO2/TiO2NTs/Ti. For comparation, IrO2 modified TiO2NTs (IrO2/TiO2NTs/Ti) were prepared by using TiO2NTs/Ti as support.

Photoelectrochemical Measurement System
The photoelectrochemical experiments were performed with a CHI 604D electrochemical analyzer (CH Instruments, USA) using a three-electrode system. In the system, Ag/AgCl in 3 M KCl, platinum wire, and prepared electrodes were used as the reference electrode, counter electrode, and working electrode, respectively. An LED light source (M365L2: 365 nm, 90 mW) was fixed 30 cm above the working electrode and modulated by Transistor-Transistor Logic (TTL) out from a SR830 lock-in amplifier. PEC detection was carried out in PBS (pH = 7.4) containing different concentrations of GSH at room temperature. The switching on and off of the light source was controlled manually.

Microstructure Analysis
The XRD patterns of Ti foil, bare TiO2NTs, and the modified TiO2NTs/Ti are shown in Figure 1.   The morphology and microstructure of the as-prepared TiO 2 NTs/Ti was characterized by SEM and the Pt-IrO 2 /TiO 2 NTs/Ti was characterized by TEM. The top view SEM image (Figure 2a) shows that the bare TiO 2 nanotubes were highly ordered, with an average diameter of 80 nm and wall thickness of 18 nm. It is also clearly observed that the tubes were open on the top. Nanomaterials 2020, 2, x FOR PEER REVIEW 4 of 10 The morphology and microstructure of the as-prepared TiO2NTs/Ti was characterized by SEM and the Pt-IrO2/TiO2NTs/Ti was characterized by TEM. The top view SEM image (Figure 2a) shows that the bare TiO2 nanotubes were highly ordered, with an average diameter of 80 nm and wall thickness of 18 nm. It is also clearly observed that the tubes were open on the top. Figure 2b shows the microstructure of TiO2NTs modified with Pt and IrO2. To confirm the presence of Pt and IrO2 on the TiO2NTs, EDS and elemental mapping were carried out. The EDS (Figure 2c) showed that atomic percentages of Pt and Ir in Pt-IrO2/TiO2NTs/Ti were 0.70% and 0.41%, respectively. The elemental mappings present the existence of Pt and IrO2 visually, as shown in Figure  2d-g. It can observe that the distributed Pt and Ir elements were homogeneous. To further investigate the composition and elemental valences of the modified electrode, XPS analysis was performed. Figure 3 shows the XPS spectrum of Pt-IrO2/TiO2NTs/Ti, and corresponding high-resolution spectra of Ti2p, O1s, Pt4f, and Ir4f (Figure 3b-e). Figure 3a shows that the sample contained Ti, O, Pt and Ir, indicating successful deposition of Pt and IrO2 on the TiO2NTs, consistent with the EDS results. In Figure 3b, two peaks at BE of 464.0 eV and 458.5 eV in Pt-IrO2/TiO2NTs/Ti were assigned to Ti2p1/2 and Ti2p3/2 respectively, indicating the existence of Ti 4+ in TiO2 [14,39,40]. In Figure 3c, the peak at BE of 529.5 eV was assigned to Ti-O [41].  To further investigate the composition and elemental valences of the modified electrode, XPS analysis was performed. Figure 3 shows the XPS spectrum of Pt-IrO 2 /TiO 2 NTs/Ti, and corresponding high-resolution spectra of Ti2p, O1s, Pt4f, and Ir4f (Figure 3b-e). Figure 3a shows that the sample contained Ti, O, Pt and Ir, indicating successful deposition of Pt and IrO 2 on the TiO 2 NTs, consistent with the EDS results. In Figure 3b, two peaks at BE of 464.0 eV and 458.5 eV in Pt-IrO 2 /TiO 2 NTs/Ti were assigned to Ti2p 1/2 and Ti2p 3/2 respectively, indicating the existence of Ti 4+ in TiO 2 [14,39,40]. In Figure 3c, the peak at BE of 529.5 eV was assigned to Ti-O [41]. Figure 3d, the satellite peaks at 75.0 eV and 71.6 eV are strong evidence for Pt4f5/2 and Pt4f7/2, which confirms the formation of metallic Pt after the photoreduction deposition process [42,43]. After deconvolution, the high-resolution Ir4f (Figure 3d) showed the peak at 64.9 eV for Ir4f5/2 and 62.0 eV for Ir4f7/2, which is the characteristic of Ir 4+ in IrO2 [41,44,45]. Consequently, Pt and IrO2 were successfully deposited on the TiO2NTs/Ti electrode, and Pt-IrO2 co-modified electrode was fabricated.

Photoelectrochemical Performance
A photocurrent-time curve can be used to characterize the separation effect of photogenerated carriers, and the increase of photocurrent indicates the low recombination probability of photogenerated electron-holes. Figure 4 shows the photocurrent-time curves of different electrodes in 0.1 M PBS solution under 365 nm LED UV light irradiation. From Figure 4a, it can be seen that the photocurrent curve of the bare TiO2NTs electrode had an anodic photocurrent spike at the initial time of irradiation, and then continuously decreased until a constant current was reached. However, the decorated TiO2NTs electrodes could generate stable photocurrent more rapidly under the same condition, as shown in Figure 4b  As displayed in Figure 3d, the satellite peaks at 75.0 eV and 71.6 eV are strong evidence for Pt4f 5/2 and Pt4f 7/2 , which confirms the formation of metallic Pt after the photoreduction deposition process [42,43]. After deconvolution, the high-resolution Ir4f (Figure 3d) showed the peak at 64.9 eV for Ir4f 5/2 and 62.0 eV for Ir4f 7/2 , which is the characteristic of Ir 4+ in IrO 2 [41,44,45]. Consequently, Pt and IrO 2 were successfully deposited on the TiO 2 NTs/Ti electrode, and Pt-IrO 2 co-modified electrode was fabricated.

Photoelectrochemical Performance
A photocurrent-time curve can be used to characterize the separation effect of photogenerated carriers, and the increase of photocurrent indicates the low recombination probability of photogenerated electron-holes. Figure 4 shows the photocurrent-time curves of different electrodes in 0.1 M PBS solution under 365 nm LED UV light irradiation. From Figure 4a, it can be seen that the photocurrent curve of the bare TiO 2 NTs electrode had an anodic photocurrent spike at the initial time of irradiation, and then continuously decreased until a constant current was reached. However, the decorated TiO 2 NTs electrodes could generate stable photocurrent more rapidly under the same condition, as shown in Figure 4b-d. recombining with holes, and promotes the migration of charges, thus improves the stability of the photocurrent (Figure 4b). For IrO2/TiO2NTs/Ti, the photoinduced holes were taken by IrO2, effectively preventing the combination of electron-hole. So the photocurrent of IrO2/TiO2NTs/Ti electrode also could quickly stabilize, as shown in Figure 4c. In the Pt-IrO2 modified TiO2 system, due to the synergistic effect of Pt and IrO2, the recombination of photogenerated electrons and holes could be effectively blocked. The Pt-IrO2/TiO2NTs/Ti electrode in Figure 4d exhibited an apparent and stable photocurrent signal compared with the TiO2NTs/Ti. However, the mono Pt-or IrO2-modified TiO2NTs/Ti exhibited lower photocurrent responses than bare TiO2NTs/Ti. The reason may be that Pt-and IrO2-loading on TiO2NTs shelter the TiO2NTs from the light illumination, which could reduce the efficiency of photogenerated carriers. Another reason may be that when Pt or IrO2 nanoparticles appeared alone on the TiO2, both photogenerated electrons and holes may transfer to Pt or IrO2 nanoparticles, making them act as recombination centers [46,47]. However, if Pt and IrO2 nanoparticles co-exist, electrons would move to Pt, while holes move to IrO2. The electron-hole recombination would be suppressed, leading to a higher photocurrent.

PEC biosensing Application for GSH
On the basis of photoelectrochemical performance, the application of the fabricated Pt-IrO2/TiO2NTs/Ti for detection of GSH was investigated. The electrode was kept in PBS at +0.3 V bias voltage under UV illumination for 5 minutesas pretreatment, and then the positive photocurrent was detected in the presence of GSH in PBS. As shown in Figure 5, the photocurrent increased with the addition of GSH under illumination, fitted with the Langmuir curve. The inset presented in Figure 5 shows that the photocurrent response of the Pt-IrO2/TiO2NTs/Ti biosensor was proportional to GSH concentration in the range of 1~10 μM with the regression equation of I-I0(μA) = 2.505 + 54.171CGSH(uM) (R 2 = 0.9932) (I represents the photocurrent obtained in the presence of GSH, and I0 is the blank photocurrent). The detection limit (LOD) of the sensor can be obtained from the formula LOD = 3 Sb/S (Sb = standard deviation of blank signal, S = sensitivity), and the calculated value was 0.8 μM.
Scheme 2 illustrates the PEC process for GSH detection by Pt-IrO2/TiO2NTs/Ti biosensor. Under the illumination of UV light, electron-hole pairs were generated in TiO2. Due to the Schottky barrier at the Pt/TiO2 interface, electrons moved from TiO2 electrode to Pt. Simultaneously, the photoinduced holes were taken by IrO2, and then oxidized GSH to GSSG. Under illumination, TiO 2 could absorb UV light and generate electron-hole pairs, and electrons from the TiO 2 electrode could transfer to Pt. So the surface of Pt nanoparticles appeared negatively charged and TiO 2 appeared positively charged. The Schottky barrier prevents electrons from recombining with holes, and promotes the migration of charges, thus improves the stability of the photocurrent (Figure 4b). For IrO 2 /TiO 2 NTs/Ti, the photoinduced holes were taken by IrO 2 , effectively preventing the combination of electron-hole. So the photocurrent of IrO 2 /TiO 2 NTs/Ti electrode also could quickly stabilize, as shown in Figure 4c. In the Pt-IrO 2 modified TiO 2 system, due to the synergistic effect of Pt and IrO 2, the recombination of photogenerated electrons and holes could be effectively blocked. The Pt-IrO 2 /TiO 2 NTs/Ti electrode in Figure 4d exhibited an apparent and stable photocurrent signal compared with the TiO 2 NTs/Ti. However, the mono Pt-or IrO 2-modified TiO 2 NTs/Ti exhibited lower photocurrent responses than bare TiO 2 NTs/Ti. The reason may be that Pt-and IrO 2-loading on TiO 2 NTs shelter the TiO 2 NTs from the light illumination, which could reduce the efficiency of photogenerated carriers. Another reason may be that when Pt or IrO 2 nanoparticles appeared alone on the TiO 2 , both photogenerated electrons and holes may transfer to Pt or IrO 2 nanoparticles, making them act as recombination centers [46,47]. However, if Pt and IrO 2 nanoparticles co-exist, electrons would move to Pt, while holes move to IrO 2 . The electron-hole recombination would be suppressed, leading to a higher photocurrent.

PEC biosensing Application for GSH
On the basis of photoelectrochemical performance, the application of the fabricated Pt-IrO 2 /TiO 2 NTs/Ti for detection of GSH was investigated. The electrode was kept in PBS at +0.3 V bias voltage under UV illumination for 5 min as pretreatment, and then the positive photocurrent was detected in the presence of GSH in PBS. As shown in Figure 5, the photocurrent increased with the addition of GSH under illumination, fitted with the Langmuir curve. The inset presented in Figure 5 shows that the photocurrent response of the Pt-IrO 2 /TiO 2 NTs/Ti biosensor was proportional to GSH concentration in the range of 1~10 µM with the regression equation of I-I 0 (µA) = 2.505 + 54.171C GSH (uM) (R 2 = 0.9932) (I represents the photocurrent obtained in the presence of GSH, and I 0 is the blank photocurrent). The detection limit (LOD) of the sensor can be obtained from the formula LOD = 3 Sb/S (Sb = standard deviation of blank signal, S = sensitivity), and the calculated value was 0.8 µM. Scheme 2. Schematic illustration of the PEC process for GSH detection by Pt-IrO2/TiO2NTs/Ti biosensor.  Table 1 summarizes the comparison of analytical performance of various GSH biosensors. The Pt-IrO2/TiO2NTs/Ti biosensor had a relatively reasonable linear range in 1-10 μM. Compared with other non-enzymatic sensors, Pt-IrO2/TiO2NTs/Ti was sensitive and could be used at relatively low GSH concentrations. Though enzymatic sensors offer high sensitivity, non-enzymatic sensors are widely recognized for their good advantages of simple operation, lack of need for expensive equipment, and high stability. The stability of the Pt-IrO2/TiO2NTs biosensor was tested by measuring photocurrent response after 30 days (see Supplementary Figure S1). The photocurrent did not decrease significantly, indicating that the electrode's good stability for GSH detection. This should contribute to the good stability of Pt and IrO2 on TiO2NTs/Ti electrodes. IrO2-Hemin-TiO2 nanowire arrays 0.01-10 0.01 [17] In summary, a PEC sensor for GSH was designed using Pt-IrO2/TiO2NTs/Ti as an electrode. Pt and IrO2 were evenly distributed on TiO2NTs/Ti. Due to the synergistic effects of Pt and IrO2, the Pt-IrO2/TiO2NTs/Ti electrode exhibited an apparent and stable photocurrent signal compared with the TiO2NTs/Ti or the mono-modified ones. The photocurrent signals of the Pt-IrO2/TiO2NT/Ti biosensor    Table 1 summarizes the comparison of analytical performance of various GSH biosensors. The Pt-IrO2/TiO2NTs/Ti biosensor had a relatively reasonable linear range in 1-10 μM. Compared with other non-enzymatic sensors, Pt-IrO2/TiO2NTs/Ti was sensitive and could be used at relatively low GSH concentrations. Though enzymatic sensors offer high sensitivity, non-enzymatic sensors are widely recognized for their good advantages of simple operation, lack of need for expensive equipment, and high stability. The stability of the Pt-IrO2/TiO2NTs biosensor was tested by Scheme 2. Schematic illustration of the PEC process for GSH detection by Pt-IrO 2 /TiO 2 NTs/Ti biosensor. Table 1 summarizes the comparison of analytical performance of various GSH biosensors. The Pt-IrO 2 /TiO 2 NTs/Ti biosensor had a relatively reasonable linear range in 1-10 µM. Compared with other non-enzymatic sensors, Pt-IrO 2 /TiO 2 NTs/Ti was sensitive and could be used at relatively low GSH concentrations. Though enzymatic sensors offer high sensitivity, non-enzymatic sensors are widely recognized for their good advantages of simple operation, lack of need for expensive equipment, and high stability. The stability of the Pt-IrO 2 /TiO 2 NTs biosensor was tested by measuring photocurrent response after 30 days (see Supplementary Figure S1). The photocurrent did not decrease significantly, indicating that the electrode's good stability for GSH detection. This should contribute to the good stability of Pt and IrO 2 on TiO 2 NTs/Ti electrodes. Enzymatic sensor IrO 2 -Hemin-TiO 2 nanowire arrays 0.01-10 0.01 [17] Nanomaterials 2020, 10, 522 8 of 10 In summary, a PEC sensor for GSH was designed using Pt-IrO 2 /TiO 2 NTs/Ti as an electrode. Pt and IrO 2 were evenly distributed on TiO 2 NTs/Ti. Due to the synergistic effects of Pt and IrO 2, the Pt-IrO 2 /TiO 2 NTs/Ti electrode exhibited an apparent and stable photocurrent signal compared with the TiO 2 NTs/Ti or the mono-modified ones. The photocurrent signals of the Pt-IrO 2 /TiO 2 NT/Ti biosensor was linear to GSH concentration in the range of 1~10 µM. Other kinds of modified TiO 2 NTs/Ti electrodes which may exhibit high PEC sensitivity under visible light are anticipated.

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