Hemin Modified TiO2 Nanoparticles with Enhanced Photoelectrocatalytic Activity for Electrochemical and Photoelectrochemical Sensing

Hemin-functionalized TiO2 nanoparticles were prepared and assembled on glassy carbon electrode through the electrostatic attraction between positively charged poly(diallyldimethylammonium chloride) and negatively charged hemin and TiO2. The scanning electron microscopy and X-ray diffraction characterization showed that the assembled hemin-TiO2 film displayed a mesoporous structure comprising plenty of TiO2 nanoparticles in rutile phase. The Fourier transform infrared spectra and UV-visible diffuse reflectance spectra indicated that hemin was successfully incorporated into TiO2 film and significantly enhanced the absorption of TiO2 film to visible light. The hemin-TiO2 film exhibited a pair of well-defined redox peaks of hemin by cyclic voltammetry, which could effectively catalyze the electroreduction of H2O2. Thus, the hemin-TiO2 modified electrode was employed as an electrochemical sensor for H2O2 determination, showing a sensitive response linearly proportional to the concentration of H2O2 from 3.0 × 10−7 to 4.7 × 10−4 mol · L−1. At the same time, the photoelectrocatalytic activity of TiO2 under visible light illumination was dramatically promoted by hemin. The hemin-TiO2 modified electrode produced a photoelectrochemical response linearly proportional to hydroquinone in the concentration range from 4.0 × 10−7 to 3.0 × 10−5 mol · L−1. © The Author(s) 2016. Published by ECS. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 License (CC BY, http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse of the work in any medium, provided the original work is properly cited. [DOI: 10.1149/2.1291609jes] All rights reserved.

Hemin is an iron-containing porphyrin, namely protoporphyrin IX Fe(III) complex, which is well-known as the active center of the hemeprotein family. Because of the reversible reaction of Fe(III)/Fe(II) redox couple, hemin has been extensively demonstrated as an efficient electrocatalyst for many small molecules such as hydrogen peroxide, 1-3 oxygen, 4 nitrite, 5 L-tyrosine 6 and artemisinin. 7 Generally, hemin is loaded on some supporting materials to avoid the molecular aggregation of hemin molecules in aqueous solution and improve the stability and activity of catalyst. 8 On the other hand, hemin is a structural analogue of chlorophyll, which can serve as a promising photosensitizer for TiO 2 photocatalyst to harvest visible light. 9,10 Moreover, the presence of Fe(III) porphyrin ring on the surface of TiO 2 can reduce the electron-hole recombination rate and act as a mediator for continuous production of enriched concentration of hydroxyl radicals to enhance the photocatalytic activity of TiO 2 . 11 TiO 2 is the most intensively studied photocatalyst for degradation of various organic pollutants. 12 In recent years, TiO 2 nanomaterials have been widely utilized for fabrication of sensing devices because of their fascinating properties such as large surface area, good biocompatibility, high stability, and unique electronic and photocatalytic performances. 13 When catalytic materials including metal nanoparticles, small molecules and biological macromolecules are immobilized on nanostructured TiO 2 , the obtained nanocomposites can act as bifunctional catalysts which not only possess the catalytic activity of introduced materials but also preserve the intrinsic photocatalytic capacity of TiO 2 . 14 Recently, TiO 2 nanomaterials coupled with hemin have been investigated for photocatalytic degradation of organic pollutants 9,11 and photoelectrochemical detection of glutathione. 15 However, these previous reports have only focused on the photocatalytic activity of hemin-TiO 2 composites.
In the present work, TiO 2 nanoparticles coupled with hemin were prepared and utilized as bifunctional sensing materials to fabricate high-performance electrochemical/photoelectrochemical sensors. In as-prepared hemin-TiO 2 film, hemin preserved its catalytic activity while mesoporous TiO 2 film provided a solid support for the loading of hemin. The composite film showed high electrocatalytic activity toward H 2 O 2 . The current was linearly proportional to the concentration of H 2 O 2 in the range of 3.0 × 10 −7 to 4.7 × 10 −4 mol · L −1 . Moreover, hemin molecule with the structure of Fe-porphyrin ring could z E-mail: zhangjd@mail.hust.edu.cn effectively enhance the absorption of TiO 2 film to visible light. The hemin-TiO 2 film exhibited high photoelectrocatalytic activity which was applied to photoelectrochemical sensing of hydroquinone (HQ) in the concentration range from 4.0 × 10 −7 to 3.0 × 10 −5 mol · L −1 .

Materials and Methods
Chemicals.-Hydrogen peroxide (30%, w/v) was obtained from Shanghai Experimental Reagent Co., China. Titanium (IV) chloride, hemin, HQ and other reagents of analytical grade were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Poly(diallyldimethylammonium chloride) solution (PDDA) was obtained from Aladdin Reagent Co. Ltd. (Shanghai, China). Phosphate buffer solution (PBS) was prepared by mixing certain amount of Na 2 HPO 4 and NaH 2 PO 4 . Double distilled water was used throughout the experiment.
Preparation of hemin-TiO 2 modified electrode.-The TiO 2 nanoparticles were prepared by a flame synthesis technique as described in a previous work. 16 The isoelectric point (IEP) of the obtained TiO 2 nanoparticles was approximately 3.4 and lower than that of other phases (including anatase, mixture of anatase and rutile), 17 which was pivotal to design novel devices for applications in sensing, catalysis and energy conversion. The resultant TiO 2 powder was dispersed in 10.0 mL of NaOH solution to give a 5 mg · mL −1 TiO 2 suspension by ultrasonic agitation. After the addition of 10 mg hemin, the mixture was ultrasonic for another 8 min to make small hemin molecules evenly dispersed in TiO 2 suspension.
The hemin-TiO 2 mixed solution was utilized to modify a glass carbon electrode (GCE) (exposed geometric area of 0.096 cm 2 ). Prior to modification, the GCE surface was polished to a mirror-like smoothness with emery papers and then cleaned successively with ethanol and deionized water. After dried with nitrogen gas, the GCE was immersed into 2% PDDA solution containing 0.5 mol · L −1 NaCl for 0.5 h, and then thoroughly rinsed with distilled water to remove the loosely adsorbed PDDA. After being dried with nitrogen gas, the GCE surface was immersed into hemin-TiO 2 mixed solution (containing 5 mg · mL −1 TiO 2 and 1 mg · mL −1 hemin) for 1 h. After thoroughly rinsed with distilled water to remove the loosely adsorbed hemin-TiO 2 , the modified electrode was dried with nitrogen gas. In this process, hemin and TiO 2 were negatively charged in alkaline solution, and positively charged PDDA was beneficial to the immobilization of Journal of The Electrochemical Society, 163 (9) B526-B532 (2016) B527 nanocomposites on electrode surface through the electrostatic attraction. For comparison, TiO 2 or hemin modified electrode was prepared in the same procedure except that pure TiO 2 or hemin solution was used instead of hemin-TiO 2 mixed solution.
Apparatus and procedure.-The morphology of TiO 2 and hemin-TiO 2 modified electrodes were characterized with a field emission scanning electron microscope (FESEM) (FEI Sirion 200, Netherland). The X-ray diffraction (XRD) pattern was measured using a X-ray diffractometer (PANalytical X'pert Pro, Netherland) equipped with a Cu Kα (λ = 1.54 Å) radiation source. The accelerating voltage and applied current were 40 kV and 40 mA, respectively. UV-visible diffuse reflectance spectra (DRS) were recorded with a UV-2550 spectrophotometer (Shimadzu, Japan). The UV-visible absorption spectra were recorded using a TU-1900 UV-visible spectrometer (Beijing Purkinje General Instrument Company, China). Fourier transform infrared (FTIR) spectroscopic analysis was carried out with an Equinox 55 FTIR spectrometer (Bruker, Germany).
The electrochemical and photoelectrochemical experiments were performed on a CHI660A electrochemical working station (Shanghai Chenhua Instrument Co. Ltd., China) in a conventional three-electrode system. The fabricated hemin-TiO 2 modified electrode, a platinum wire, and a saturated calomel electrode (SCE) served as the working, auxiliary and reference electrodes, respectively. All voltammetric and amperometric experiments were carried out in deaerated solutions in an atmosphere of nitrogen. The photoelectrochemical measurements were performed in air-saturated solutions as described previously. 18 A xenon lamp (PLS-SXE300, Perfect Co., China) with an optical filter (λ > 420 nm) was used as the visible light irradiation source, and the distance between the lamp and working electrode surface was 15 cm.
The photodegradation experiments for HQ were carried out in a quartz beaker containing 100 mL of 2 × 10 −4 mol · L −1 HQ aqueous solution. A 15-W UV lamp with a major emission wavelength at 254 nm was employed as the UV irradiation source. After irradiating for a certain time, 1 mL of sample was taken out of the photoreactor and mixed with 9 mL PBS, and then analyzed using the proposed photoelectrochemical sensor.

Results and Discussion
Characterization of hemin-TiO 2 composite film.-The surface morphology of pure TiO 2 and hemin-TiO 2 composite film modified electrode was characterized by FESEM. It was observed that for pure TiO 2 film, plenty of TiO 2 nanoparticles in the size of 10−50 nm were assembled on the electrode surface which formed a relatively rich and uniform mesoporous film structure (Fig. 1A). While hemin was coupled with TiO 2 , the characteristic of TiO 2 nanoparticles and mesoporous film structure were not destroyed (Fig. 1B). Obviously, the mesoporous film structure of TiO 2 cannot only provide an excellent matrix for hemin incorporation but also offer many active adsorption sites for catalytic reactions. 19 The crystalline nature of the TiO 2 film was investigated by XRD. As shown in curve a in Fig. 2, the main diffraction peaks assigned to the rutile phase structure of TiO 2 (JCPDS 76-1940) were clearly observed, in accord with the previous report. 20 For comparison, the XRD pattern of hemin-TiO 2 film is depicted in curve b in Fig. 2. Consistent with SEM observation, the coupling of hemin did not change the crystal structure of TiO 2 . bending vibration, and the band 2923 cm −1 assigned to the C-H stretching vibration of methyl. The obvious absorption peaks at 1563 cm −1 might correspond to the C=C-H and amide band II. Moreover, the broad peak around 1652 cm −1 might be assigned to C=C-H and amide band I or -NH 2 bending vibration and skeletal vibration of aromatic ring. 22 Thus, the coupling of hemin with TiO 2 in the composite film was successfully confirmed by FTIR spectroscopic analysis. The incorporation of hemin obviously influenced the absorption of TiO 2 in the visible light region. It was observed that pure TiO 2 film mainly absorbed UV light at a wavelength less than 380 nm but exhibited weak absorption in the visible light region (curve a in Fig. 4). For hemin, the absorption curve was characterized by a Soret peak at ∼388 nm along with a group of weak absorption bands between 500 and 600 nm attributed to Q-bands (curve b in Fig. 4). While hemin was coupled with TiO 2 , a redshift in the absorption onset value was observed and the absorption in the visible region was significantly improved (curve c in Fig. 4). The enhanced absorption of TiO 2 film to visible light by hemin should be due to the structure of Fe-porphyrin ring in hemin molecules.  Fig. 5). The formal potential estimated as the average value of the anodic and cathodic peak potentials is −0.33 V (vs. SCE), which is close to those obtained with hemin-adsorbed carbon felt and hemin-functionalized carbon nanotubes. 4,23 Obviously, the redox peaks are attributed to the mono-electron transfer process concerning the Fe(III)/Fe(II) redox couple coordinated in the porphyrinic ring of hemin. The mesoporous structure of TiO 2 not only plays an important role for effective immobilization of hemin molecules on the electrode surface but also promotes the electron transfer between hemin and electrode. The effects of scan rate and pH on the voltammetric behavior of hemin-TiO 2 film were investigated. As shown in Fig.  6A, the cathodic peak potential of the hemin-TiO 2 film shifted negatively with increasing the scan rate. Moreover, the cathodic peak current (i pc ) was linearly enhanced with the increase of the scan rate (ν). The linear regression equation was expressed as i pc (μA) = −0.0361ν (mV · s −1 )−1.2573 (correlation coefficient r = 0.996). This result indicates that the electrochemical reaction of hemin on hemin-TiO 2 modified electrode is a surface-controlled process.

Electrochemical behavior of hemin-TiO
On the other hand, the cathodic peak potential of hemin-TiO 2 film shifted negatively with increasing the solution pH from 6.0 to 8.0 (Fig. 6B). A linear relationship was found between the cathodic peak potential (E pc ) and pH, which could be expressed by E pc (V) = −0.0421pH-0.100 (correlation coefficient r = 0.996). The slope of 0.042 V/pH is lower than the theoretical value of 0.059 V/pH for a reversible one proton coupled single-electron transfer, implying that protons are not only involved in the interfacial charge compensation associated with the electron transfer process. 4 The solution pH can cause changes in protonation state of the propionate side chains attached to the protoporphyrin macro-cycle and the ligation state of the iron center of hemin. 24 H 2 O 2 .-Fig. 7 displays the CVs of various modified electrodes recorded in deaerated PBS without and with 0.1 mmol · L −1 H 2 O 2 . On pure TiO 2 modified electrode (Fig. 7A), the CV curve did not show obvious change after H 2 O 2 was added. When hemin modified electrode was applied (Fig. 7B), the presence of H 2 O 2 led to a slight enhancement in cathodic current. In comparison, when H 2 O 2 was added into the solution, the reduction peak current of hemin-TiO 2 modified electrode was dramatically enhanced, accompanied by disappearance of oxidation peak current (Fig. 7C). This result is similar to our previous observation on a graphene-hemin modified graphite electrode, 25 demonstrating the high electrocatalytic activity of hemin-TiO 2 toward the reduction of H 2 O 2 . The hemin-TiO 2 modified electrode was explored as an electrochemical sensor for quantitation of H 2 O 2 concentration by chronoamperometry. Although the reduction peak potential for H 2 O 2 displayed in CV was around −0.32 V (curve b in Fig. 7C), the applied potential for amperometric sensing of H 2 O 2 should be more positive to decrease the background current and minimize the response of common interference species. 26,27 We studied the influence of the applied potential on the amperometric response of the sensor to H 2 O 2 . As shown in Fig.  8A, the current response toward H 2 O 2 was obviously enhanced when the applied potential was shifted from −0.10 to −0.15 V. When the applied potential was further negatively shifted from −0.15 to −0.35 V, the background current was markedly increased whereas the current response toward H 2 O 2 decreased. Therefore, we selected −0.15 V as the working potential for amperometric sensing of H 2 O 2 in the following experiments. Fig. 8B illustrates the chronoamperometric response of the hemin-TiO 2 modified electrode to the successive injection of H 2 O 2 into PBS under constant passage of high purity nitrogen gas. It was seen that upon each addition of H 2 O 2 , the amperometric current was increased sharply and then reached to a steady value within a response time less than 5 s. With increasing the concentration of H 2 O 2 from 3.0 × 10 −7 to 4.7 × 10 −4 mol · L −1 , a linearly enhanced amperometric response was obtained (Fig. 8C) r = 0.999). The limit of detection (3S/N) was estimated to be 7.2 × 10 −8 mol · L −1 , which was lower than previously reported results. 1,[27][28][29] For comparison, the amperometric responses of pure TiO 2 and hemin modified electrodes to H 2 O 2 were also studied under the same experimental conditions. As shown in Fig. 8D, the TiO 2 modified electrode did not show any response to the addition of H 2 O 2 while hemin modified electrode responded to H 2 O 2 . The amperometric current on hemin modified electrode was found to be increased linearly with increasing the concentration of H 2 O 2 from 5.0 × 10 −6 and 2.8 × 10 −4 mol · L −1 (inset of Fig. 8D). The linear relationship could be expressed as I (μA) = −0.026 [H 2 O 2 ] (μmol · L −1 )-0.003 (correlation coefficient r = 0.998), and the detection limit (3S/N) was estimated to be 1.9 × 10 −7 mol · L −1 . Obviously, hemin-TiO 2 modified electrode exhibited higher sensitivity and lower limit of detection for H 2 O 2 determination than hemin modified electrode, suggesting the high performance of the proposed sensor based on TiO 2 -supported hemin.

Electrocatalytic activity of hemin-TiO 2 and amperometric sensing of
The reproducibility of such an electrochemical sensor was investigated by recording the current responses of ten independently prepared hemin-TiO 2 modified electrodes in deaerated PBS containing 1.0 × 10 −5 mol · L −1 H 2 O 2 . The relative standard deviation (RSD) was 2.1%, showing a good reproducibility. The RSD of the sensor for 10 successive measurements was 2.8%, indicating a good repeatability. The stability of the developed sensor was investigated by recording the current response every four days. When not in use, the sensor was stored in 4 • C. The result indicated that the response did not show obvious change after sixteen days, demonstrating that the sensor owned a high stability.
Moreover, the applicability of the developed sensor was evaluated for the determination of H 2 O 2 concentration in practical honey samples purchased from a supermarket in Wuhan City. Prior to the electrochemical measurement, the diluted honey solution was obtained by dissolving 2.0 g of the honey sample into 10.0 mL distilled water. Then 1.0 mL of the sample solution was transferred to 9.0 mL PBS (pH 7.0) to record the amperometric current on hemin-TiO 2 modified electrode. Because no H 2 O 2 was detected in the original honey sample, we carried out the electrochemical determination using the standard addition method by spiking different amounts of standard H 2 O 2 into the sample. As can be seen from Table I, the recovery values of the proposed method were in the range from 98.6% to 102.4%, indicating the feasibility of the hemin-TiO 2 based electrochemical sensor for the determination of H 2 O 2 in real samples.
Photoelectrochemical sensing of hydroquinone.-Considering that the photoelectrocatalytic activity of semiconducting materials could be evaluated by photocurrent, we measured the photocurrent response of hemin-TiO 2 modified electrode in 0.1 mol · L −1 PBS (pH 7.0) under visible light illumination. As shown in curve a in Fig. 9A, the hemin-TiO 2 modified electrode responded sensitively to the visible light irradiation. When the lamp was switched on, the current was rapidly increased until a nearly steady-state photocurrent value was  reached. While the lamp was switched off, the current was quickly decreased to the original low dark-current value. This confirms the high photoelectrocatalytic activity of hemin-TiO 2 . In contrast, the pure TiO 2 film only displayed a low photoelectrocatalytic activity under visible light illumination (curve b in Fig. 9A), on which the photocurrent value was only ca. 1/3 of that on the hemin-TiO 2 modified electrode. This is consistent with the enhanced absorption of TiO 2 to visible light by hemin as illustrated in Fig. 4. When hemin-TiO 2 is irradiated under visible light, the porphyrin moiety of hemin is excited instead of TiO 2 to generate electron-hole pairs (electron in the excited singlet or triplet state of hemin and hole in the ground state of hemin). The excited state of hemin rapidly transfers electrons into the conduction band of TiO 2 , while TiO 2 works as the electron trapper and hinders the electron-hole recombination, significantly improving the photoefficiency. 30 In addition, the iron metal ion chelated to porphyrin ring takes up the excited energy of material and causes strong intramolecular perturbations. As a result, an excited triplet state with longer lifetime is created, which provides enough time for the transfer of electrons from the excited hemin molecule to the TiO 2 conduction band. 31 Moreover, when electron donor is added to consume the holes located on the excited state of hemin, the electrons may quickly be injected into the conduction band of the TiO 2 and subsequently transferred to the GC electrode, resulting in enhanced photocurrent.
Since hemin-TiO 2 possessed high photoelectrocatalytic activity, we employed the hemin-TiO 2 modified electrode to develop a visible light-driven photoelectrochemical sensor for the quantitative determination of hydroquinone (HQ), a common environmental pollutant. Fig. 9B illustrates the responses of HQ at different concentrations on the hemin-TiO 2 -based sensor. It was seen that the photocurrent increased with the increase of HQ concentration, which could be attributed to the quick oxidation of adsorbed HQ molecules by photogenerated holes on hemin-TiO 2 . However, the photocurrent response for HQ at higher concentration became difficult to reach the steady state even though the irradiation time was prolonged. To ensure the fast photoelectrochemical sensing but avoid the problem of decayed photocurrent, we recorded the photocurrent responses at the same irradiation period and determined the transient photocurrent values at the middle moment of this period for quantitative analysis. 32 The photocurrent difference ( PI) before and after adding HQ was found to be linearly proportional to the concentration of HQ ranging from 4.0 × 10 −7 to 3.0 × 10 −5 mol · L −1 . The linear regression equation was expressed as PI/μA = 0.233C/μM−0.025 (correlation coefficient r = 0.998). The detection limit (3S/N) was estimated to be 9.8 × 10 −8 mol · L −1 .
The proposed photoelectrochemical sensor was applied to monitoring the degradation of HQ treated by UV irradiation. As shown in Fig. 9C, the photocurrent of HQ on the sensor was decreased with increasing the degradation time, indicating that the degraded HQ molecules were unable to consume the photogenerated holes on hemin-TiO 2 film. The decay percentages of photoelectrochemical response toward HQ after 30-min and 60-min degradation were calculated to be 9.2% and 14.7% respectively, close to the degradation efficiency of 9.0% and 14.6% determined by the traditional spectrometry. Therefore, such a photoelectrochemical sensor provided a valid and sensitive method for monitoring HQ degradation.

Conclusions
In this work, hemin-functionalized TiO 2 nanoparticles were found to simultaneously possess both the catalytic properties of hemin and TiO 2 . Due to the electrocatalytic activity of hemin toward H 2 O 2 and mesoporous structure of TiO 2 for hemin loading, the hemin-TiO 2 modified electrodes could be utilized as a H 2 O 2 sensor, which displayed a high sensitivity, good reproducibility and high stability. Meanwhile, the sensitization of TiO 2 by hemin remarkably promoted the photoelectrocatalytic activity of TiO 2 to visible light. Based on the high photoelectrocatalytic activity of hemin-TiO 2 , a photoelectrochemical sensor for the determination of HQ was developed. Thus, hemin-TiO 2 composites were successfully demonstrated in developing electrochemical and photoelectrochemical sensing devices.