Editable TiO2 Nanomaterial-Modified Paper in Situ for Highly Efficient

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Biological and Medical Applications of Materials and Interfaces

Editable TiO2 nanomaterials modified paper in-situ for highly efficient detection of carcinoembryonic antigen by photoelectrochemical method Li Li, Ting Wang, Yan Zhang, Caixia Xu, Lina Zhang, Xin Cheng, Hong Liu, Xiaodong Chen, and Jinghua Yu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b03632 • Publication Date (Web): 11 Apr 2018 Downloaded from http://pubs.acs.org on April 11, 2018

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ACS Applied Materials & Interfaces

Editable TiO2 nanomaterials modified paper in-situ for highly efficient detection of carcinoembryonic antigen by photoelectrochemical method Li Li,† Ting Wang,‡ Yan Zhang,† Caixia Xu,§ Lina Zhang,*,# Xin Cheng,# Hong Liu,§ Xiaodong Chen,*,‡ and Jinghua Yu*†,§

†

School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022,

P.R. China. ‡

School of Materials Science and Engineering, Nanyang Technological University,

Singapore 639798 §

Institute for Advanced Interdisciplinary Research, University of Jinan, Jinan 250022,

China. #

Shandong Provincial Key Laboratory of Preparation and Measurement of Building

Materials, University of Jinan, Jinan 250022, P.R. China.

Abstract In this work, a versatile photoelectrochemical paper-based sensor based on N-carbon dots/TiO2-Pt modified paper in-situ is developed for sensitive detection of carcinoembryonic antigen (CEA) in clinical serum samples. Interconnected cellulose fibers on paper provide a porous, 3D, and flexible substrate for photoelectrochemical sensing. In-situ modification of N-carbon dots/TiO2-Pt with editable structures on paper significantly increases conductivity, widens adsorption range, and enhances photoelectrochemical ability, which enables the higher sensitivity and flexibility

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compared to traditional rigid sensors. Based on this novel protocol, a miniaturized and portable origami device realizes the CEA determination with a low detection limit of 1.0 pg mL-1 and a wide linear range from 0.002 to 200 ng mL-1. Thanks to the good biocompatibility, the paper-based device provides a new avenue for detection of CEA on cell surface which is promising for portable diagnostics in early tumor warning. Keywords: paper-based sensor, photoelectrochemical, TiO2 nanoparticles, in-situ modification, early tumor warning


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1. Introduction Paper is made of randomly interconnected cellulose fibers with attractive features of high porosity, excellent flexibility and 3D structure, which is employed as intriguing support for flexible energy storage devices,1 primary battery,2 and piezoelectric nanogenerators.3 In particular, paper-based biosensors4-6 are drawing increasing attention both in laboratory and clinical test because of their low-cost, portable, and degradability

characteristics.

Among

these

biosensors,

paper-based

photoelectrochemical biosensors7-9 are identified as the most promising analytical detection technology due to their simple instruments, high selectivity, and sensitivity. Despite the advantages, paper fibers own poor electrical conductivity and limited functionality, which are adverse to achieve high-performance photoelectrochemical devices. Rational modification of functional nanomaterials is a potent strategy to achieve high performance and multifunctional devices.10-16 Therefore, to obtain a high efficient flexible paper-based photoelectrochemical device, we need to design an efficient photoactive nanomaterial and develop a way to readily immobilize nanomaterials on cellulose fibers as well. Among various photoactive nanomaterials, TiO2 nanoparticles (NPs) based materials are most promising due to their high resistance

to

photocorrosion,

low

cost,

attractive

catalytic

and

optical

performances.17-20 Recently, biosensors based on the TiO2 NPs generally modify TiO2 NPs on the surface of rigid electrode utilizing the template-mediated synthesis or the drop-casting method.21,22 These strategies greatly limit the effective area of the 3 ACS Paragon Plus Environment


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working electrode and run counter to the purpose of the personal small and flexible electronic devices. Herein, we propose a method for synthesis of TiO2 NPs-based hierarchical structures and in-situ modification of them on paper fibers. We firstly modified a dense Pt NPs layer23 to significantly enhance the conductivity of the cellulose fibers (Pt/PWE) and also lower the recombination probability of electron-hole pairs through formation of the Pt/TiO2 heterojunction24 and N-doped carbon dots/TiO2-Pt.25-27 Meanwhile, seed-mediate growth method was firstly applied for preparing TiO2 NPs in situ on fibers which ensured the stability of immobilized TiO2 NPs. The increased conductivity, widened adsorption range, and enhanced stability are expected to boost the photoelectrochemical efficiency of the flexible collapsible paper-based platform. The

paper-based

photoelectrochemical

device

is

then

applied

in

carcinoembryonic antigen (CEA) detection which is the most common tumor markers related with variety of cancers. The biosensor principle is that the specific recognition of CEA hinders the photoelectron transfer and decreases the photocurrent intensity (Scheme 1). The developed biosensor showed a detection limit at 1.0 pg mL-1 and a wide linear response region from 0.002 to 200 ng mL-1 which fully covered the concentration range of CEA in human serum. This work not only opens up new possibilities for preparing the TiO2 NPs based flexible electronic platforms, but also widens the application potentials of the paper-based photoelectrochemical devices for early diagnosis of cancers and personalized diagnostics.


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Scheme 1. Schematic illustration of the preparation procedure of the photoelectrochemical biosensor (a-c); (d) the schematic diagram of the flexible paper-based platform; (e) the detection mechanism of the biosensor for CEA on the surface of MCF-7 cells and the corresponding signals (f), number 1 and 2 were the photocurrent intensity without (c) or with target presence (e), respectively.

2. Materials and methods 2.1. Reagents and materials TiCl3 aqueous solution was bought from Alfa Aesar. CEA and H2PtCl6 were purchased from Beijing Keybiotech Company, Ltd. (China). The target aptamer sequences for CEA was 5’-ATA CCA GCT TAT TCA ATT-NH2-3’, which was provided from Sangon Biological Engineering Technology & Company Ltd. (Shanghai, China). 2.2. Fabrication of the TiO2-Pt/PWE Before the preparation of TiO2-Pt/PWE, the Pt/PWE was fabricated via a redox reaction by utilizing H2PtCl6 and ascorbic acid (AA) as raw materials described in our previous report.28 Then seed-mediate growth strategy was used for fabrication of the TiO2-Pt/PWE. 5 ACS Paragon Plus Environment


Synthesis of TiO2 seeds: 20.0 mL of ultrapure water, HCl, and 0.2 mL of TiCl3 solution were mixed at 80 °C for 2 h. After washed with water and ethanol for three times, the TiO2 seeds were obtained. Then the obtained TiO2 seeds were dried at 450 °C in air for 2 h. Process of TiO2 seeds growth: The as-prepared TiO2 seeds (15 µL) were moved onto the Pt/PWE by a micropipette and equilibrated at 37 °C. The process above-mentioned was repeated for three times. Then the paper was put in a beaker containing 20.0 mL of water, 1~2 g of NaCl, 0.2 mL of TiCl3, 600 µL of HCl, and incubated at 80 °C for 0.5~10 h. Finally, the TiO2-Pt/PWE was obtained. 2.3. Fabrication of the target aptamer-conjugated N-doped carbon dots/TiO2-Pt/PWE The N-doped carbon dots were firstly prepared according to the previous report29 and used as light-sensitive material to sensitize TiO2-Pt/PWE. 5 µL of mixture solution containing 3-aminopropyltriethoxysilane, water, and ethanol was added into the Pt/PWE. The resulted TiO2-Pt/PWE with rich -NH2 functional groups was washed with ethanol and deionized to remove the free 3-aminopropyltriethoxysilane. Then carbon dots solution containing glutaraldehyde (2.5%) was put in with standing for about 50 min. Then, the functionalized electrode was cleaned twice, which was abbreviated with NPWE. Next, 10 µL of target aptamer and 2.5% glutaraldehyde solution were applied to the paper working zone at 4 °C overnight. As a result, the target aptamer was connected to the carbon dots by connecting of the glutaraldehyde. After thoroughly washed with water, some possible remaining aptamer-binding sites of the NPWE were blocked with 4.0 µL of 2 mM hexanethiol for 40 min. 2.4. Photoelectrochemical assay for the detection of CEA 6 ACS Paragon Plus Environment

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20 µL of CEA solutions with different concentrations were added into the target aptamer-conjugated NPWE and incubated at 37 °C for 1 h. Then the biosensor was finally washed with ultrapure water to perform the photoelectrochemical measurements in 20 µL of phosphate buffered solution (pH 7.4) containing 0.1 M of AA using a home-built photoelectrochemical system. A 500 W Xe arc lamp armed with a monochromator was adopted as the light source. A CHI660D electrochemistry workstation was adopted to record the current-time responds. The operating steps for detecting CEA on cell surface were similar with that in clinical serum samples. The differences were that 20 µL of CEA solutions with different concentrations were replaced with 20 µL of MCF-7 cells. 3. Results and discussion 3.1. Structural and photoelectrochemical properties evaluation of the TiO2-Pt/PWE The successful modification of nanomaterials on cellulose fibers was characterized using scanning electron microscopy (SEM). The bare paper sample zone (inset of Figure 1a) showed porous and interconnected network which provided abundant sites30 to anchor Pt and TiO2 NPs. After chemical deposition of Pt, the surface of paper fiber was uniformly covered with a continuous and thin Pt layer (Pt/PWE) (Figure 1a), which not only increased the surface area of the working zone but also accelerated the photogenerated electrons transfer. Then, an in-situ seed-mediated growth method is utilized for preparing the TiO2-Pt/PWE through controlling experiment parameters. It is explained that seeds could minimize self-nucleation and benefit the formation of uniform TiO2 particles on fibers.31-34 Compared with Figure 7 ACS Paragon Plus Environment


1c-d, and Figure S1a-d, the reaction time for 2 h with the 0.01 mg mL-1 of TiO2 seeds as the initiator (Figure 1b) is more beneficial to grow uniform TiO2 NPs on the Pt/PWE. However, the obtained TiO2-Pt/PWE in Figure 1b still not possess distinct particle morphology, which is adverse to achieving high photocurrent intensity. Therefore, to further regulate the morphology of TiO2 NPs, different amounts of HCl and NaCl were added into the growth-promoting solution. It is expected that the addition of HCl and NaCl will lower the degrees of supersaturation which might retard the homogeneous nucleation rate of particles in solution.35,36

Figure 1. SEM images of Pt/PWE (a); Pt and TiO2 NPs modified PWE (TiO2-Pt/PWE) with 0.01 mg mL-1 of TiO2 seeds as the initiator for 2 h (b), 4 h (c) or 10 h (d); 600 µL of HCl and 1 g of NaCl added (e); 600 µL of HCl and 2 g of NaCl added (f), subsequently; the EDS data of the Pt/PWE (g) and TiO2-Pt/PWE (h), respectively; (i) XRD diffraction patterns of the bare PWE, Pt/PWE, and TiO2-Pt/PWE. Inset: SEM image of the bare PWE.


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As shown in Figure 1e and 1f, the structure of TiO2 NPs became more and more distinct, especially when more amount of NaCl was added into the growth-promoting solution (Figure 1f). Finally, the TiO2-Pt with editable structures modified paper was prepared successfully. Then the structure of TiO2 NPs based materials is characterized by the energy-dispersed spectrum (EDS) and X-ray diffraction (XRD) technique. As shown in Figure 1g, the Pt/PWE showed obvious peaks of Pt. After TiO2 coating, the peaks of Ti appeared (Figure 1h). Besides, the XRD pattern of Pt/PWE (Figure 1i) showed two characteristic diffraction peaks in 40 and 46.8°, which matched well with the standard pattern of Pt (JCPDS No. 65-2868).37 After TiO2 NPs coating on the Pt/PWE (Figure 1i), five diffraction peaks in the range of 25 to 56° were detected which were assigned to the (101), (004), (200), (105), and (211) diffractions of the anatase TiO2, respectively (JCPDS No. 86-1156). These results further testify the successful synthesis of the TiO2-Pt/PWE. It is worth noting that the proposed approach in this work endows the device with features of flexibility and high specific surface area which could not be achieved by rigid template or direct dripping methods. To select the most efficient nanomaterials for photoelectrochemical application, the photoelectrochemical properties of TiO2-Pt/PWE with different morphologies were investigated (Figure S2). The TiO2-Pt/PWE with sesame balls-like structure (Figure 1e) exhibited much better performances (number 4 of Figure S2a) than TiO2 with small particles (particles in Figure 1b-d, and 1f, corresponding to trial number 1, 2, 3, and 5 of Figure S2a). Hence, sesame balls-like TiO2-Pt/PWE was selected as the 9 ACS Paragon Plus Environment


promising substrate. Besides, the merits of paper-based photoelectrochemical device were further verified by comparing the photoelectrochemical intensity and stability of TiO2 NPs on different substrates. The TiO2-Pt/PWE shows the highest photoelectrochemical performances (Figure S2b) and stability (inset of Figure S2b) compared with that of TiO2-Pt/FTO and TiO2-Pt/ITO surfaces. It is explained that paper fibers with porous and 3D interconnected structures provide abundant sites for the TiO2 NPs immobilization and enhance the photoelectrochemical efficiency. 3.2. Structural characterization of the N-doped carbon dots

Figure 2. (a) UV-vis absorption and FL, XPS (b), (c) C 1s spectra and N 1s spectra (d) of the N-doped carbon dots. Inset: TEM image of the N-doped carbon dots.


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In this work, N-doped carbon dot were selected as green sensitizer to further enhance the photoelectrochemical efficiency of the TiO2-Pt/PWE with sesame balls-like structure due to its outstanding optoelectronic performances and low toxicity. Therefore, the prepared N-doped carbon dot was characterized before use. As shown in Transmission electron microscopy (TEM) image (the inset of Figure 2a), the carbon dots show good distribution with diameters in the range of 3-7 nm. The optical properties and surface composition were characterized using UV-vis absorption, fluorescent (FL) spectrum (Figure 2a) and X-ray photoelectron spectroscopy (XPS). As shown in the UV-vis absorption spectrum (Figure 2a), the N-doped carbon dots have a broad absorption below 700 nm, indicating that they are the promising light absorbers to facilitate the light absorption efficiency of TiO2 NPs in UV-vis region. The FL spectrum (Figure 2a) showed that the carbon dots owned an emission peak at 495 nm (excitation wavelength 360 nm). As shown in the XPS survey scans of carbon dots (Figure 2b), three strong peaks of C 1s, N 1s, and O 1s at 284.0, 400.0, and 530.5 eV were observed. The high-resolved C 1s XPS spectrum contains four obvious peaks at 285, 286.1, 286.7, and 288.2 eV (Figure 2c), belonging to C–C, C–N, C–O, and C=N/C=O, respectively.38 Besides, N1s XPS spectrum (Figure 2d) matchs well with previous reported works,39 proving the successful preparation of the N-doped carbon dots. 3.3. Mechanism analysis The band alignment of the materials plays a vital role in photoelectrochemical measurements. To test the feasibility of photoelectrochemical sensing, cyclic 11 ACS Paragon Plus Environment


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voltammetric (CV) method is adopted to investigate the band edge of the N-doped carbon dots. The test was conducted in an inert atmosphere using three electrodes system. The conduction band (CB) and valence band (VB) positions of the N-doped carbon dots nanomaterials were obtained using the following equations:40,41 ECB/LUMO = - (Ered + 4.71) eV

(1)

EVB/HOMO = - (Eox + 4.71) eV

(2)

where the Ered and Eox are the onset reduction and oxidation potential, respectively. As shown in Figure S3, the Ered and Eox of N-doped carbon dots was -0.5 and 0.72 V, respectively. Therefore, the CB and VB of N-doped carbon dots were calculated as -4.21 and -5.43 eV, respectively, according to eqs 1 & 2. Hence, we testify that the carbon dots enable photogenerated electrons transfer toward TiO2 NPs in theory (Figure 3a).42,43 Besides, the photoelectrochemical efficiency of the paper prepared during lay-by-lay modification process was investigated to verify the real effect of each component. The TiO2-Pt/PWE showed favorable photocurrent response (Figure S2b). When the N-doped carbon dots were further fixed on the TiO2-Pt/PWE (NPWE), a boosting photocurrent was observed (Figure S4), thus demonstrating that the N-doped carbon dot was an excellent photosensitive reagent to effectively intensify the photocurrent response of TiO2.


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Figure 3. The photoelectrochemical response mechanism (a); (b) EIS responses of (1) bare PWE, (2) Pt/PWE, (3) TiO2-Pt/PWE, (4) N-doped carbon dots, and target aptamer (5) added onto the TiO2-Pt/PWE, after CEA (6) were fixed on the electrode in 0.1 M KCl + 5.0 mM Fe(CN)64-/3-, where Cdl, Ret, Rs, and Zw, represent the double-layer capacitance, electron transfer resistance, ohmic resistance of the electrolyte, Warburg impedance, respectively; photocurrent responses (c) and calibration curve of the proposed paper based sensing platform towards target antigen with different concentrations (d). The error bars represent the standard deviation of seven repeated tests.

3.4. EIS characterization of the biosensor Electrochemical impedance spectroscopy (EIS) is helpful for understanding the chemical formations/processes on electrode surface. Hence, EIS is utilized to understand the status of electrode surface during modification and reveal the sensing mechanism.44 As shown in Figure 3b, the Pt/PWE showed a smaller electron-transfer resistance (Ret) (curve 2) than bare PWE (curve 1) due to the increment of conductivity and surface area after Pt NPs modification. After TiO2 NPs were further 13 ACS Paragon Plus Environment


modified on the surface of the Pt/PWE in situ, the Ret showed a relatively large semicircle domain (curve 3). As N-doped carbon dots (curve 4) and target aptamer (curve 5) were added onto the electrode surface in sequence, the Ret increased gradually. When CEA (curve 6) was subsequently added into the PWE, a sharp increased semicircle domain appeared, suggesting that the successful binding of CEA with high steric hindrance. For biosensing application, this steric hindrance is expected to obstruct the electron transfer process and decrease the photocurrent intensity. 3.5. Detection capability for CEA CEA is regarded as one of the most widely used serum biomarkers and can provide quantitative indications for diseases. In particular, it gives prediction of metastases and evaluation of curative effect. Therefore, the TiO2 based paper device was used for quantitative detection of CEA in clinical serum samples as well as the surface of MCF-7 cells (Scheme 1). As shown in Figure 3c, under the optimal conditions (Figure S5), the photocurrent intensity decreased with the increment of different CEA concentrations. The calibration plot shows a good linear relationship between photoelectrochemical response and the logarithmic value of CEA concentrations ranging from 2.0 pg mL-1 to 200 ng mL-1 with a correlation coefficiency of 0.997. The linear regression is described as: I (µA) = 157.61 - 42.76 lgCCEA (ng mL-1) with a detection limit of 1 pg mL-1, which is comparable with previous reported sensors (Table 1). Besides, to further prove the fascinating performances, such as high sensitivity, low cost, flexibility, and fine biocompatibility of the developed 14 ACS Paragon Plus Environment

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Table 1 Analytical performance of different CEA biosensors Linear range

Detection limit

(ng mL-1)

(ng mL-1)

0.001-90

0.00033

45

Electrochemical method

0.003-600

0.0008

46

Electrochemiluminescence

0.001-10

0.0006

47

0.0005-100

0.00016

48

0.0005-8

0.00028

49

0.002-200

0.001

This work

Detection means

References

Photoelectrochemical method

Photoelectrochemical method Photoelectrochemical method Photoelectrochemical method

paper-based device, we attempt using this device to detect the CEA, the very helpful biomarkers at MCF-7 cells surface. As shown in Figure S4, the photoelectrochemical intensity is gradually reduced with increasing the concentration of MCF-7 cells, which suggested that the CEA is high express at the surface of MCF-7 cells. Besides, to further testify the accuracy of the cyto-sensor, PC 3 cells are selected as the control experiment because of no expression of CEA at the cells surface. The photoelectrochemical responses have no evident changes compared with NPWE without cells added (Figure S4), indicating the developed biosensor owing unique accuracy and satisfying selectivity, paving a way for flexible paper-based photoelectrochemical diagnostic device using in clinical and remote regions. Meanwhile, in this work, the reliability and accuracy of the developed method for 15 ACS Paragon Plus Environment


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Figure 4. Detection of target antigen (CEA) with different methods. The serum sample was come from cancer patients (numbers 1-5) and healthy persons (numbers 6-10), respectively.

detecting CEA in clinical serum samples were also investigated using the proposed method and reference values. The reference methods were commercial ELISA kit and electrochemiluminescence single-analyte tests (ECL) (Electrochemiluminescent Analyzer, ROCHE E601, Switzerland). As shown in Figure 4 and Table S1, the developed method exhibited an excellent correlation compared with the two reference values with relative errors less than 4.35% (relative to ELISA kit) and 4.89% (relative to ECL) for cancer patients and 4.28% (relative to ELISA kit) and 5.49% (relative to ECL) for healthy people, further indicating the acceptable accuracy between the three techniques. 3.6. Evaluation of nanomaterials biotoxicity For

a

further

biological

application

point

of

view,

a

standard

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay was 16 ACS Paragon Plus Environment

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Figure 5. Microscopy images of MCF-7 cell (stained with Calcein AM) after incubation without (a, b) and with TiO2 NPs (c, d), nitrogen-doped carbon dots (e, f) in the culture dish for 12 h. (a, c, e) bright-field, (b, d, f) fluorescence.

employed to evaluate cytotoxicity of the TiO2 NPs and N-doped carbon dots nanomaterials through incubating MCF-7 cells with different materials. Figure 5 (a, c, e) were optic microscopy images of MCF-7 cells and their corresponding fluorescence microscopy images (b, d, f) after incubated without (a, b) and with TiO2 (c, d) and nitrogen-doped carbon dots (e, f) nanomaterials for 12 h, which were stained with Calcein AM (green representing living cells). From the microscopy images we can see that the MCF-7 cells exihibit well morphologies after incubated with TiO2/nitrogen-doped carbon dots nanomaterials, demonstrating the low toxicity



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of these materials for live cells, which paves a way for the developed biosensor to detect specific biomarkers on the surface of the cells. 4. Conclusion We

have

demonstrated

a

protocol

for

fabricating

TiO2

NPs

modified

photoelectrochemical flexible paper-based device with high stability, high specificity, lightweight, and low cost. Interlaced cellulose fibers provide a porous, 3D, and flexible substrate. Meanwhile, in-situ modification of N-doped carbon dots/TiO2-Pt significantly increases conductivity, widens adsorption range, and enhances photoelectrochemical ability. The TiO2 modified paper successfully realized the CEA detection based on photoelectrochemical signal decrease due to CEA binding. The good compatibility also enables the sensitive detection of CEA at MCF-7 cells surfaces. It is expected that the TiO2 modified paper platforms can be further functionalized and used for other flexible, portable, eco-friendly, and miniaturized devices, such as self-cleaning paper-based device, light-printable rewritable paper, and self-powered paper electronics.

Associated content Supporting Information Fabrication of the paper based device, SEM images of the TiO2 NPs modified paper substrates, photoelectrochemical response of the TiO2-Pt/PWE with different structures, UV-vis absorption spectra of the solution obtained from TiO2 functionalized Pt/ITO and Pt/FTO treated with ultrasonic for 20 min; results 18 ACS Paragon Plus Environment

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comparation of different methods. This material is available free of charge via the Internet at http://pubs.acs.org.

Author information Corresponding Author *E-mail: [email protected] *E-mail: [email protected] *E-mail: [email protected]. Phone: +86-531-82767161.

Notes The authors declare no competing financial interest.

Acknowledgements This work was financially supported by the program for Taishan Scholer of Shandong province (ts201712048), and the National Natural Science Foundation of China (21775055, 21475052).



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