Photoelectrochemical Bioanalysis Platform for Cells Monitoring Based

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A Photoelectrochemical Bioanalysis Platform for Cells Monitoring Based on Dual Signal Amplification Using in Situ Generation of Electron Acceptor Coupled with Heterojunction Ruyan Li, Yue Zhang, Wenwen Tu, and Zhihui Dai ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06107 • Publication Date (Web): 16 Jun 2017 Downloaded from http://pubs.acs.org on June 18, 2017

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ACS Applied Materials & Interfaces

A Photoelectrochemical Bioanalysis Platform for Cells Monitoring Based on Dual Signal Amplification Using In Situ Generation of Electron

Acceptor

Coupled

with

Heterojunction Ruyan Li, Yue Zhang, Wenwen Tu,* and Zhihui Dai* Jiangsu Collaborative Innovation Center of Biomedical Functional Materials and Jiangsu Key Laboratory of Biofunctional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing, 210023, P. R. China

ABSTRACT Using in situ generation of electron acceptor coupled with heterojunction as dual signal amplification, a simple photoelectrochemical (PEC) bioanalysis platform was designed. The synergic effect between the PEC activities of carbon nitride (C3N4) nanosheets and PbS quantum dots (QDs) achieved almost 9 fold photocurrent intensity increment, compared with the C3N4 alone. After the G-quadruplex/hemin/Pt NPs with catalase-like activity towards H2O2 was introduced, oxygen was in situ generated and acted as electron donor, improving charge separation efficiency and further enhancing photocurrent response. The dually amplified signal made enough sensitivity for monitoring H2O2 released from live cells. The photocathode was prepared by the stepwise assembly of C3N4 nanosheets and PbS QDs on indium tin oxide (ITO) electrode, which was characterized by scanning electron microscope. A

ACS Paragon Plus Environment


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signal-on protocol was achieved for H2O2 detection in vitro due to the relevance of photocurrent on the concentration of H2O2. Under the optimized condition, the fabricated PEC bioanalysis platform exhibited a linear range of 10 to 7000 µM with a detection limit of 1.05 µM at S/N of 3. Besides, the bioanalysis platform displayed good selectivity against other reductive biological species. Using HepG2 cells as a model, a dual signal amplifying PEC bioanalysis platform for monitoring cells was developed. The bioanalysis platform was successfully applied to the detection of H2O2 release from live cells, which provided a novel method for cells monitoring and would have prospect in clinical assay. KEYWORDS Photoelectrochemical, Cells monitoring, Dual signal amplification, Heterojunction, Enzymatic catalysis, Bioanalysis INTRODUCTION

Semiconductor carbon nitride (C3N4), as one of the most typical graphene-like two-dimensional layered nanomaterials, has aroused increasing interest in photocatalysis,1 electrocatalysis2 and biosensing3 due to its unique optical and electronic properties. Nevertheless, the pure C3N4-based PEC biosensing platforms have been rarely reported owing to their low photoelectric conversion efficiency, which is limited by its poor absorption in visible light and high recombination rate of photogenerated electron-hole pairs.4 To improve the PEC performance of C3N4, many strategies such as coupling with Au NPs,5 carbon dots,6 graphene7 have been proposed. However, constructing a heterojunction composite with another semiconductor materials was the most effective means. Some heterojunctions incorporating C3N4 have been developed for photocatalysts.8-11 For example, HSbO3/C3N4 heterojunction showed much higher photocatalytic activity than that of pure C3N4, which was mainly


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ascribed to the increase of electron-hole separations.8 Furthermore, a C-TiO2/C3N4 heterojunction exhibited 3.8-fold enhancement of photocatalytic activity compared to that of pure C3N4. The conduction band edge of C3N4 is more negative than that of TiO2, leading to the transfer of electrons from C3N4 to TiO2 easily before recombination.10 When the heterojunction was formed, a built-in electric field was created by the space charge layer, which separated the electrons-holes upon illumination12 and enhanced photocurrent response. In this work, in order to promote photoelectric conversion efficiency of C3N4 nanosheets, PbS quantum dots (QDs) was integrated by layer-by-layer assembly to construct C3N4/PbS heterogeneous structure for the generation of the remarkably enhanced photocurrent, which was used to amplify signal of our developed PEC bioanalysis platform. To the best of our knowledge, the coupling of p-type semiconductor (PbS QDs) with n-type semiconductor (C3N4 nanosheets) for constructing a PEC bioanalysis platform has not been reported previously. PbS QDs is an interesting p-type semiconductor with broad absorption in visible light region due to its narrow band gap energy,13 suggesting its suitability to be a photoelectrochemically active material under visible light irradiation. The coupling of PbS QDs and C3N4 nanosheets formed a junction between them and an internal electric field was built up by the space charge layer,12 which much improved charge separation efficiency and promoted the photocurrent response. As one of the most active reactive oxygen species (ROS) in live cells, hydrogen peroxide (H2O2) participates in numerous biological processes such as signaling, protein folding, cell apoptosis, and so on.14,15 The accumulation of excess of H2O2 in the live cells will induce oxidative stress, which is considered as a major source of genomic damage16 and further caused aging and some diseases including Parkinson’s disease,17 Alzheimer’s disease,18 diabetes19 and cancer.20 Due to the importance of



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H2O2 in live cells, diverse techniques including colorimetry,21,22 surface-enhanced Raman

spectroscopy,23,24

fluorescence

spectroscopy25,26

and

electrochemical

technique27,28 have been developed to probe the level of H2O2 in live cells. Fluorescent analysis is the most common technique, however, it suffered from the complicated probe synthesis.29 Electrochemical measurement has high overpotential and undesirable background signal. Other techniques are limited by low sensitivity or sophisticated equipment. PEC technique could overcome the drawbacks of the above methods due to the complete separation of the excitation source (light) and the detection signal (current) which leads to low background signal and high sensitivity.30-32 Tang et al have designed a series of PEC biosensing platforms for immunoassay,33-36 which exhibited excellent analytical performance. Nevertheless, PEC bioanalysis of H2O2 in cells were rarely reported. Yu et al proposed a PEC cyto-analysis for the detection of H2O2 released from tumor cells based on in situ hydroxyl radicals (•OH) cleaving DNA approach.37 But the preparation process of biosensor was complicated and the stability was yet to be improved. Zhang et al set up a new PEC analysis platform for H2O2 sensing and this photocathodic detection strategy presented efficient analysis performance.38 Whereas, the preparation of PEC sensing substrate suffered from high temperature (400℃). Therefore, the exploration of new PEC bioanalysis platform for the detection of H2O2 released from cells is urgently desired. In this work, the C3N4 nanosheets coupled with PbS QDs by layer-by-layer assembly to form C3N4/PbS heterogeneous structure. The C3N4/PbS composites as PEC substrate was explored for constructing a PEC bioanalysis platform for the first time. Due to the typical type I band alignment of C3N4/PbS heterojunction, the composites demonstrated the supreme PEC performance compared with that of the


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pure C3N4 nanosheets and PbS QDs. The G-quadruplex/hemin/Pt NPs with catalase-like activity was introduced to further amplify signal, which could catalyze the decomposition of H2O2 to produce oxygen in situ.39 Oxygen was an efficient electron acceptor,39,40 which captured the conduction band (CB) electrons of the irradiated PbS QDs and inhibited the recombination of electron-hole pairs, leading to the further improvement of the photocurrent response. The dually amplified signal guaranteed enough sensitivity for monitoring H2O2 released from live cells. Using HepG2 cells as a model, a dual signal amplifying PEC bioanalysis platform for monitoring cells based on in situ generation of electron acceptor (oxygen) coupled with heterojunction was designed (Scheme 1). The PEC bioanalysis platform exhibited good performance. The linear range (10-7000 µM) was suitable for the PEC bioanalysis of H2O2 in biological samples, since the local extracellular concentrations of H2O2 was elevated to as high as 10-50 µM under the pathological conditions.28 The proposed PEC bioanalysis strategy gave a novel and alternative method for cells monitoring and extended the application of C3N4/PbS composites in PEC biosensing devices.

Scheme 1. Fabrication procedure of the PEC bioanalysis platform and monitoring of



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H2O2 released from cells. EXPERIMENTAL SECTION

Synthesis of C3N4 Nanosheets and PbS QDs. The C3N4 nanosheets was synthesized according to the previously reported work with a slight modification.41 To obtain the C3N4 nanosheets, bulk C3N4 was first prepared. In detail, 5 g melamine was heated to 600 ℃ at a ramp rate of 3 ℃/min, and keep for 2 h. After cooling down to room temperature, the obtained yellow product was bulk C3N4. The C3N4 nanosheets was prepared by liquid exfoliation of alkali-treated bulk C3N4. In detail, 100 mg bulk C3N4 was mixed with 30 mL concentrated NH3·H2O, and then it was transferred into 45 mL Teflon cup, followed by heated in a sealed autoclave at 130 ℃ overnight. After cooling down to room temperature, the precipitate was collected and washed with ultrapure water several times to remove adsorbed NH3 molecules. Followed by, the collected product was redispersed in 15 mL ultrapure water and ultrasound continuously for about 8 h. And then the suspension was centrifuged at 5000 rpm for 10 min to remove unexfoliated parts. The as-obtained nanosheets was collected and dried at 60 °C. PbS QDs were prepared via a simple method according to the previous works.13,39 Briefly, 17 µL MPA was added into 25 mL Pb(NO3)2 aqueous solution (4.0 mM), and then the pH of this solution was adjusted to 11 by 1.0 M NaOH. After eliminating oxygen through blowing nitrogen for 30 min, 2.0 mL of 15 mM Na2S was injected dropwise. The solution rapidly became dark-brown, and then keeping the mixture reacting under a nitrogen atmosphere for 4 h. The as-prepared PbS QDs were stored at 4 °C before use. Preparation of Pt NPs and G-quadruplex/hemin/Pt NPs. Poly (diallyldimethylammonium chloride) (PDDA)-capped Pt NPs were prepared by reducing HPtCl6 with


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NaBH4 as described in the previous report.39 All of the glassware was immersed in aqua regia overnight and washed carefully before use. In detail, 20 mL H2PtCl6 (1 mM) solution and 5 mL PDDA (2%) solution were added into 50 mL ultrapure water under stirring. A few minutes later, 20 mL ice-cold, freshly prepared NaBH4 aqueous solution (1.0 mg/mL) was added to the mixed solution slowly, and then the solution gradually became brown. After stirring at room temperature for 12 h, the impurities were removed by dialysis. Finally, the PDDA-capped Pt NPs were obtained. As for the

preparation

of

G-quadruplex/hemin/Pt

NPs,39

10

µL

of

10

mM

tris(2-carboxyethyl)phosphine (TCEP) was added to 10 µL of 100 µΜ report DNA to activate the thiol group of the report DNA. 980 µL of Pt NPs was mixed with activated report DNA and the mixed solution was stirred at room temperature for 24 h for the adsorption of report DNA on Pt NPs through Pt-S bond. After that, 300 µL of 10 mM Tris-HCl buffered saline (pH=7.4, CNaCl = 0.1M, CKCl = 0.05 M) was added to the mixture and kept stirring for 2 h at 4 ℃. After adding 300 µL of 5 µM hemin, the solution was kept stirring for another 6 h. The resulting solution was centrifuged and redispersed in 1 mL of 10 mM Tris-HCl buffered saline (pH=7.4, CNaCl=0.1M, CKCl=0.05 M) and stored at 4 ℃ for further use. Construction of PEC Bioanalysis Platform. Before construction of the bioanalysis platform, a piece of bulked ITO (sheet resistance 20−25 Ω/square) was incised to small pieces of rectangular ITO. The ITO glasses were washed with acetone, 1 M NaOH (the solvent was the mixture of water and ethanol with the volume ratio of 1:1) and ultrapure water in order by ultrasonic treatment for 15 min and dried at 60 °C, and then the nonconductive rubberized fabric with hollow-carved circle of ~0.28 cm2 (the diameter was 0.6 cm) was pasted on the small piece of rectangular ITO to obtain the modified electrode (Figure S1A). The ITO electrode was coated with 20 µL of C3N4



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nanosheets. After being dried on the vacuum drying oven at 30 °C, 5 µL of PbS QDs solution was dripped to the electrode surface and dried at 30 °C to ensure the effective adsorption of PbS QDs on C3N4 nanosheets. Followed by, 20 µL of help DNA (1 µM) was immobilized onto the surface of the PbS QDs/C3N4/ITO electrode via classical coupling reaction between carbonyl groups on the surface of PbS QDs and amino groups on the terminal of help DNA. After washing the electrode to remove the unabsorbed help DNA, the nonspecific sites were blocked with 20 µL monoethanolamine (MEA) (1 mM) for 1h, which could reduce the nonspecific adsorption. Subsequently, 20 µL of G-quadruplex/hemin/Pt NPs was spread on the electrode surface and incubated at 37 ℃ for hybridization of help DNA with a segment bases of G-quadruplex/hemin/Pt NPs. Finally, it was washed with washing solution thoroughly to obtain the PEC bioanalysis platform. PEC Measurement Procedure. High pure nitrogen was used to remove existing oxygen by bubbling for 30 min throughout the electrolyte and the photocurrent of as-prepared G-quadruplex/hemin/Pt NPs/help DNA/PbS QDs/C3N4/ITO electrode (Figure S1B) was measured and defined as I0. And then, a certain concentration of H2O2 was injected quickly and the photocurrent of the electrode was recorded 2 minutes later and defined as I. Different electrodes were used for continuous detection of H2O2 from low concentration to high value. All of the photocurrent measurements were carried out under 405 nm irradiation at a constant potential of -0.15 V in supporting electrolyte solution without special instructions. At a negative potential (-0.15 V), the negatively charged electric field of the electrode surface generated static repulsion force to prompt the negatively-charged oligonucleotide to stand upright,42,43 which might reduce the nonspecific adsorption. All the error bars in this work originated from 3 times of parallel measurement.


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Cells Cultures and Determination of H2O2 Released from Live Cells. HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin and 1% streptomycin, and incubated at 37 ℃ under a humidified atmosphere of 5% CO2 and 95% air. The cells in the logarithmic phase of growth were washed thrice with sterile PBS (10 mM, pH=7.4) and collected through centrifugation at 1000 rpm for 5 min. The collected sediments were resuspended in N2-saturated sterile PBS to obtain the cells suspension, and then 5 µg/mL of lipopolysaccharide (LPS) was injected quickly into the cells suspension to stimulate HepG2 cells for the generation of H2O2. After centrifugation, the upper PBS containing H2O2 was injected quickly into the N2-saturated supporting electrolyte solution to record the photocurrent for the determination of H2O2 from HepG2 cells. RESULTS AND DISCUSSION

Characterization of Materials. The AFM image of the as-prepared C3N4 nanosheets was shown in Figure 1A, the thickness of the synthesized C3N4 nanosheets was about 1 nm. Considering the theoretical thickness of 0.33 nm for a monolayer C3N4,44 it was about 3 layers of C3N4 nanosheets. The size of them was estimated to be several tens of nanometers. XRD was used to analyze the crystalline phases of C3N4 nanosheets (Figure S2A). Two distinct diffraction peaks at 13.0° and 27.4° appeared in the XRD pattern of bulk C3N4 (curve a), which confirmed the graphitic-like layer structure. The peak at 13.0°and the peak at 27.4° were assigned to (100) peak and (002) peak, corresponding to in-planar ordering of tri-striazine units and interlayer stacking of conjugated aromatic systems, respectively.45 While, the (100) peak disappeared (curve b) after ultrasonic processing, proving the bulk C3N4 had been successfully exfoliated to nanosheets as we expected.45 The change of chemical structure of C3N4 before and



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after exfoliation was further confirmed by the FT-IR spectra (Figure S2B). In the spectrum of bulk C3N4 (curve a), the broad peaks at around 3500 to 3000 cm-1 corresponding to the uncondensed terminal amino groups (-NH2 or =NH groups) could be observed clearly.46 Several characteristic absorption bands between 1700 and 1000 cm-1 were the typical stretching vibration modes of C=N and C-N heterocycles.46 The sharp peak appeared at 807 cm−1 was originated from the typical breathing mode of striazine ring system of C3N4.46 Nevertheless, the FTIR spectrum of C3N4 nanosheets showed the disappearance of several stretching vibrations of connected units of C-N(-C)-C or C-NH-C located between 1000 and 1700 cm-1 (curve b), which could be ascribed to partial break of triazine rings with their small sizes.41 Therefore, the FT-IR spectra further demonstrated the successful preparation of C3N4 nanosheets. When the bulk C3N4 was exfoliated to nanosheets, the water-solubility and PEC response was tremendously improved (Figure S2C and Figure S2D). The C3N4 nanosheets aqueous solution kept unchanged after several months, which proved good stability of the C3N4 nanosheets. A small photocurrent of ~60 nA was observed at the bulk C3N4 modified ITO electrode (Figure S2D, curve a). After the bulk C3N4 was exfoliate to C3N4 nanosheets, an enhanced photocurrent of ~140 nA occurred (Figure S2D, curve b). Therefore, it is necessary to exfoliate bulk C3N4 to C3N4 nanosheets. The HRTEM image of PbS QDs was shown in Figure 1B, the PbS QDs with spherical morphology were about 4 nm (see inset). In addition, the HRTEM image of an individual PbS QD showed a lattice spacing of ~0.17 nm, which was assigned to the (222) crystal planes of cubic PbS.13 The PbS QDs was passivated by MPA and the existence of carboxyl groups was further confirmed by FI-IR spectrum. An obvious characteristic peak at ~3446 cm-1 corresponding to –OH stretching, a peak at ~1638


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cm-1 due to C=O stretching vibration, and an evident peak at ~1389 cm-1 attributing to the symmetric COO- stretching appeared on the FI-IR spectrum of PbS QDs (Figure S3). These peaks indicated the existence of carboxyl groups on the surfaces of PbS QDs. The morphological structures of C3N4 nanosheets modified ITO electrodes were observed in SEM images. Figure 1C showed that the C3N4 nanosheets on the ITO electrode surface displayed a typical slate-like morphology, which was caused by the aggregation of C3N4 nanosheets on the electrode surface. The obtained C3N4 nanosheets with large specific surface area could provide more effective anchor sites for loading PbS QDs. Accordingly, the SEM image of C3N4/PbS QDs composites modified ITO electrode (Figure 1D) demonstrated that the C3N4 nanosheets were decorated with PbS QDs of high density, which was beneficial for developing PEC platform.

Figure 1. (A) AFM image of C3N4 nanosheets. (B) HRTEM image of PbS QDs. Inset: amplified image of PbS QDs. (C) SEM image of C3N4 nanosheets modified ITO electrode. (D) SEM image of C3N4/PbS composites modified ITO electrode.



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C3N4 nanosheets, PbS QDs and C3N4/PbS composites were further characterized by UV-vis absorption and fluorescence spectroscopy. As shown in Figure 2A, the UV-vis absorption spectrum of C3N4 nanosheets showed a absorption peak at ~302 nm (curve a), which was consisted with the previous reports.47,48 Whereas the absorption intensity of C3N4 nanosheets aqueous solution in the range of visible light was relatively weaker than that of ultraviolet light. When the C3N4 nanosheets were coupled with PbS QDs, the absorption in the visible region was stronger than that of C3N4 nanosheets (curve b), which could improve the absorbance and further enhance photocurrent response in the range of visible light.

Figure 2. (A) UV-vis spectra of the C3N4 nanosheets (a) and C3N4/PbS composites (b). (B) FL spectra of C3N4 nanosheets (a), PbS QDs (b) and C3N4/PbS QDs composites (c). Lower inset: amplified FL spectrum of PbS QDs. Upper inset: photographs of the C3N4 nanosheets solution taken under visible-light (left) and UV-light (right). Under UV light irradiation, the C3N4 nanosheets aqueous solution presented as blue, while it was milk white under visible-light irradiation (The upper inset of Figure 2B). The deeper color under UV light irradiation implied the excellent fluorescent properties of C3N4 nanosheets. Meanwhile, the solution exhibited strong blue emission at about 438 nm under the excitation of 360 nm (Figure 2B, curve a). The PbS QDs showed slight fluorescence emission under the same condition (The lower


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inset of Figure 2B). While, the fluorescence intensity decreased as the addition of optimized number of PbS QDs (Figure 2B, curve c), suggesting that the recombination rate of the electron-hole pairs was decreased.49 This phenomenon indicates the assembly of PbS QDs on C3N4 nanosheets. The decline of the recombination odds of the generated electron-hole pairs in the composites was advantageous for promoting the photocurrent response.

Figure 3. Nyquist plots (A) of C3N4/ITO (a), PbS QDs/C3N4/ITO (b), help DNA/PbS QDs/C3N4/ITO (c), G-quadruplex/hemin/Pt NPs/help DNA/PbS QDs/C3N4/ITO (d) electrodes. Photocurrent responses (B) of C3N4/ITO (a), PbS QDs/C3N4/ITO (b), help DNA/PbS QDs/C3N4/ITO (c), G-quadruplex/hemin/Pt NPs/help DNA/PbS QDs /C3N4/ITO

(d)

electrodes

in

N2-saturated

Tris-HCl

buffered

saline

and

G-quadruplex/hemin/Pt NPs /help DNA/PbS QDs/C3N4/ITO electrode in N2-saturated Tris-HCl buffered saline containing 100 µM H2O2 (e). Characterization of the PEC Bioanalysis Platform. The stepwise assembly process of the PEC bioanalysis platform was analyzed by the electrochemical impedance spectroscopy (EIS) (Figure 3A). When the ITO electrode was modified with C3N4 nanosheets, an obvious semicircle appeared in the Nyquist plot of EIS (curve a). The electron transfer resistance (Ret) reflected by the semicircle diameter is related to the electron transfer kinetics of the (Fe(CN)6)3-/4- redox probe. After the PbS



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QDs further coated onto the C3N4 nanosheets modified ITO electrode, the Ret value increased (curve b) owing to that the negatively charged MPA-capped PbS QDs prevented the access of redox probe to the electrode surface. Subsequently, help DNA was modified on the electrode surface through the EDC coupling reaction, leading to the increase of Ret (curve c). This was caused by the poor conductivity and negative charge of help DNA. After MEA blocking and the hybridization of help DNA with a segment of DNA in the Pt NPs/G-quadruplex/hemin for the capture of Pt NPs/G-quadruplex/hemin on the electrode surface, the Ret increased gradually (curve d), suggesting that the steric effect and the increased negative charge density would further restrain the transfer of negatively charged probe to the surface of electrode. The changes of Ret value of the different modified electrodes verified the successful fabrication of the PEC bioanalysis platform. PEC Response of the PEC Bioanalysis Platform. The PEC response of the stepwise assembly process was recorded in N2-saturated Tris-HCl buffered saline (Figure 3B). A small photocurrent of ~8 nA was observed at the C3N4 nanosheets modified ITO electrode (curve a). After the PbS QDs was further modified on the C3N4/ITO electrode, an enhanced photocurrent of ~165 nA appeared (curve b). This might be caused by the formation of heterojunction between C3N4 nanosheets and PbS QDs, which accelerated the photoinduced electron transfer and improved the photovoltaic conversion efficiency.40 When the help DNA was attached on the surface of the electrode through amide bond formed by the –COOH groups on the surfaces of PbS QDs and the –NH2 at the terminal of help DNA, the photocurrent response declined to ~120 nA due to that the generated steric hindrance impeded the electron transfer between the modified electrode and electrolyte (curve c). After the immobilization of MEA and Pt NPs/G-quadruplex/hemin, the photocurrent response


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decreased to ~30 nA owing to that the increased steric hindrance effect further blocked photogenerated electron transfer (curve d). Upon addition of H2O2 with the concentration of 100 µM, the photocurrent response increased sharply to ~680 nA, which was attributed to that the catalytic decomposition of H2O2 produced an effective electron acceptor39 to improve the photocurrent response (curve e). The enhanced photocurrent response (~680 nA) might guarantee enough sensitivity for PEC determination of H2O2 released from live cells. Optimization of Condition for PEC Bioanalysis Platform. The photocurrent responses of the electrodes prepared with different concentration of C3N4 nanosheets were studied (Figure S4A). The photocurrent response promoted as the concentration of C3N4 nanosheets solution increased from 0.125 to 0.75 mg/mL, suggesting that more C3N4 nanosheets could provide more electron-hole pairs to take part in the PEC process. While the concentration further increased to 1.25 mg/mL, the photocurrent response leveled off. Thus, 0.75 mg/mL was selected as the optimal concentration. The dosage of PbS QDs takes an important role for signal amplification (Figure S4B). The photocurrent intensity rose with increasing the volume of PbS ODs from 0 to 5 µL, and then it reduced gradually after 5 µL. It might be due to that the excessive deposition of PbS QDs on the C3N4/ITO electrode could inhibit electrons and holes transport. Therefore, we chose 5 µL as the deposition volume for further experiment. The irradiation wavelength was also an important parameter to influence the PEC response (Figure S4C). The photocurrent response decreased as the excitation wavelength increased from 365 to 470 nm. The photocurrent at 405 nm was 78.5% of that at 385 nm, while the photocurrent at 430 nm was only 46.3% of that at 385 nm. The photocurrent response at 430 nm was too weak to provide sufficient sensitivity for PEC detection. The photocurrent response under ultraviolet light illumination



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were higher than that of visible light illumination, the short wavelength means high energy which might damage the biomolecules. Considering the two aspects, 405 nm was more suitable for PEC determination. The PEC performance was also dependent on the bias potential. The photocurrent response improved remarkably as the applied potential fell from 0.2 to -0.15 V, and then the photocurrent response increased slowly after -0.15 V (Figure S4D). Since the low applied potential was beneficial for eliminating the interference of other species released from live cells. Taking into account the two aspects, -0.15 V was fixed as the bias potential for PEC bioanalysis. The concentration of help DNA affected the analytical performance of the proposed bioanalysis platform. The photocurrent change value (∆I=I-I0, the concentration of H2O2 was 100 µM) rose with increasing the concentration of help DNA from 0.25 to 1 µM. While the concentration further promote to 1.5 µM, it leveled off (Figure S4E). Thus, 1µM was selected as the optimal concentration of help DNA. Both Pt NPs and G-quadruplex/hemin exhibited the catalytic activity toward H2O2 for the in situ generation of oxygen39 which could enhance photocurrent response (Figure S4F, columns b and c). After combining Pt NPs with G-quadruplex/hemin to form Pt NPs/G-quadruplex/hemin, the photocurrent promoted compared with that of Pt NPs or G-quadruplex/hemin alone, indicating the cooperative effect of their catalytic activity. Although H2O2 also could induce the obvious photocurrent (column a) as an electron acceptor, all of catalase modified electrodes showed larger photocurrent than that of the electrode without modification of any catalase, suggesting that oxygen acted as a more effective electron acceptor than H2O2 for the generation of photocurrent. PEC Behavior. To evaluate the PEC performance of C3N4 nanosheets, PbS QDs


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and C3N4/PbS QDs, the photocurrent responses of various electrodes were measured in 0.1 M air-saturated Tris-HCl buffered saline at a bias potential of -0.15V under visible light (λ= 405nm) irradiation (Figure 4A). A small photocurrent of ~55 nA was observed at the PbS QDs modified electrode (curve a). When the ITO electrode was modified by C3N4 nanosheets alone, it exhibited a photocurrent of ~140 nA (curve b). Whereas a dramatic enhancement of photocurrent (~1450 nA) was observed (curve c) after the C3N4 nanosheets modified electrode was functionalized with PbS QDs. It achieved ~25 times (curves a and c) and ~9 times (curves b and c) enhancement of photocurrent intensity respectively, confirming the synergic effect between the PEC activities of C3N4 and PbS QDs.

Figure 4. (A) Photocurrent responses of PbS QDs/ITO (a), C3N4/ITO (b) and PbS QDs/C3N4/ITO (c) electrodes in 0.1 M air-saturated Tris-HCl buffered saline. (B) Photocurrent responses of the PbS QDs/C3N4/ITO electrode in N2-saturated (a), air-saturated (b) and O2-saturated (c) Tris-HCl buffered saline.

Scheme 2. Schematic illustration of the possible charge transfer for PEC signal



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amplification. The possible mechanism of signal amplification was displayed in Scheme 2. First of all, the conduction band (CB) potential and valence band (VB) potential of C3N4 nanosheets were estimated to -1.22 and +1.57 eV (Figures S5A and S5B), which was similar to the previous literature.50 Meanwhile, the CB and VB potentials of PbS QDs were estimated to be -0.83 and 0.34 eV (Figures S5C and S5D), corresponding with the previous works.13,39 The CB and VB potential of C3N4 nanosheets were -1.02 and +1.77 eV vs normal hydrogen electrode (NHE) and the CB and VB potential of PbS QDs was -0.63 and +0.54 eV vs NHE, respectively. Under light irradiation, both C3N4 nanosheets and PbS QDs could harvest high-energy photons, resulting in the transfer of the photogenerated electrons from VB to CB for the generation of electron-hole pairs. When the p-type PbS and n-type C3N4 nanosheets were in contact to form heterojunction, the space charge layer created a built-in electric field, which separated the electrons-holes.12 Subsequently, the photogenerated electrons could diffuse from C3N4 nanosheets to PbS, since the CB potential of C3N4 nanosheets was more negative than that of PbS. While the difference of VB potential would favor the holes transfer from the VB of C3N4 nanosheets to PbS. Therefore, the intrinsic straddling (Type I) band structure of C3N4/PbS composites was beneficial for the migration of photo-induced electrons and holes.39 The migration could be enhanced by the inner electric field established at the heterojunction interfaces, leading to the effective separation of electron-hole pairs and improvement of the photoelectric conversion efficiency.12 Furthermore, compared with the reduction potential of oxygen (0.815 eV vs NHE),39 the CB edge potential of PbS QDs was more negative, which was beneficial to charge transfer. Thus, oxygen as an excellent electron acceptor captured ·-

the CB electrons of the irradiated PbS QDs.39 O2 received electron and turned to O2 .


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As shown in Figure 4B, the PbS QDs/C3N4 electrodes were sensitive to dissolved oxygen in air-saturated Tris-HCl buffered saline (curve b), and of special note was the photocurrent enhancement of 23 times ( curves a and c), owing to the existence of enough oxygen.

Figure 5. (A) Photocurrent responses of the PEC biosensor toward H2O2 at different concentrations: (a) 0, (b) 10, (c) 40, (d) 70, (e) 100, (f) 400, (g) 700, (h) 1000, (i) 4000, (j) 7000, (k) 10000 µM. (B) The photocurrent changes versus different concentrations of H2O2 ranging from 10 to 10000 µM. The inset is the linear relationship between photocurrent change and the logarithm value of H2O2 concentration. Analytical performance. The G-quadruplex/hemin/Pt NPs as a member of catalase mimetics exhibited catalase-like activity for catalyzing the decomposition of H2O2 to oxygen which was an efficient electron acceptor for enhancing photocurrent response of PbS/C3N4 modified electrode. Thus, a photocurrent-based biosensor for H2O2 detection was developed under the optimized condition. The photocurrent response was found to promote with increasing the concentration of H2O2 (Figure 5A). The increment of photocurrent (∆I=I-I0) displayed a good linear relationship versus the logarithm of concentration for H2O2 increasing from 10 to 7000 µM. The linear regression equation was ∆I (nA) = 583.48lgC-542.16 with a correlation coefficient of 0.9996 (Figure 5B). The detection limit was estimated to be 1.05 µM at S/N of 3. This linear range was wider or the detection limit was lower than those of some other



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methods with the similar materials (Table S1). The linear range was suitable for the PEC bioanalysis of H2O2 in biological samples, since the local extracellular concentrations of H2O2 was elevated to as high as 10-50 µM under the pathological conditions.28

Figure 6. (A) The interference effects of various species. (50-fold mass ratio of urea, cysteine, 10-fold mass ratio of glucose, citric acid, 100 µM of interfering agents mentioned above together with 100 µM H2O2). (B) Photocurrent responses of G-quadruplex/hemin/Pt NPs/help DNA/PbS QDs/C3N4/ITO electrode in N2-saturated Tris-HCl buffered saline before and after the addition of LPS (a), the supernatant obtained from cell suspension without LPS (b) and with LPS (c). Moreover, the relative standard deviations (RSD) of 6.54% was estimated from the parallel measurements of five freshly prepared G-quadruplex/hemin/Pt NPs/help DNA/PbS QDs/C3N4/ITO electrode in 100 µM H2O2, suggesting the good fabrication reproducibility of the PEC bioanalysis platform. Additionally, the selectivity of the PEC bioanalysis platform for H2O2 at 100 µM was evaluated (Figure 6A). The photocurrent response of other species, such as urea, cysteine (cys), glucose (glc), and citric acid (CA) were lower than 8.0% of the photocurrent response of H2O2, although the concentration of H2O2 was lower than the other species. Meanwhile, the photocurrent response referred to the mixture of H2O2 and the same concentration


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level of other species declined slightly, compared with that of 100 µM H2O2, indicating that the existence of other species could not obviously influence the determination of H2O2. The results confirmed good selectivity of the PEC bioanalysis platform. PEC Determination of H2O2 Released from Live Cells. The prepared PEC bioanalysis platform was used to monitor H2O2 released from live cells. As shown in Figure 6B, the photocurrent response of the G-quadruplex/hemin/Pt NPs/help DNA/PbS QDs/C3N4/ ITO electrode exhibited no obvious change after the addition 5 µg/mL LPS (curve a), suggesting that the LPS could not interfere the photocurrent response. The HepG2 cells were dispersed in the N2-saturated PBS to generate cells suspensions. 5 µg/mL of LPS was injected into the cells suspensions for stimulating HepG2 cells to generate H2O2. And then, the supernatant containing H2O2 released from live cells was collected by centrifugalization. The photocurrent response increased after the addition of the above supernatant with slowly stirred (curve c), while no obvious change of photocurrent response was obtained at the electrode after the addition of supernatant without treatment of LPS (curve b). The increased photocurrent was caused by the existence of electron acceptor (O2) generated from the catalytic decomposition of H2O2. The photocurrent density change for these cells (~107) was estimated to be 45 nA, corresponding to pM level of H2O2 being released from each cell (under the stimulation of LPS). Nevertheless, the similar low concentration of H2O2 in vitro could not induce the obvious photocurrent change of PbS QDs/C3N4/ITO (Figure S6). Therefore, the PEC bioanalysis platform was successfully applied to monitor H2O2 released from live cells owing to that the dually amplified signal made enough sensitivity. CONCLUSIONS



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A simple PEC bioanalysis platform for monitoring the H2O2 released from cells was designed based on dual signal amplification by use of C3N4/PbS heterojunction and in situ generation of electron acceptor. The formation of heterojunction between C3N4 nanosheets and PbS QDs accelerated the photoinduced electron transfer and improved the photovoltaic conversion efficiency, leading to the enhancement of photocurrent response. After immobilization of G-quadruplex/hemin/Pt NPs, a mimic enzyme of H2O2, the catalytic decomposition of H2O2 produced an effective electron acceptor (O2) in situ to further improve the photocurrent response. The dually amplified signal might guarantee enough sensitivity for monitoring H2O2 released from live cells. The changes of Ret value of the different modified electrodes verified the successful fabrication of the PEC bioanalysis platform. The PEC bioanalysis platform with good sensitivity and excellent anti-interference was used to detect H2O2 in vitro. Furthermore, the bioanalysis platform was successfully applied to monitor H2O2 release from live cells, which would provide a novel and alternative method for cells monitoring and demonstrate the C3N4/PbS composites have promising prospect in PEC biosensing devices. Nevertheless, the biggest challenge will be in situ and on-line monitoring of live cells in vivo by using this proposed bioanalysis platform. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Materials and Reagents. XRD patterns, FT-IR spectra, optimization, possible charge transfer schematic diagram, catalase effect and selectivity (PDF) AUTHOR INFORMATION


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Corresponding Authors *Tel./Fax: +86-25-85891051. E-mail: [email protected], [email protected]. ORCID Zhihui Dai: 0000-0001-7049-7217 Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China for the project (21625502, 21475062) and Natural Science Research of Jiangsu Higher Education Institutions of China (15KJB150016). We appreciate the financial support from the Priority Academic Program Development of Jiangsu Higher Education Institutions. REFERENCES (1) Niu, P.; Zhang, L. L.; Liu, G.; Cheng, H. M. Graphene-Like Carbon Nitride Nanosheets for Improved Photocatalytic Activities. Adv. Funct. Mater. 2012, 22, 4763−4770. (2) Zheng, Y.; Jiao, Y.; Zhu, Y.; Cai, Q.; Vasileff, A.; Li, L. H.; Han, Y.; Chen, Y.; Qiao, S. Z. Molecule-Level g-C3N4 Coordinated Transition Metals as a New Class of Electrocatalysts for Oxygen Electrode Reactions. J. Am. Chem. Soc. 2017, 139, 3336−3339. (3) Feng, Q. M.; Shen, Y. Z.; Li, M. X.; Zhang, Z. L.; Zhao, W.; Xu, J. J.; Chen, H. Y. Dual-Wavelength Electrochemiluminescence Ratiometry Based on Resonance Energy Transfer between Au Nanoparticles Functionalized g-C3N4 Nanosheet and Ru(bpy)32+ for microRNA Detection. Anal. Chem. 2016, 88, 937−944. (4) Yin, H. S.; Zhou, Y. L.; Li, B. C.; Li, X.; Yang, Z. Q.; Ai, S. Y.; Zhang, X. S.



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Photoelectrochemical Bioanalysis Platform for Cells Monitoring Based

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