Peptide-Based Photoelectrochemical Cytosensor Using a Hollow

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Peptide-Based Photoelectrochemical Cytosensor Using a HollowTiO2/EG/ZnIn2S4 Co-Sensitized Structure for Ultrasensitive Detection of Early Apoptotic Cells and Drug Evaluation Rong Wu, Gao-Chao Fan, Li-Ping Jiang, and Jun-Jie Zhu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 12 Jan 2018 Downloaded from http://pubs.acs.org on January 12, 2018

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Peptide-Based Photoelectrochemical Cytosensor Using a Hollow-TiO2/EG/ZnIn2S4 Co-Sensitized Structure for Ultrasensitive Detection of Early Apoptotic Cells and Drug Evaluation Rong Wu§, Gao-Chao Fan§, Li-Ping Jiang*, and Jun-Jie Zhu*

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, People’s Republic of China

KEYWORDS: photoelectrochemistry, cytosensing, co-sensitized, apoptotic cell, drug evaluation

ABSTRACT: The ability to rapidly detect apoptotic cells and accurately evaluate therapeutic effects is significant in cancer research. To address this target, a biocompatible, ultrasensitive photoelectrochemical (PEC) cytosensing platform was developed based on electrochemically reduced graphene (EG)/ZnIn2S4 co-sensitized TiO2 coupled with specific recognition between apoptotic cells and phosphatidylserine (PS)-binding peptide (PSBP). In this strategy, HL-60 cells were selected as a model, and C005, nilotinib and imatinib were selected as apoptosis inducers to show cytosensing performance. In particular, a TiO2 photoactive substrate was designed as hollow spheres to enhance the PEC performance. Graphene was electrodeposited on the hollow TiO2-modified electrode to accelerate electron transfer and increase conductivity, followed by in situ growth of ZnIn2S4 nanocrystals as photosensitiz1

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ers via successive ionic layer adsorption and reaction (SILAR) method, forming a TiO2/EG/ZnIn2S4 cosensitized structure that was used as a PEC matrix to immobilize PSBP for the recognition of early apoptotic cells. The detection of apoptotic cells was based on steric hindrance originating from apoptotic cell capture to induce an obvious decrease in the photocurrent signal. The ultrahigh sensitivity of the cytosensor resulted from enhanced PEC performance, bioactivity, and high binding affinity between PSBP and the apoptotic cells. Compared with other assays, incorporate toxic elements were avoided, such as Cd, Ru, and Te, which ensured normal cell growth and are appropriate for cell analysis. The designed PEC cytosensor showed a low detection limit of apoptotic cells (as low as three cells), a wide linear range from 1×103-5×107 cells/mL and an accurate evaluation of therapeutic effects. It also exhibited good specificity, reproducibility, and stability.

INTRODUCTION Apoptosis, also known as programmed cell death, plays an important role in the etiology of leukemia.1 Recently, selective apoptosis induction in leukemia tissue has become an effective chemotherapeutic strategy.2 Exploring the early stages of apoptosis pushes the envelope for solutions in early instances of real-time monitoring of death kinetics. Given the importance of exploring early apoptosis, detecting apoptotic cells and evaluating drug effects, various techniques have been developed for analyzing apoptosis, such as electron microscopy,3 the terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL),4 flow cytometry,5 microfluidic devices,6 electrochemistry (EC),7,8 and electrochemiluminescence (ECL).9,10 Although these methods have been used in some systems, several involve inadequacies, including slow speed, high cost, high technical expertise, and sophisticated instrumentation. Thus, it is essential to develop a more sensitive, selective and economical method for apoptotic cell detection and drug evaluation.

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Photoelectrochemical (PEC) detection is a recently developed technique for rapid and sensitive biological assays, wherein white light is used to excite photoactive materials to produce photocurrent as the detection signal.11,12 Because of the complete separation of the excitation signal and detection signal, the PEC method has lower background signal and noteworthy advantages in sensitivity compared to the other conventional methods mentioned above. Owing to its simplicity, low cost, and simple sample preparation, PEC cytosensors have also attracted attention and have been utilized for apoptotic cell detection. Notably, photoactive materials and recognition molecules play important roles in the performance of PEC cytosensors. For a PEC sensing system, high photocurrent intensity and less electronhole recombination are essential for ultrasensitive detection. Currently, most of the photoactive materials used in PEC sensors incorporate toxic elements, such as Cd, Se, and Te, which may induce cell apoptosis and are inappropriate for cytoanalysis.13-15 However, low-toxicity photoactive materials usually exhibit poor photocurrent performance.16,17 To achieve high photocurrent performance for lowtoxicity photoactive materials, a co-sensitized structure with cascade band-edge levels that is composed of basic photoactive materials and two or more sensitizers has been developed.18 Coupling different band gap semiconductors can not only promote the utilization of white light but also facilitate charge separation, due to promotion of the photocurrent conversion efficiency.15,19 Additionally, among the various cytosensors for apoptotic cell detection developed thus far, most focus on the recognition of antibodies at the cell surface, which has shortcomings in terms of expense, speed and immunogenicity.7-9 In this work, the phosphatidylserine (PS)-binding peptide (PSBP) was utilized to recognize and capture early apoptotic cells with high affinity to PS.20,21 Thanks to its specific recognition, speed and low cost, a peptide-based PEC cytosensor with a co-sensitized structure would certainly be desirable. However, this superior strategy has not appeared in any PEC cytosensor until now. Here, we presented a novel PEC assay for apoptotic cell detection and drug evaluation based on a peptide-modified hollow-TiO2/electrochemically reduced graphene (EG)/ZnIn2S4 co-sensitized PEC matrix, 3

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as illustrated in scheme 1. Nontoxic and stable TiO2 nanoparticles were chosen as the photoactive substrate. To obtain high photocurrent intensity, a hollow-sphere morphology was designed. Low-toxicity ZnIn2S4 nanocrystals were deposited as a photosensitizer. The EG film acted as an electronic relay between ZnIn2S4 and TiO2 to accelerate electron transfer. Initially, hollow TiO2 nanoparticles were coated onto a bare indium−tin oxide (ITO) electrode, and the EG film was formed after electrodeposition. Then, ZnIn2S4 nanocrystals were deposited on the hollow-TiO2/EG electrode using successive adsorption and reaction of S2-, In3+ and Zn2+ ions, forming a hollow-TiO2/EG/ZnIn2S4 co-sensitized structure to enhance photocurrent intensity. Next, PSBP was immobilized on the electrode using a coupling reaction between amino and carbonyl groups. After bovine serum albumin (BSA) blocked the unbound sites of the PSBP-modified electrode, the PEC cytosensing electrode was successfully fabricated. Human leukemic HL-60 cells, as a model, were exposed to an apoptosis inducer, which resulted in cell apoptosis with translocation of PS (Scheme S1). For apoptotic cell detection, apoptotic cell suspensions with different concentrations were cultured on the cytosensing electrode via specific recognition between PSBP and PS. Steric hindrance led to a remarkably decreased photocurrent signal. Hollow-TiO2/EG/ZnIn2S4 hybrid structure was originally designed for excellent photocurrent performance and PSBP was utilized for specific recognition, which were benefit to rapid early apoptotic cell detection and accurate therapeutic effect evaluation. The designed PEC cytosensor exhibited ultrahigh sensitivity, reproducibility, specificity, biocompatibility and stability. Scheme 1. Construction Process of the Peptide-Based PEC Cytosensor.

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EXPERIMENT SECTION Preparation of Hollow TiO2 Spheres. The SiO2 spheres were prepared through Stöber’s method.22 The TiO2 shell on the SiO2 surface was obtained via the versatile kinetics-controlled coating method.23 The SiO2@TiO2 spheres were dried at 100 °C for 12 h and then calcined at 550 °C in air atmosphere for 1 h to improve crystallinity and remove the organic species. Finally, the hollow TiO2 spheres were obtained directly etching with HF. The detailed description was presented in the Supporting Information. Preparation of the Hollow-TiO2/EG/ZIS Electrode. Prior to use, ITO slices were cleaned by ultrasonic treatment for 15 min in acetone, 1 M NaOH of water/ethanol mixture (1:1, v/v), and water in succession and dried at 80 °C. Next, a 1.25 mg/mL hollow-TiO2 homogeneous suspension was prepared in 5 mL of DI water, and 20 µL of the suspension was dropped onto a piece of an ITO slice. After air drying, the film was annealed at 300 °C for 1 h in air and then naturally cooled to room temperature. An EG film was obtained through electrodeposition using a graphene oxide (GO) aqueous solution as a precursor. Electrochemical reduction was performed on the deposition solutions with magnetic stirring and N2 bubbling on a CHI 660D electrochemical workstation (CH Instruments, Shanghai) using a threeelectrode system,24 and the detailed description was presented in the Supporting Information. The deposition of ZnIn2S4 nanocrystals on the ITO/hollow-TiO2/EG electrode was obtained using successive ionic layer adsorption and reaction (SILAR) method with some modifications.25 The ITO/hollow-TiO2/EG electrode was first dipped into a 0.1 M Zn(NO3)2 methanol solution, a 0.1 M Na2S methanol/water mixture (1:1, v/v), a 0.1 M InCl3 methanol solution, and a 0.1 M Na2S methanol/water mixture (1:1, v/v) for 2 min, successively, followed by a rinse with methanol. This SILAR cycle was repeated four times. The ITO/hollow-TiO2/EG/ ZnIn2S4 electrode was annealed at 180 °C for 1 h in air after air drying to yield the ITO/hollow-TiO2/EG/ZnIn2S4 electrode.

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Fabrication of the PEC Cytosensor. Briefly, 20 µL of MPA aqueous solution was dropped onto the ITO/hollow-TiO2/EG/ ZnIn2S4 electrode and was allowed to incubate at 4 °C for 2 h to introduce the carboxyl group. After being rinsed with D-PBS to remove the excessive MPA, the electrode was covered with 20 µL of aqueous solution containing 20 mM EDC and 10 mM NHS for 1 h at room temperature, and then washed with D-PBS. Next, 20 µL of 1 mg/mL PSBP (FNFRLKAGAKIRFGRGC) was dropped onto the electrode and incubated at 4 °C for 12 h, which allowed the amidogen group of the peptide to react with previously activated carboxyl group on the electrode surface. Subsequently, the electrode was rinsed with D-PBS and incubated with 20 µL of 1 mg/mL BSA at room temperature for 1 h to block nonspecific binding sites. After being rinsed with D-PBS, the electrode was employed as a PEC cytosensor. Cell Culture and Induction of Apoptosis. HL-60 cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (FCS, Sigma), penicillin (100 µg/mL), and streptomycin (100 µg/mL) in an incubator (5% CO2, 37 ℃). At the logarithmic growth phase, the HL-60 cells were collected and redispersed in fresh culture medium with the concentration of 5×105 cells/mL. Then, 4 mL of HL-60 cell suspension was treated with apoptosis inducers (1 µL/mL C005, 50µM nilotinib, and 50µM imatinib) for 0, 4, 8, 12, 16, and 24 h, respectively. After that, the apoptotic cells were washed trice with D-PBS. For drug evaluation, apoptotic cells with the concentration of 1×107 cells/mL were prepared. Also, for apoptotic cell detection, the apoptotic cell suspension with various concentrations treated with C005 for 16 h was obtained from this stock. Microscopy Imaging of Cells. Confocal laser scanning microscopy (CLSM) performances were carried out using a Leica TCS SP5 microscope. The cells on the cytosensor were incubated with Hoechst 33342 or DAPI in medium for 15 min, and then washed with D-PBS for microscopy imaging. Hoechst 33342 was excited at 405 nm with an argon ion laser, and the emission was collected from 435 to 480 nm. 6

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DAPI was excited at 405 nm with an argon ion laser, and the emission was collected at 461 nm. All images were digitized and analyzed by Leica Application Suite Advanced Fluorescence (LAS-AF) software. Flow Cytometric Analysis. HL-60 cells were seeded on 6-well plates and incubated in medium for 4 h at 37 °C. After the treatment with C005 for 4, 16, and 24 h, HL-60 cells were washed with sterile cold D-PBS and resuspended in 500 µL of binding buffer. After the treatment with nilotinib for 4, 8,16, and 24 h, HL-60 cells were washed with sterile cold D-PBS and resuspended in 500 µL of binding buffer. After the treatment with imatinib for 4, 8, 20, and 24 h, HL-60 cells were washed with sterile cold DPBS and resuspended in 500 µL of binding buffer. Nilotinib and imatinib were dispersed in Dimethyl sulfoxide (DMSO). Then, the apoptotic cells were stained with annexin V-FITC/PI according to the manufacturer’s instructions, and subjected to perform flow cytometric assay using Cytomics FC500 Flow Cytometry (Beckman Coulter Ltd.). The data were analyzed using FCS Express V3. PEC Detection of Apoptotic HL-60 Cells and Evaluation of Anticancer Drugs. After fabrication of the PEC cytosensor, 20 µL of apoptotic cell suspension at a certain concentration was dropped onto the electrode and incubated at 37 °C for 1 h for apoptotic cell capture. The electrode was rinsed with incubation buffer to remove the non-captured cells. Thus, apoptotic cells could be detected indirectly by observing variations in the photocurrent signal. The PEC cytosensing platform could be utilized to evaluate anticancer drugs indirectly. PEC Measurements. PEC detection was carried out at room temperature in the substrate solution, which was 0.1 M AA in phosphate-buffered saline (PBS, 0.1 M, pH 7.4). The substrate solution was deoxygenated by bubbling nitrogen for 15 min before PEC measurement, which acted as a sacrificial electron donor. The excitation light source was white light, which was generated by a xenon lamp with a spectral range from 200 to 2500 nm and switched on and off every 10 s. The external voltage was 0.0 V. 7

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RESULTS AND DISCUSSION Characterization of the Hollow TiO2 Spheres. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to characterize the synthesis of hollow TiO2 spheres, as shown in Figure S1. Colloidal SiO2 nanoparticles were prepared using the well-known Stöber method and a uniform diameter of ~350 nm was observed (Figure S1B, S2, in the Supporting Information).21 Then, SiO2@TiO2 core−shell particles with a shell thickness of ~100 nm were obtained via a versatile kinetics-controlled coating method, and the TiO2 shells were amorphous (Figure S1C).23 Next, as shown in Figure S1D and S1F, the obtained nanoparticles were annealed at 500 °C for 1 h in air for crystaline transfer and particle growth, which was an essential step for etching. After etching with 1% HF solution, the SiO2 nanoparticles were removed, and the hollow TiO2 nanoparticles were synthesized successfully. TEM and SEM images of the hollow TiO2 nanoparticles (Figure 1A, S1E and S1G) indicated that the nanoparticles were composed of hundreds of smaller nanoparticles (~20 nm) and had a mesoporous structure. X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) patterns were utilized to analyze valence states and crystal structures, respectively, as shown in Figure 1 and S3. XPS of O 1s for hollow TiO2 (Figure 1B) showed three main peaks at binding energies of 527.5, 528.2, and 529.6 eV, which were assigned to the lattice oxygen O−Ti3+, O−Ti4+ and hydroxyl oxygen (O−H) on the surface. Correspondingly, two sets of peaks were observed in the Ti 2p XPS spectra (Figure 1C), which were assigned to Ti3+ (Ti 2p1/2 at 462.4 eV, Ti 2p3/2 at 456.8 eV) and Ti4+ (Ti 2p1/2 at 461.4 eV, Ti 2p3/2 at 456.1 eV), respectively.26 It was suggested that oxygen vacancies (Ti3+ sites) were distributed on hollow TiO2 nanoparticles. XRD patterns (Figure 1D) revealed the crystal structures of the nanoparticles.27 Hollow TiO2 nanoparticles contained two crystalline phases: an anatase phase and rutile phase. It was confirmed that hollow TiO2 nanoparticles have a mixed-crystalline structure.

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Figure 1. Characterization of hollow TiO2 nanoparticles. (A) TEM image of synthesized hollow TiO2 nanoparticles. (B) O1s XPS core-level spectrum for hollow TiO2 nanoparticles. (C) Ti2p XPS core-level spectrum for hollow TiO2 nanoparticles. (D) XRD pattern of mixed anatase-rutile systems. PEC Properties of the Hollow-TiO2/EG/ZIS Co-Sensitized Structure. As shown in Figure S4, the hollow mixed-crystalline TiO2 nanoparticles showed excellent photocurrent performance. However, a wide energy band gap limited their utilization of white light. Coupling of EG with hollow TiO2 was a good choice to accelerate electron transfer and enhance photovoltaic performance.28,29 ZnIn2S4 semiconductor with a narrower energy band gap showed a wider absorption range extending to the visible light region. Thus, the hollow-TiO2/EG/ZnIn2S4 hybrid structure could further extend the absorption range and promote photocurrent intensity.30 The photocurrent intensity of the ITO/hollow-TiO2/EG/ZnIn2S4 electrode was affected by the thickness of the hollow TiO2 film, the ratio of rutile to anatase, the amount of EG and the deposition of ZnIn2S4 nanocrystals. Thus, the corresponding optimal preparation conditions should be investigated. The thick9

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ness of the hollow TiO2 film could be changed by addition of a hollow TiO2 suspension at different concentrations while the area of the ITO electrode (0.25 cm2) and the applied volume of the hollow TiO2 suspension (20 µL) were fixed. Figure 2A shows that the ITO/hollow-TiO2 electrode fabricated with a 1.25 mg/mL suspension annealed at 450 °C for 1 h in air exhibited the highest photocurrent intensity. Increasing the concentration of hollow TiO2 suspension offers additional hollow TiO2 to absorb light and enhance photocurrent intensity. However, excessive hollow TiO2 with additional surface recombination centers evidently increased the diffusion resistance for electron motion, which led to gradually decreased photocurrent intensity.31,32 The ratio could be optimized by changing the calcination temperature with a fixed calcination time.27 Figure S5 shows XRD patterns of hollow TiO2 and P25, which exhibited changes in anatase-rutile mixed-crystalline structure. When the temperature increased, the rutile structure content increased. The crystalline phase influenced the absorption band of the semiconductor and the energy gap, which affected photocurrent intensity.33 Figure 2B shows that the ITO/hollow-TiO2 electrode annealed at 300 °C for 1 h in air exhibited the highest photocurrent intensity. For electrodeposition of the EG film, the sweep segments of the cyclic voltammetric reduction determined the amount of EG at a rate of 0.1 V s-1. As Figure 2C shows, 32 segments resulted in the appropriate amount of EG for the ITO/hollow-TiO2/EG electrode to exhibit the highest photocurrent intensity. Excessive EG prevented hollow-TiO2 from absorbing ultraviolet light, which decreased the photocurrent intensity. The deposition of ZnIn2S4 nanocrystals could be adjusted by SILAR cycles. Figure 2D shows the photocurrent intensity of ITO/hollow-TiO2/EG/ZnIn2S4 electrodes prepared with different SILAR cycles of ZnIn2S4 nanocrystals. As the fourth SILAR cycles was reached, ZnIn2S4 nanocrystals gradually accumulated and the ITO/hollow-TiO2/EG/ZnIn2S4 electrode exhibited the highest photocurrent intensity due to greater white light absorption and the generation of additional electron hole. Excessive deposition of ZnIn2S4 nanocrystals decreased the photocurrent, for reasons similar to those stated above for hollow TiO2.

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Figure 2. Relationship between photocurrent and (A) concentration of hollow-TiO2 suspension, (B) calcination temperature of hollow-TiO2, (C) sweep segment of EG, and (D) coating number of ZnIn2S4. Error bars show standard deviation of three parallel measurements.

PEC Characterization of the Cytosensor. Photocurrent response can be used to monitor the fabrication of cytosensors, as shown in Figure 3A. The ITO/hollow-TiO2 electrode generated an appropriate photocurrent intensity (curve a, I = 42.57 µA). After EG film electrodeposition, photocurrent intensity slightly increased (curve b, I = 62.21 µA) and was higher than the ITO/hollow-TiO2 electrode due to extension of the absorption range and acceleration of electron transfer. After ZnIn2S4 nanocrystals deposition, the photocurrent intensity increased (curve c, I = 213.6 µA) and was much higher than the ITO/hollow-TiO2/EG electron due to the formation of a hollow-TiO2/EG/ZnIn2S4 co-sensitized hybrid structure. The photocurrent decreased after PSBP immobilization (curve d, I = 177.4 µA) and after BSA blocking (curve e, I = 154.3 µA), which could be explained by the weak charge-transfer abilities of PSBP and BSA. After apoptotic cells were incubated with the cytosensing electrode, a decrease in photocurrent was observed (curve f, I = 81.57 µA) due to the increase in steric hindrance, which prevented 11

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the diffusion of AA to the ITO/hollow-TiO2/EG/ ZnIn2S4 electrode surface and reaction with the photogenerated holes. These results demonstrated the successful fabrication of the PEC cytosensor.

Figure 3. (A) Photocurrent response and (B) EIS of (a) ITO/hollow TiO2 electrode, (b) after EG film electrodeposition, (c) after ZnIn2S4 nanocrystals deposition, (d) after PSBP immobilization, (e) after BSA blocking, and (f) after incubation of cytosensing electrode with apoptotic cells. Inset of part B: the partial enlarged drawing and the electrical equivalent circuit applied to fit the impedance spectra; Rs, Zw, Ret, and Cdl represent ohmic resistance of the electrolyte, Warburg impedance, electron-transfer resistance, and the double layer capacitance, respectively.

EIS Characterization of the Cytosensor. Electrochemical impedance spectroscopy (EIS) can also monitor the assembly of the cytosensor, which is an effective method for exploring the interface properties of electrode. Figure 3B displays Nyquist plots of the impedance spectroscopy of the electrode formed step-by-step. The inset shows the partial enlarged drawing and the corresponding equivalent circuit. Generally, the impedance spectrum included a semicircular portion (the electron-transfer-limited process) and linear portion (the diffusion-limited process). For the ITO/hollow-TiO2 electrode, the electron-transfer resistance (Ret) was relatively small (curve a), indicating low electron transfer resistance. The redox probe [Fe (CN)6]4-/3- could exchange the charge rapidly. After electrodeposition of the EG film, Ret decreased slightly due to increased conductivity (curve b). After ZnIn2S4 nanocrystal deposition, the low conductivity of ZnIn2S4 nanocrystals resulted in increased Ret (curve c). When the 12

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ITO/hollow-TiO2/EG/ZnIn2S4 electrode was immobilized with PSBP, Ret increased due to the low conductivity of PSBP (curve d). With the blocking of BSA, Ret increased continuously (curve e), which also weakened the transfer of the redox probe [Fe (CN)6]4-/3-. After the apoptotic HL-60 cells were incubated on the cytosensing electrode, Ret increased remarkably (curve f) due to dramatic blocking of electron transfer. Accordingly, the stepwise characterization of EIS also suggested successful fabrication of the cytosensor. SEM Characterization of the Cytosensor. The surface morphology of the electrode throughout the assembly process was characterized by SEM. Figure 4 displays SEM images of ITO, ITO/hollow-TiO2, ITO/hollow-TiO2/EG and the ITO/hollow-TiO2/EG/ZnIn2S4 electrode surface. Figure 4A shows that the bare ITO electrode was covered with a mass of indium tin oxide nanoclusters. As shown in Figure 4B, abundant hollow-TiO2 nanoparticles with a diameter of ~600 nm were coated on the ITO electrode surface, and these nanoparticles formed a mesoporous film. After EG electrodeposition, as shown in Figure 4C, a very thin film was obtained and hollow-TiO2 nanoparticles were clearly visible under the film, indicating successful fabrication of the ITO/hollow-TiO2/EG electrode and showing that the interlayer EG could not seriously affect light absorption. With four SILAR cycles of ZnIn2S4 nanocrystal deposition, the electrode surface became rough and abundant nanoparticles with smaller diameter were produced, as shown in Figure 4D, demonstrating the successful decoration of ZnIn2S4 nanocrystals. Figure S6 shows the EDS spectrum of the hollow-TiO2/EG/ZnIn2S4 electrode. The electrode indicator elements Ti, C, S, In and Zn were observed in the spectrum; other elements (Si, Sn, etc.) were from the ITO slice. Hence, SEM characterization indicated successful development of the cytosensor.

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Figure 4. SEM images of (A) ITO, (B) ITO/hollow TiO2 electrode, (C) after EG film electrodeposition, (D) after ZnIn2S4 nanocrystal deposition. Characterization of the Cell-Electrode Interface. To explore the biocompatibility of the electrode, normal HL-60 cells were seeded onto the TiO2/EG/ZnIn2S4 electrode surface for an additional 12 h and 24 h of incubation. Next, normal HL-60 cells and apoptotic cells on the electrode were incubated with Hoechst 33342, which stains apoptotic cells to exhibit blue fluorescence. As displayed in Figure S7 in the Supporting Information, no fluorescence signal was detected and the electrode exhibited good biocompatibility for normal HL-60 cells over 12 h and 24 h (Figure S7A, 7B). In contrast, apoptotic cells were stained and exhibited blue fluorescence (Figure S7C). For hollow-TiO2/EG/ZnIn2S4/PSBP PEC cytosensors, the ability to capture apoptotic cells was directly demonstrated through SEM and CLSM. SEM images of the fixed cells showed that the apoptotic HL-60 cells preserved unabridged morphologies and tight junctions contacting the electrode interface (Figure S8A). Similarly, interface characterization between electrode and cells was also tested by CLSM, as shown in Figure S8B, which confirmed that apoptotic cells were successfully captured by the electrode. 14

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Scheme 2. Photogenerated Electron−Hole Transfer Mechanism of the Cytosensing System for the Detection of Apoptotic Cells.

PEC Mechanism of Cytosensing. TiO2 semiconductors are extensively used in solar cells due to their photoelectric activity with high stability, environmental safety, and low cost. However, rapid electronhole recombination, poor electrical conductivity and low light absorption efficiency limit their practical applications. In the present study, hollow, mixed-crystalline TiO2 spheres and a co-sensitized structure were designed as a PEC cytosensing platform to obtain a stable, obvious photocurrent signal. The obtained TiO2 nanoparticles appeared to be more attractive than solid or monocrystalline TiO2 due to their mesoporous structure, multiple interparticle scattering, oxygen vacancies, and broad light absorption. 26,34,35

The easily prepared hollow TiO2 nanoparticles were suitable for the photoelectrode substrate, and

were comparable to commercial Degussa TiO2 P25. The general consensus places the bandgaps of rutile and anatase TiO2 at 3.03 and 3.20 eV, respectively. As shown in Scheme 2, when the PEC electrode was excited by white light, the electrons in the valence band (VB) of the semiconductor materials absorbed the energy of photon and moved to the conduction band (CB), leading to the formation the electron-hole pairs. For the mixed-crystalline TiO2 sphere, the electron affinity of anatase was higher than rutile material, and photogenerated electrons flow from rutile to anatase.33 For a co-sensitized hybrid structure, ultrafast transfer of electrons and effective prevention of charge recombination originated from the cas15

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cade band-edge levels. Moreover, the hollow TiO2 substrate and ZnIn2S4 nanocrystals possessed different energy band gaps, which increased the utilization of light and facilitated charge separation. The EG interlayer, according to low electron-hole recombination and fast electron transfer of graphene, accelerated electron transfer and increased the conductivity of the hollow-TiO2/EG/ZnIn2S4 electrode, resulted in enhanced photovoltaic performances. The electron was finally transferred to external circuit, forming photocurrent as detection signal. Ascorbic acid (AA) in the electrolyte acted as electron donor and reacted with hole for preventing the electron-hole pair recombination of semiconductor materials followed by the formation of oxidative product (AA+) and ground state of semiconductor materials. For cytosensing, PSBP was immobilized on the PEC matrix through covalent binding, which was essential to sensitively capture and detect early apoptotic cells. In contrast, with the traditional protein macromolecule annexin V, PSBP has low molecular weight and preserved high binding affinity to the PS of apoptotic cells without Ca2+, which improved its specificity and anti-interference ability.20 Once apoptotic cells were captured on the electrode, photocurrent intensity obviously decreased due to increased steric hindrance, which weakened the light absorption efficiency and prevented diffusion of AA onto the ITO/hollow-TiO2/EG/ ZnIn2S4 electrode surface and reaction with the photogenerated holes. Two main factors contributed to the high sensitivity of the proposed cytoassay. Before incubation of apoptotic cells, the cytosensor showed a stable and obvious photocurrent response via the hollow-TiO2/EG/ ZnIn2S4 co-sensitized structure. For apoptotic cells bound to the electrode surface, the presence of cells evidently increased the steric hindrance of electrode, which led to a significantly decreased photocurrent response.

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Figure 5. Evaluation of anticancer drugs via time-course for apoptotic cells induced by C005 at various time points. (A) Photocurrent intensity of the cytosensor capturing apoptotic cells, induced by C005 for time points 0, 4, 8, 12, 16, 20, and 24 h. (B) Relationship between the photocurrent and C005 incubation time. (C) Flow cytograms representing apoptosis assays based on annexin V-FITC and PI staining of HL-60 cells: i) control, ii) 4 h, iii) 16 h, and iv) 24 h.

Evaluation of Anticancer Drugs via the PEC Cytosensing Platform. The detection of apoptotic cells was based on photocurrent change produced by steric hindrance. The photocurrent response of the cytosensor was directly associated with the number of apoptotic cells. Based on the proposed PEC cytosensor, apoptotic cells induced by C005, nilotinib and imatinib were detected and the therapeutic effects of anticancer drugs for HL-60 cells were indirectly evaluated. As shown in Figure 5A and 5B, with treatment of C005 for 4 h, the photocurrent signal obviously decreases, implying that some HL-60 cells were exposed to PS and were in the early stage of apoptosis. As the treatment time proceeded to 16 h, a maximal decrease in photocurrent signal was observed, revealing the remarkable apoptotic state of HL-60 cells. At 24 h, the photocurrent signal increased slightly, possibly due to a decline in therapeutic effects. Further extension of reaction time would not generate more apoptotic cells, known as drug resistance. The cell proliferation would restart after a decline in therapeutic effects, leading to an increased photocurrent.8,36 Taken together, the PEC platform showed that the apoptotic inducer C005 had obvious therapeutic effects and 16 h was the optimal treatment time. To confirm these experiment results, HL-60 17

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cells (1×107cells/mL) treated with C005 for various time points (4 h, 16 h, 24 h) were further analyzed by flow cytometry analysis. As shown in Figure 5C, for 4 h, the percentages of early- [annexin V (+)/PI (-)] and late- [annexin V (+)/PI (+)] stage apoptotic cells were only approximately 8.1% and 0.6%. The early stage accounted for 86.4% and the late stage accounted for 10.8% at 16 h. At 24 h, the early apoptotic cells had decreased and the late apoptotic cells increased, consistent with PEC cytosensor detection. Different from C005, nilotinib and imatinib showed inequable therapeutic effects for HL-60 cells. As shown in Figure S9A and S9B in Supporting Information, with treatment of nilotinib for 4 h, the photocurrent signal obviously decreases. As the treatment time proceeded to 24 h, a continue decrease in photocurrent signal was observed, revealing the remarkable apoptotic state of HL-60 cells. However, for imatinib, as the treatment time proceeded to 20 h, a maximal decrease in photocurrent signal was observed, revealing the optimal treatment time (Figure S9C and S9D in Supporting Information). Similarly, flow cytometry analysis was performed to confirm these therapeutic effects of nilotinib and imatinib evaluated via PEC platform. As shown in Figure S10C-F in Supporting Information, with treatment of nilotinib, as the incubation time goes on, normal cells continually decreased. While, with treatment of imatinib, obvious minimum signal of normal cells was observed at 20 h (Figure S10G-J in Supporting Information), which was consistent with PEC cytosensor detection. Therefore, the proposed PEC cytosensing platform can rapidly detect apoptotic cells and accurately evaluate therapeutic effects of drug with great sensitivity and high stability. Detection of Apoptotic Cells Induced by Apoptosis Inducers. Figure 6A shows the photocurrent response of the cytosensor after capturing apoptotic cells with different concentrations. With increased concentrations, more apoptotic cells bound to the electrode surface, resulting in a gradual decrease in photocurrent signal. As shown in Figure 6B, the photocurrent response decreased in a linear manner with

logarithmically increasing apoptotic cell concentration from 1×103 cells/mL to 5×107 cells/mL.

The regression equation was ∆I = 38.58−14.7 log c (cells/mL) and its correlation coefficient was 0.997. 18

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Due to the apoptosis rate of 86.4%, the detection limit (S/N=3) for apoptotic cell concentration was estimated at 158 cells/mL. Given the test volume (20 µL), the PEC cytosensor achieved the detection limit of only 3 apoptotic HL-60 cells. Compared to other methods, as shown in Table S1, a lower detection limit and a wider linear range were obtained from the proposed PEC cytosensor, demonstrating its superiority.

Figure 6. Detection of apoptotic cells. (A) Photocurrent response of (a-k) 0, 1×103, 5×103, 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107 and 5×107 cells/mL and (B) calibration curve. Error bars show the standard deviations of three parallel measurements.

Selectivity, Stability, and Reproducibility of the Cytosensor. To evaluate the selectivity of the proposed PEC cytosensor, cell suspensions without cells, with normal cells and apoptotic cells were investigated via photocurrent intensity under the same conditions. As shown in Figure 7A, unlike the cytosensor with normal cells or without cells, the cytosensor with apoptotic cells showed obviously lower photocurrent intensity, which indicated that the designed PEC cytosensor had good selectivity. Within 400 s, the photocurrent response of the hollow-TiO2/EG/ZnIn2S4 electrode showed no obvious change (Figure 7B). After the cytosensor was stored in D-PBS at 4 °C in a refrigerator for over 2 weeks, no obvious change in photocurrent signal was obtained when detecting 5×107 cells/mL, which also suggested the stability of the cytosensor. The reproducibility of the designed cytosensor was evaluated by analyzing five independently fabricated sensing electrodes. The photocurrent response offered a relative stand-

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ard deviation (RSD) of 3.6% toward 5×107 cells/mL, which indicated the acceptable reproducibility of this assay.

Figure 7. (A) Selectivity and (B) stability of the PEC biosensor. The concentration of cells was 5×107 cells/mL. Error bars show the standard deviations of five replicate determinations.

CONCLUSION In summary, a highly biocompatible, sensitive, and selective cytosensor was developed based on a PSBP-modified PEC matrix that rapidly detected early apoptotic cells and accurately evaluated therapeutic effects. Based on the hollow, mixed-crystalline structure with oxygen vacancies, the prepared TiO2 spheres showed excellent photocurrent performance. The EG interlayer accelerated electron transfer and increased conductivity. The deposition of low-toxicity ZnIn2S4 nanocrystals further extended the absorption range and prevented electron-hole recombination. The designed TiO2/EG/ZnIn2S4 cosensitized structure promoted the photocurrent intensity and stability. PSBP preserved a high binding affinity to PS, which resulted in sensitive and selective capture of apoptotic cells. Steric hindrance prevented white light absorption, electron transfer, and the diffusion of AA onto the electrode, which obviously reduced the photocurrent signal. Compared with other methods, the designed low-cost, convenient PEC cytosensor exhibited a low detection limit, a broad detection range, and an accurate drug evaluation. Thus, the proposed PEC cytosensor provided a promising strategy for apoptosis research and a valid therapeutic schedule for other diseases and drugs. 20 ACS Paragon Plus Environment

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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experiment details and additional TEM, SEM, XRD, CLSM, and PEC data. (pdf) AUTHOR INFORMATION

Corresponding Author * E-mail: [email protected] (L.P.J.) * E-mail: [email protected] (J.J.Z.) Author Contributions §W.R. and G.C.F. contributed equally to this work. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We gratefully appreciate funding from the National Natural Science Foundation of China (21475057, 21603099, and 21775070), and the China Postdoctoral Science Foundation (2017T100352). REFERENCES (1) Tilborg, G. A. F. V.; Mulder, W. J. M.; Chin, P. T. K.; Storm, G.; Reutelingsperger, C. P.; Nicolay, K.; Strijkers, G. J. Annexin A5-Conjugated Quantum Dots with a Paramagnetic Lipidic Coating for the Multimodal Detection of Apoptotic Cells. J. Bioconjugate Chem. 2006, 17, 865–868.

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