Ultrasensitive Cytosensor Based on Self-Enhanced

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Ultrasensitive Cytosensor Based on Self-enhanced Electrochemiluminescent Ruthenium-Silica Composite Nanoparticles for Efficient Drug Screening with Cell Apoptosis Monitoring Wenbin Liang, Ying Zhuo, Chengyi Xiong, Yingning Zheng, Yaqin Chai, and Ruo Yuan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03822 • Publication Date (Web): 20 Nov 2015 Downloaded from http://pubs.acs.org on November 26, 2015

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Analytical Chemistry

Ultrasensitive Cytosensor Based on Self-enhanced Electrochemiluminescent Ruthenium-Silica Composite Nanoparticles for Efficient Drug Screening with Cell Apoptosis Monitoring Wenbin Liang,1,2 Ying Zhuo,1 Chengyi Xiong,1 Yingning Zheng,1 Yaqin Chai*,1 and Ruo Yuan*,1 1

Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University),

Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China 2

Department of Clinical Biochemistry, Laboratory Sciences, Third Military Medical University,

30 Gaotanyan Street, Shapingba District, Chongqing 400038, PR China

ABSTRACT: The self-enhanced electrochemiluminescence (ECL) with high sensitivity could be an effective method for anticancer drug screening with cell apoptosis monitoring. Here we reported an ultrasensitive ECL cytosensor for cell apoptosis monitoring by using self-enhanced electrochemiluminescent ruthenium-silica composite nanoparticles (Ru-N-SiNPs) labeled annexin V as signal probes. The Ru-N-SiNPs were firstly synthesized through simple hydrolysis of a novel precursor containing luminescent and intra-coreactant groups in one molecule, which presented higher emission efficiency and enhanced ECL intensity due to the shorter electron-transfer path and less energy loss. Moreover, the as-proposed ECL cytosensor was successfully used to investigate efficiency of paclitaxel toward MDA-MB-231 breast cancer cell in the range from 1 nM to 200 nM with a detection limit of 0.3 nM and a correlation coefficient of 0.9917. The im-

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proved accuracy and excellent dynamic range revealed the potential applications in biomolecules diagnostics and cells detections, especially in living and complex systems.

KEYWORDS: electrochemiluminescence, cytosensor, self-enhancement, ruthenium-silica composite nanoparticles ■ INTRODUCTION Cancer has become a major cause of morbidity and mortality throughout the world and the situation would worsen in the next decade due to the population growth, ageing and spread of risk factors, such as tobacco, obesity and infection.1 Current cancer therapy typically included taking anticancer drug to regulate cells apoptosis, using radiation to kill cancer cells or applying surgery to remove the tumor, in which taking anticancer drug was the simple, efficient and economical approach for cancer therapy with minimum side effects, especially for the early-stage cancer. Moreover, anticancer drug was also requisite to prevent recurrence of cancer effectively when radiation or surgery was employed.2 Currently, it was one of the efficient and reliable approaches to discover new anticancer drug and evaluate the effectiveness of potential anticancer drugs by cells apoptosis monitoring after the living cells treated with anticancer drugs. Recent investigations indicated that phosphatidylserine, an important phospholipid membrane component, would be translocated from the cytoplasmic leaflet to the extracellular leaflet during apoptosis, which provided a good biological marker for apoptotic cells. Taking the advantage of specific binding between annexin V and phosphatidylserine, annexin V as a phospholopid-binding protein with high affinity has been labeled with different signal molecule to act as detection probes for cells apoptosis monitoring. Commonly, fluorescence-labeled annexin V has been widely employed to detect the translocated phosphatidylserine.3-5 However, improvements were still required, as the-


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se fluorescent strategies remained laborious, time-consuming and expensive.6-9 Recently, electrochemiluminescence (ECL) bioassay has attracted considerable attentions due to their high sensitivity, controllability of ECL reaction, low background and cost-effectiveness, which has been widely employed in clinical diagnosis and pharmaceutical analysis.10-12 In these regards, it may be a desirable approach to detect the translocated phosphatidylserine by ECL cytosensor for cell apoptosis monitoring. However, no ECL cytosensor has been reported until now for anticancer drug screening by cell apoptosis monitoring to the best of our knowledge. Commonly, ECL cytosensor have been employed for sensitive detection of cancer cells with sandwiched format.13-15 Signal probes containing luminophore and specific binding protein played the most important role in these sandwiched ECL cytosensor. For example, Wu and coworkers have reported an ECL cytosensor to detect cancer cell by using tris (2, 2'-bipyridyl-4,4'dicarboxylato) ruthenium (II) (Ru(dcbpy)32+) labeled specific binding protein as signal probes13. With rapid development of nanotechnology, a variety of nanomaterials with larger surface area have been widely used as nanocarriers to improve the amount of luminophore labeled on signal probes for improving the sensitivity of ECL cytosensor. Specially, silica nanoparticles were demonstrated to be a good nanocarrier due to their good biocompatibility and simple synthesis, which was widely employed for immobilizing large amount of luminophore by doping the luminophore in the nanoparticles.16, 17 Li and co-workers have reported an ECL cytosensor to detect cancer cells with improved sensitivity by using Ru(dcbpy)32+ doped silica nanoparticles (Ru@SiNPs) to label specific binding protein as signal probes14. However, the ECL intensity of pure Ru@SiNPs was still not strong enough. The coreactant, which could significantly increase ECL intensity through inter-molecular interaction, was essential in these Ru(dcbpy)32+-based ECL systems by adding these coreactant in the testing solution.18-22 Although these Ru@SiNPs-


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based ECL systems with coreactant showed improved sensitivity, these strategies were inefficient to detect cells apoptosis for anticancer drug screening due to possible leakage of Ru(dcbpy)32+ and potential biotoxicity of Ru(dcbpy)32+ and coreactant. Recently, we have proposed the selfenhanced ECL ruthenium complex containing intra-coreactant and luminophore in one molecule for the fabrications of biosensors with simple operation, higher efficiency and enhanced ECL intensity without adding of any more inter-coreactant in the testing solution.23, 24 Due to the shorter electron-transfer path and less energy loss, the intra-coreactant with intra-molecular reactions have allowed improved efficiency and enhanced ECL emission compared with the common inter-coreactant with inter-molecular reactions. These self-enhanced approaches provided a new perspective to construct an efficient and suitable ECL system for anticancer drug screening by cell apoptosis monitoring. Herein, a novel self-enhanced precursor containing two regions of intra-coreactant and Ru(dcbpy)32+ in one molecule was designed to prepare self-enhanced electrochemiluminescent ruthenium-silica composite nanoparticles (Ru-N-SiNPs) for the first time. And then, the Ru-NSiNPs were labeled on annexin V (annexin V/Ru-N-SiNPs) to act as signal probes for anticancer drug screening based on ECL cytosensor (Scheme 1). Briefly, the sensing interface of ECL cytosensor was fabricated by modifying Con A onto gold nanoparticles (AuNPs) coated electrode surface to capture cells based on the specific interaction between Con A and glycoproteins on cell membrane. Then the fabricated cytosensor was carried out for the anticancer drug screening by cells apoptosis monitoring with MDA-MB-231 breast cancer cell and paclitaxel as a research model, where paclitaxel was a medicine used to treat breast cancer normally by increasing cancer cells apoptosis. The responses of ECL cytosensor would be increased with increasing concentration of paclitaxel, because of the higher ratio of apoptotic cells captured on the cytosensor sur-


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face and more annexin V/Ru-N-SiNPs signal probes combined on the apoptotic cells. Furthermore, the applicability of the as-proposed ECL cytosensor was investigated and compared with that from laser confocal microscope.

Scheme 1. Schematic diagrams of Ru-N-SiNPs labeled annexin V and ECL cytosensor.

■ EXPERIMENTAL METHODS Reagents and Materials. Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were obtained from Gibco Laboratories Life Technologies Inc. (Grand Island, NY, USA). Fluorescein isothiocyanate isomer I (FITC), 4',6-diamidino-2phenylindole (DAPI), Con A, paclitaxel, 3-[2-(2-aminoethylamino) ethylamino] propyltrimethoxysilane (NNN-TES), 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (sulfo-NHS), tetraethylorthosiliconte (TEOS), Triton X-100, cyclohexane, 1-hexanol, NH3·H2O, isopropyl β-D-1-thiogalactopyranoside (IPTG), dimethyl-


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sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), albumin from bovine serum (BSA) and chloroauric acid (HAuCl4) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Tris (2, 2'-bipyridyl-4,4'-dicarboxylato) ruthenium (II) (Ru(dcbpy)32+) was purchased from Suna Technology Inc. (Suzhou, China). Phycoerythrin conjugated annexin V (annexin V-PE) and FITC conjugated Con A (FITC-Con A) were obtained from Invitrogen Life Technologies Inc. (Carlsbad, CA, USA). Immunol staining blocking buffer, phosphate buffered saline (PBS), phosphate buffered saline with tween-20 (PBST) and immnol fluresce staining secondary antibody dilution buffer were received from Beyotime Institute of Biotechnology Inc. (Jiangshu, China). Ultrapure water was used throughout the study. Apparatus. Electrochemistry and electrochemiluminescence (ECL) measurements were performed with a model MPI-E II multifunctional electrochemical and chemiluminescent analytical system (Xi’an Remax Electronic Science and Technology Co., Xi’an, China). The voltage of the photomultiplier tube (PMT) was biased at 800 V, and scan rate was 100 mV/s in the process of ECL detection. A series of optical filters were placed between the electrode and PMT in this ECL system for ECL spectrum, which was calculated with the maximum ECL intensity during the cyclic potential sweep with these optical filter range from 275 nm to 825 nm. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements were performed with a CHI 660e electrochemical workstation (CH Instruments Inc., Shanghai, China). In the electrochemistry and ECL emission measuremetns, a conventional three-electrode system was employed with a Ag/AgCl (saturated KCl) as the reference electrode, a platinum wire as auxiliary electrode, and a bare or modified glass carbon electrode (GCE, diameter of 3 mm) as working electrode, respectively. Transmission electron microscopy (TEM) was carried out using H600 transmission electron microscope (Hitachi Co., Tokyo, Japan) at an acceleration voltage of 80


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kV. The nanomaterials were deposited onto the conventional copper micro-grids covered with carbon foil and dried in vacuum desiccators for TEM characterizations. Scanning electron microscope (SEM) was carried out by S4800 scanning electron microscope (Hitachi Co., Tokyo, Japan) at an acceleration voltage of 15 kV. The photophysical characterizations were carried out with a UV-2550 spectrophotometer and a RF-5301PC fluorescence spectrophotometer (Shimadzu Co., Tokyo, Japan) with standard quartz cuvettes (open-top type, 10 mm optical path length and 3.5 mL volume). X-ray photoelectron spectroscopy (XPS) measurements were invited by VG Scientific ESCALAB 250 spectrometer (Thermoelectricity Instruments Inc., USA) with Al Kα X-ray (1486.6 eV) as the light source. Absorbance reading in MTT assay was performed using multiskan spectrum microplate reader (Thermo Electron Corp., NC, USA). Laser confocal microscopy measurements were performed with PE labelled annexin V, FITC labelled Con A and DEPI by laser confocal microscope (Leica Onc., Heidelbery, Germany). Cell culture. The MDA-MB-231 cell line was obtained from the cell bank of the Committee on Type Culture Collection of Chinese Academy of Science (Shanghai, China). According to the manufacturer’s instructions, the MDA-MB-231 cells were cultured in DMEM containing 10% FBS, 100 U/mL penicillin and 100 U/mL streptomycin, and maintained in a humidified atmosphere with 5% CO2 at 37 °C. The MDA-MB-231 cells were treated with different concentrations of palitaxel ranged from 0 nM to 200 nM for 24 h. The palitaxel concentrations and experimental details were described in the text and figure legends. Synthesis of Ru-N-SiNPs. Ru(dcbpy)32+ was firstly conjugated with NNN-TES to form the precursor of Ru-N-SiNPs by EDC/sulfo-NHS reaction as showed in Figure S3. Briefly, 1 mL Ru(dcbpy)32+ solution (2 mM) was reacted with EDC (0.2 M) and sulfo-NHS (0.05 M) for 15 min at room temperature to active the carboxyl groups on Ru(dcbpy)32+. And then, 400 µL acti-


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vated Ru(dcbpy)32+ was reacted with 150 µL NNN-TES at room temperature for 2 h to conjugate Ru(dcbpy)32+ with NNN-TES for forming the precursor, NNN-TES-Ru(dcbpy)32+. At the same time, the W/O micro-emulsion was prepared with the protocol by mixing 1.77 mL of TX-100, 7.5 mL of cyclohexane and 1.8 mL of 1-hexanol with continuous magnetic stirring for 30 min. And then, 500 µL of NNN-TES-Ru(dcbpy)32+ and 30 µL TEOS were added into the mixture with rapid stirring for additional 20 min to wrap NNN-TES-Ru(dcbpy)32+ and TEOS into the microemulsion cell. Followed with an equilibration, 100 µL of NH3·H2O (25~28%) was added to assist the hydrolysis of NNN-TES-Ru(dcbpy)32+ and TEOS, and the micro-emulsion system was remained with continuous stirring for additional 48 h. The Ru@SiNPs and silica nanoparticles (SiNPs) were synthesized by W/O micro-emulsion method also as showed in supporting information. Preparation of the Ru-N-SiNPs labeled annexin V. Ru-N-SiNPs were labeled onto annexin V by EDC/NHS reaction. Briefly, 20 µL prepared Ru-N-SiNPs were added into 1 mL annexin V (5 mg/mL) with continues stirring for 15 min. And then, 100 µL EDC (0.2 M) and 100 µL sulfoNHS (0.05 M) was added into the mixture and the reaction was remained at room temperature for 2 h for conjugation. The obtained Ru-N-SiNPs labeled annexin V (annexin V/Ru-N-SiNPs) were washed and centrifuged four times to remove the unlabeled annexin V and related chemicals. The prepared annexin V/Ru-N-SiNPs were dispersed in PBST with 2 mg/mL BSA for 2 h to block the non-specific sites. Ru@SiNPs labeled annexin V (annexin V/Ru@SiNPs) and Ru(dcbpy)32+ labeled annexin V (annexin V/Ru(dcbpy)32+) used in the control systems were prepared with the similar protocols. Fabrication of the cytosensor’s sensing interface. Before the modification, the bare GCE was polished with 0.3 and 0.05 µm aluminum slurry, and ultrasonicated with ethanol and water, re-


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spectively. After cleaned with abundant water, the GCE was modified with AuNPs based on electrodeposition in HAuCl4 solution (10 mg/mL) at - 0.2 V for 30 s. Then, the AuNPs modified GCE was dipped into the solution containing 1 mg/mL Con A for 2 h to absorb Con A onto the AuNPs layer. Finally, Con A modified electrode was immersed in PBST with 2 mg/mL BSA to block the non-specific sites to obtain BSA/Con A/AuNPs modified electrode. Measurement procedure. The ECL measurement was based on a sandwich-type assay. Firstly, about 1×106 MDA-MB-231 cells were reduced with 0, 1, 2, 5, 10, 20, 50, 100 and 200 nM paclitaxel respectively for 24 h. After three minutes digestion with 0.5 % trypsin, the cells were dispersed in culture medium and the prepared cytosensor was incubated with the reduced cells at 37 °C for 40 min to capture the cells onto the cytosensor surface. The resultant cytosensor was incubated in the PBST with annexin V/Ru-N-SiNPs at 37 °C for 30 min. Every resultant electrode was washed with PBST after each step. Finally, the ECL intensity of the cytosensor was investigated with a model MPI-E II multifunctional electrochemical and chemiluminescent analytical system in PBS at room temperature. The laser confocal microscopy assays were performed with annexin V-PE, Con A-FITC and DAPI following the manufacturer’s instructions. Briefly, the MDA-MB-231 cells were firstly arrayed on the adhesive slides. After 24 h incubation for the cells adhesion, paclitaxel was added into the culture medium to concentrations ranged from 0 nM to 200 nM, and the cells were cultured for additional 24 h. And then, all these slides were fixed with 10% formaldehydum polymerisatum, blocked with immunol staining blocking buffer and washed with TBST buffer for at least 5 times. Immediately, the slides were incubated with annexin V-PE and Con A-FITC (diluted 1:1000 in immnol fluresce staining secondary antibody dilution buffer), and dyed with


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DAPI. After carefully washing with TBST buffer for at least 5 times, the slides were viewed by laser confocal microscopy. ■ RESULTS AND DISCUSSION Morphology, Structure and Photophysical Characterizations of Ru-N-SiNPs. The morphology of Ru-N-SiNPs was characterized by SEM, which presented a spherical structure with an average diameter of 120 ± 11 nm (Figure 1A), suggesting the well nucleation and dispersion of Ru-N-SiNPs. Small salient points on the surface would increase the surface area, which was conducive to further modification. Furthermore, XPS was employed for the elemental analysis of the proposed Ru-N-SiNPs with SiNPs and Ru@SiNPs as controls. As expected, all these characteristic peaks for Si2p (99.9 eV and 102.4 eV), Ru3p (284.8 eV), O1s (532.2 eV), N1s (399.6 eV) and C1s (284.8 eV) core-level regions could be obviously observed in the XPS analysis of prepared Ru-N-SiNPs, respectively, where the Si2p core levels were mainly derived from the SiNPs, and Ru3p, N1s and C1s core levels indicated the presence of Ru(dcbpy)32+ groups. Importantly, the coexisted characteristic peaks for the Si2p of Si-C and Si-O in Ru-N-SiNPs on 99.9 eV and 102.4 eV respectively suggested C^N linker has connected with Si-O nanostructure by Si-C chemical bond (Figure 1B).25 Based on the ratio of different elements detected by elemental analysis, contents of Ru(dcbpy)32+ in Ru-N-SiNPs was 15.08%. The combination between Ru(dcbpy)32+ and C^N linker could be confirmed furthermore by amide group characteristic wavenumber (1717.36) with infrared spectrum (Figure 1C). At the same time, the characteristic wavenumbers of C-C (1032.55 for saturated groups), and C-H (2851.88 and 2920.43) mainly attributed from carbon chain, and C=N (2335.67 for heterocyclic groups) and C-C (1468.09 for heterocyclic groups) mainly came from Ru(dcbpy)32+. All of these results confirmed that the Ru-


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N-SiNPs showed spherical nanoparticles and Ru(dcbpy)32+ group has combined onto the Si-O nanostructure by C^N linker (Figure 1D).

Figure 1. Characteristics and schematic diagrams of the structure and morphology for the Ru-N-SiNPs. (A) SEM image of Ru-N-SiNPs; (B) Si2p region of XPS analysis for Ru-N-SiNPs, SiNPs and Ru@SiNPs; (C) Infrared spectrum characterization for Ru-N-SiNPs from 1100 to 2900 cm-1; (D) schematic diagrams of the structure and mor-


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phology for the Ru-N-SiNPs; (E) UV-vis adsorption spectra characterizations of SiNPs, Ru(dcbpy)32+, Ru@SiNPs and Ru-N-SiNPs, respectively (range from 200 to 800 nm); (F) normalized photoluminescence and ECL emission spectra of Ru-N-SiNPs (inset: images for the solutions of Ru-N-SiNPs under UV-light (λ = 365 nm) irradiation (orange) and visible-light irradiation (light-yellow), respectively).

Furthermore, the photophysical characterizations of Ru-N-SiNPs were carried out with UV-vis absorption spectra, fluorescence and ECL measurements. As showed in Figure 1E, the characteristic absorption spectra of Ru(dcbpy)32+ was 489 nm, whereas characteristic absorption spectra of Ru-N-SiNPs and Ru@SiNPs were 461 nm and 467 nm, respectively. The blue shift of characteristic absorption for Ru-N-SiNPs and Ru@SiNPs may be attribute to the Si-O group decreased the interaction between Ru(dcbpy)32+ and the surrounding water molecules by increasing steric hindrance effect.26 An obvious ECL emission with peak signals could be obtained for Ru-NSiNPs, which was characterized by ECL spectra with maximum emission peak at 635 nm. It was essentially close to the fluorescent emission spectra (optimal excitation at 470 nm and emission peak at 594 nm) (Figure 1F). However, there was 41 nm red shift of ECL emission, which indicated radical ion and triplet-triplet annihilation lead to some excimer formation32-35. In addition, inset of Figure 1F presented the images of Ru-N-SiNPs solutions under UV-light irradiation (orange) and visible-light irradiation (light yellow), respectively. The Biocompatibility of Ru-N-SiNPs. To characterize the biocompatibility of Ru-N-SiNPs, the MTT assay was employed with Ru@SiNPs and Ru(dcbpy)32+ as controls. Here, the biocompatibility was demonstrated by the survival fraction, which was calculated by the equation: survival fraction = I

Itest -Iblack control -Iblack

×100% (Itest was the mean intensity of the test wells, Iblack was the mean in-

tensity of the black wells and Icontrol was the mean intensity of the control wells). Research details and results were showed in the supporting information (Figure S4). It could be found the survival


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fraction above 90% was received for the proposed Ru-N-SiNPs, which was obviously higher than survival fraction of 69% for Ru@SiNPs and 53% for Ru(dcbpy)32+, respectively, indicating the good biocompatibility of Ru-N-SiNPs. Probable Self-enhanced ECL Mechanism of Ru-N-SiNPs. The electrochemical (Figure 2A curve a, dot line) and ECL-potential (Figure 2A curve b, solid line) responses of Ru-N-SiNPs were showed in Figure 2. There was a significant ECL emission with peak intensity on 1.52 V, when Ru(dcbpy)32+ was oxidized to Ru(dcbpy)33+ and secondary amine (-NH-) in intracoreactant was oxidized to radical cation (-N·+H-). To conform the important role of intracoreactant in Ru-N-SiNPs, their enhancement of ECL emission was compared with the enhancement of Ru(dcbpy)32+-based ECL systems enhanced by the similar secondary amine as inter-coreactant (Figure 2B), where the enhancement was obtained by the ratio of the ECL intensity of Ru(dcbpy)32+ with/without coreactant (Intx/Int0). Specially, the Intx/Int0 of Ru-N-SiNPs was obtained by the ratio of ECL intensity between Ru-N-SiNPs and Ru@SiNPs, where Ru@SiNPs could be recognized as the Ru-N-SiNPs without intra-coreactant. An Intx/Int0 of 1195 was obtained for Ru-N-SiNPs, which was at least 100-folds higher than the Intx/Int0 of Ru(dcbpy)32+based ECL systems with diethylamine (1.82) or diethanolamine (7.00) as coreactant respectively, indicating the higher efficiency of intra-coreactant because of its more effective reactions with the shorter electron-transfer path, higher reaction speed and a lower energy loss.


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Figure 2. (A) Cyclic voltammograms (a, dot line) and ECL-potential (b, solid line) of Ru-N-SiNPs modified GCE in PBS with potential ranged from 0.2 V to 1.6 V. (B) Comparison between the ECL emission of Ru-N-SiNPs and Ru(dcbpy)32+-based ECL systems enhanced by 25 mM diethylamine or diethanolamine as inter-coreactant.

On the basis of the previous results, a schematic illustration for the self-enhanced ECL mechanism of the Ru-N-SiNPs could be demonstrated as showed in Scheme 2, which included three main reactions: (1) the oxidation of secondary amine (-NH-) in intra-coreactant to radical cation (-N·+H-) and Ru(dcbpy)32+ to Ru(dcbpy)33+ taken place on the electrode surface; (2) the excited form of Ru(dcbpy)3*2+ generated by the reaction between radical cation (-N˙+H-) and Ru(dcbpy)33+; (3) ECL emission generated when Ru(dcbpy)3*2+ goes back to Ru(dcbpy)32+.


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Scheme 2. Schematic illustration for the mechanism model of Ru-N-SiNPs with intra-coreactant.

Electrochemical Characterizations of the Cytosensor. To characterize the fabrication of the cytosensor, CV measurements were employed step by step in 0.1 M PBS with 5 mM [Fe(CN)6]3/4-

at the potential ranged from 0.2 V to 0.6 V with a scan rate of 50 mV/s (Figure 3A). A pair of

well-defined redox peaks of [Fe(CN)6]3-/4- could be seen for the bare GCE (curve a). After the modification of AuNPs, the peak currents were increased obviously because of the good conductivity and electron transfer ability of AuNPs (curve b). When Con A was immobilized onto the electrode surface based on the interaction between -SH or -NH2 of Con A and AuNPs, the redox currents decreased obviously (curve c). A further decrease of peak current was found after the


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blocking with albumin from BSA (curve d). Finally, the peak current decreased furthermore after the fabricated cytosensor incubated with cells. The reason was that Con A, BSA and cells on the electrode would retard the electron transfer due to the increased steric hindrance effect. At the same time, another useful electrochemical measurement, EIS, was also used to characterize the fabrication of the cytosensor and capture of cells as shown in Figure 3B. In the EIS measurements, the electron transfer resistance (Ret) is the key parameter for the electron transfer kinetics at the electrode interface, which was similar to the semicircle diameter in EIS response with form of Nyquist plot. For the bare GCE (curve a), a normal EIS response with Ret about 50 Ω was received in 0.1 M PBS with 5 mM [Fe(CN)6]3-/4- at a frequency ranged from 5 × 10-2 to 1 × 106 Hz in a given open circuit voltage with amplitude of 10 mV. Further EIS determinations were employed on the same conditions. When AuNPs were deposited onto the electrode surface, an obvious decreasing of Ret was obtained (curve b, Ret ≈ 20 Ω). An increased Ret about 100 Ω was found due to the modification of Con A (curve c). After blocking with BSA (curve d), the Ret increased to about 150 Ω. Furthermore, the Ret increased to about 1000 Ω after the capture of cells (curve e), indicating the capture of cells on the proposed cytosensor.


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Figure 3. CV (A) and EIS (B) of the stepwise modified electrodes in 5 mM [Fe(CN)6]3-/4- containing 0.1 M KCl for (a) bare GCE, (b) AuNPs/GCE, (c) Con A/AuNPs/GCE, (d) BSA/Con A/AuNPs/GCE and (e) cells/BSA/Con A/AuNPs/GCE, respectively.

Comparison of different signal probes. To investigate the efficiency of annexin V/Ru-N-SiNPs as signal probe, other two kinds of annexin V functionalized probes including annexin V/Ru(dcbpy)32+ and annexin V/Ru@SiNPs were employed as controls. Here, the same batch of cytosensor was prepared and incubated with paxlitaxel treated cells, and then incubated with these different annexin V functionalized probes including annexin V/Ru(dcbpy)32+ (Figure 4A), annexin V/Ru@SiNPs (Figure 4B) and annexin V/Ru-N-SiNPs (Figure 4C), respectively. It could be seen in Figure 4A that the ECL response of cytosensor with annexin V/Ru(dcbpy)32+ as signal probe was about 203 a.u. compared with the background value. As showed in Figure 4B, an enhanced ECL response, about 1058 a.u. was received by the cytosensor with annexin V/Ru@SiNPs, which was attributed to the increased amount of Ru(dcbpy)32+ in Ru@SiNPs. For the proposed cytosensor with annexin V/Ru-N-SiNPs (Figure 4C), an obviously enhanced ECL emission about 1767 a.u. was obtained, indicating the great ECL efficiency of our proposed RuN-SiNPs and cytosensor with annexin V/Ru-N-SiNPs as signal probe. Thus, these results indicated the proposed annexin V/Ru-N-SiNPs could be utilized as signal probe for ultrasensitive determinations.


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Figure 4. ECL profiles of the cytosensor by using various annexin V functionalized signal probes: (A) annexin V/Ru(dcbpy)32+ with 25 mM TPrA as inter-coreactant, (B) annexin V/Ru@SiNPs with 25 mM TPrA as intercoreactant, and (C) annexin V/Ru-N-SiNPs based on a sandwich-type assay for cells treated with 20 nM paxlitaxel.

Application of cytosensor based on the Ru-N-SiNPs for anticancer drug screening. The apoptosis of paclitaxel treated cells was firstly detected by laser confocal microscope, which is a standard method to observe the luminescence signals from cells. The laser confocal microscope images of MDA-MB-231 cell which were treated with or without 20 nM paclitaxel, were showed in Figure 5 A and B respectively (the red fluorescent responses in these images were resulted from the annexin V-PE stained apoptotic cells). It could be seen that the treated cells (Figure 5A) showed obviously higher fluorescent responses from annexin V-PE than that of the untreated cells (Figure 5B), indicating the higher apoptotic ratio due to the treatment of paclitaxel. These cells were also detected by the proposed cytosensor. As showed in Figure 5C, there was a maximum ECL intensity about 300 a.u. for the cancer cells untreated with paxlitacel (curve a), whereas a maximum ECL intensity about 1800 a.u. was obtained for the cells treated with 20 nM paxlitaxel (curve b), indicating the proposed cytosensor could be used to detect cells apoptosis, which was remarkable for the anticancer drug screening by cell apoptosis monitoring.


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To validate the performance of the proposed cytosensor in anticancer drug screening, the proposed cytosensors were employed for the determination of cells reduced with 0, 1, 2, 5, 10, 20, 50, 100 and 200 nM paclitaxel respectively (Figure 5D). Paclitaxel with higher concentration would lead to increased cell’s apoptosis, increased binding of annexin V/Ru-N-SiNPs, and increased ECL signals. It could be found that the ECL intensity increased accordingly to the concentrations of paclitaxel in the range from 1 nM to 200 nM with a detection limit of 0.3 nM and a correlation coefficient of 0.9917, which indicated that the proposed cytosensor can be used for quantitative anticancer drug screening with cell apoptosis monitoring.

Figure 5. (A) Laser confocal microscope image for annexin V-PE stained apoptotic cells without treatment of paclitaxel; (B) Laser confocal microscope image for annexin V-PE stained apoptotic cells with treatment of 20 nM paclitaxel; (C) ECL signals of the cytosensor reacted with cells untreated (a)/treated (b) with 20 nM paxlitaxel; (D)


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Calibration curve for the relationship between ECL intensity based on the proposed cytosensor and logarithm of concentrations of paclitaxel (insert: ECL-time curves of the cytosensor for the determination of cells reduced with 1, 2, 5, 10, 20, 50, 100 and 200 nM paclitaxel, respectively).

To evaluate the applicability of the proposed cytosensor, laser confocal microscope was employed as a control to detect these MDA-MB-231 cell reduced with different concentration of paclitaxel respectively (Figure S5). The fluorescent images with blue, green and red color could be observed from DAPI stained cell nuclear, Con A-FITC stained cell membrane and annexin VPE stained apoptotic cells, respectively. In addition, these images were overlaid as the polychrome images (PCI). It could be seen that fluorescent intensity of DAPI and Con A-FITC was similar, and fluorescent intensity of annexin V-PE was increased with increasing concentrations of paxlitaxel. These results also indicated the increased cell apoptosis with increasing concentration of paxlitaxel. Here, an Apoptotic Index was calculated with ten different images by equation 1, in which the signal degree was the fluorescent intensity of annexin V-PE with 0 - 4 levels.27 Apoptotic Index = ∑

Cell Numberi × Signal degree Totle Cell Number

eq. 1

The Apoptotic Index increased obviously with increasing concentrations of paclitaxel from 1 nM to 200 nM with a correlation coefficient of 0.9272, which was lower than that received by the proposed cytosensor (Figure 6 A), indicating the well performance and potential application of the proposed cytosensor for the detection of cells apoptosis and anticancer drug screening by cell apoptosis monitoring.


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Figure 6 (A) a, calibration curve for the relationship between ECL intensity based on the proposed cytosensor and logarithm of concentrations for paclitaxel; b, calibration curve for the relationship between Apoptotic Indexes based on laser confocal microscope and logarithm of concentrations for paclitaxel; (B) ECL stability of the proposed cyrosensor to cells treated with various concentration of paclitaxel.

Stability and Repeatability of the Proposed Cytosensor. Stability as one of the important factors was investigated by employing one cytosensor for consecutive cyclic potentials scans, which was evaluated for the proposed cytosensor incubated with cells induced with various concentration of paclitaxel (Figure 6B). It could be found that relative stable curves could be obtained for the proposed cytosensor on every concentration of paclitaxel, indicating the excellent stability of the proposed cytosensor. The repeatability of the cytosensor was evaluated by using 10 different cytosensor incubated with paxlitaxel treated cells. The relative standard deviation (RSD) of the detection was 3.6% indicating the acceptable repeatability of the proposed cytosensor for the application in anticancer drug screening. ■ CONCLUSION In summary, a novel ECL cytosensor with a sandwiched format was developed by utilizing the Ru-N-SiNPs for quantitative anticancer drug screening with cell apoptosis monitoring. The Ru-


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N-SiNPs showed more than 100 folds higher ECL emission than those enhanced by similar intercoreactant due to the higher efficiency of intra-coreactant. Using MDA-MB-231 cell-paclitaxel as a model, the cytosensor with Ru-N-SiNPs labeled annexin V as signal probe outperformed laser scanning confocal microscopy on anticancer drug screening with cell apoptosis monitoring. The success in the establishment of the as-proposed ECL cytosensor with Ru-N-SiNPs could provide an efficient tool for anticancer drug screening by cells apoptosis monitoring and open a new way for ultrasensitive biomolecules diagnostics, cells detections and cell function researches, especially in living and complex systems. ■ ASSOCIATED CONTENT Supporting Information. Experimental details for synthesis of Ru@SiNPs and SiNPs, MTT assay, morphology study of the sensing interface and supplementary figures and tables. ■ AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (Ruo Yuan); [email protected] (Yaqin Chai) Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation (NNSF) of China (21575116, 51473136, 21275119 and 81301518), and the Fundamental Research Funds for the Central Universities (XDJK2015A002), China.


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■ ABBREVIATIONS annexin V-PE, phycoerythrin conjugated annexin V; AuNPs, gold nanoparticles; BSA, albumin from bovine serum; Con A, concanavalin A; CV, cyclic voltammetry; DEPI, 4',6-diamidino-2phenylindole; DMEM, dulbecco’s modified eagle’s medium; DMSO, dimethylsulfoxide; ECL, electrochemiluminescence; EDC, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride; EIS, electrochemical impedance spectroscopy; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate isomer I; GCE, glassy carbon electrode; Intx/Int0, the ratio of the ECL intensity of 25 mM Ru(dcbpy)32+ with/without coreactant; NNN-TES, N-(3-trimethoxysilylpropyl) diethylenetriamine; PBS, phosphate buffered saline; PBST, phosphate buffered saline with tween-20; PCI, polychrome images; PE, phycoerythrin; PMT, photomultiplier tube; Ru(dcbpy)32+, tris (2, 2’-bipyridyl-4,4’-dicarboxylato) ruthenium (II); Ru-N-SiNPs, the proposed self-enhanced electrochemiluminescent ruthenium-silica nanoparticle; SEM, scanning electron microscope; Ru@SiNPs, Ru(dcbpy)32+ doped silica nanoparticles; sulfo-NHS, N-hydroxysulfosuccinimide; TEM, transmission electron microscopy; TEOS, tetraethylorthosiliconte; TPrA, tripropylamine; XPS, X-ray photoelectron spectroscopy. ■ REFERENCES (1) Bernard, W. S.; Christopher, P. W. World cancer repot 2014. World Health Organization, Lyon, 2014; pp 18-20. (2) Lisa B. P.; James, O. B. Adv. Drug Delivery Rev. 2012, 64, 206-212. (3) Helena, M.; Claudio, P.; Antonio, A. R.; Arben, M.; Carme, N. Nanoscale 2015, 7, 40974104. (4) Antony, K.; Kong, M. L.; Valentina, R. N.; Srinivas, N.; Kelvin, C.; Georges, E. G.; George, Q. L. J. Pharm. Pharm. Sci. 2015, 4, 424-433.


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A graphic entry for the Table of Contents (TOC)

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Ultrasensitive Cytosensor Based on Self-Enhanced

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