Assay of Single-Cell Apoptosis by Ensemble and Single-Molecule

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Assay of Single Cell Apoptosis by Ensemble and Single Molecule Fluorescence Methods: AnnexinV/ PEG Functionalized Quantum Dots as Probes Lingao Ruan, Mei Ge, Xiangyi Huang, and Jicun Ren Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01749 • Publication Date (Web): 31 Jul 2018 Downloaded from http://pubs.acs.org on August 1, 2018

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Assay of Single Cell Apoptosis by Ensemble and Single Molecule Fluorescence Methods: Annexin-V/ PEG Functionalized Quantum Dots as Probes Lingao Ruan†,‡, Mei Ge‡, Xiangyi Huang*,†, Jicun Ren*,† † School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, People’s Republic of China ‡ Shanghai Laiyi Center for Biopharmaceutical R&D, 200 Niudun Road, Shanghai, 201203, People’s Republic of China

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ABSTRACT: Apoptosis plays a critical role in many biological processes and the etiology of various diseases of the immune system. The study of apoptosis would allow both improving the diagnosis of certain diseases and serving as a target of drug screening. In this paper, we developed a sensitive assay of single cell apoptosis using semiconductor quantum dots (QDs) as fluorescent labeling probes. The principle of this assay is based on the detection of phosphatidylserine (PS) exposed on the plasma membrane during the drug-induced apoptosis. The QDs-labeled Annexin V (AV) were prepared to specifically target PS on the membrane of apoptotic cells, and PS was detected by fluorescent imaging, flow cytometer and single molecule fluorescence correlation spectroscopy (FCS). We developed the procedures for conjugation of QDs to AV and for purification of their conjugates by gel chromatography. The obtained conjugates were characterized by FCS, capillary electrophoresis and Zeta potential analyzer. We studied the nonspecific adsorption of cells to different surface modified QDs, and found that the nonspecific adsorption effects were significantly reduced by modification of QDs with polyethylene glycol (PEG) in detection of apoptosis. In this assay, the results obtained by flow cytometry were consistent with the commercial test kit. Furthermore, a home-built single molecule FCS system was developed to in situ study the drug-induced apoptosis. We observed the significant change in the diffusion coefficients of QDs on cells during the progress of cell apoptosis. Compared with conventional methods, the fluorescent methods represented here possess high sensitivity due to the use of high photo stability and brightness QDs as labeling probes, and provide the temp-spatial information on a single apoptotic cell.


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INTRODUCTION Apoptosis is a form of programmed cell death in the physiological or pathological conditions, and is a very important bioprocess to regulate cell number by counterbalancing cell proliferation.1,2 Apoptosis plays a key function in quantities of cellular events, for instance, morphogenetic death of cells during embryogenesis.3 It also plays a critical role in a range of bioprocesses, which mainly include the etiology of various diseases of the immune system4 and the screening of apoptosis-related drugs.5 Therefore, the study of apoptosis is necessitated in various basic and clinical researches.6 Apoptotic cell death is a highly regulated process, which is characterized by stereotypical morphological changes of the cellular architecture. The characters, including cell shrinkage, plasma membrane blebbing,7 cell detachment,8 externalization of phosphatidylserine (PS),9 nuclear condensation,10 activated caspases 3/611 and ultimately DNA fragmentation,12 demonstrate the possibility of apoptosis.13 Recently, various methods are introduced for the analysis of cell apoptosis, and their principles are mainly based on the variation of morphology,14 caspases activation,15 and DNA fragmentation16 during cell apoptosis. Electron microscopy (EM) is generally used to detect the morphological change of apoptotic cells.17 The EM method provides visible morphological information, and it works as the gold standard for the detection of apoptosis. Conventional methods such as DNA-electrophoresis, Western blot or colorimetric enzyme-linked immunosorbent assays are widely used to detect cell apoptosis, but these methods cannot distinguish apoptotic cells from viable and necrotic cells.18,19 Florescence methods like flow cytometry20 and fluorescence microscopy21 are alternative methods for exploring cell apoptosis. In the florescence methods, Annexin V (AV), a specific phospholipid-binding protein, has been widely applied to monitor the progression of


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apoptosis22−24 when a fluorescent dye such as fluorescein isothiocyanate (FITC) or Alexa Fluor® 488 is labeled to AV. Early apoptotic cells show AV-positive and propidium iodide (PI)negative, whereas end-stage apoptotic cells show AV/PI-double-positive. However, other methods such as fluorescence labeled antibodies assays are necessary to verify the stages of apoptosis. These assays need a large number of cells and could not explore single apoptotic cells. Furthermore, the conventional detection techniques above cannot acquire temp-spatial information on a single apoptotic cell and real-time monitor apoptotic processes. So far, organic fluorescent dyes are widely used in the fluorescent analysis of apoptosis, but they have certain drawback such as insufficient photo stability and low brightness per particle,24 which limit to obtain temp-spatial information on a single apoptotic cell and long-time monitor apoptotic processes. When compared to organic fluorescent dyes, semiconductor quantum dots (QDs) have unique physical and chemical properties25 such as narrow, size-dependent fluorescence emission,26 good photo stability,27 and high quantum yield.28 Currently, QDs are extensively applied in fluorescence based methods, such as fluorescence imaging,29 flow cytometry,30 fluorescent spectroscopy and microarrays analysis.26 AV-quantum dot conjugates have been successfully used to distinguish and image the apoptotic cells.31−35 In this paper, we demonstrated a sensitive assay of single cell apoptosis using QDs-labeled AV conjugates (QD-AV) as optical probe. The principle of this assay is based on the detection of PS exposed on the plasma membrane during the drug-induced apoptosis. The QD-AV were prepared to specifically target PS on the membrane of apoptotic cells, and PS was detected by fluorescent imaging, flow cytometer and single molecule fluorescence correlation spectroscopy (FCS). We studied the nonspecific adsorption of cells to different surface modified QDs, and found that the nonspecific adsorption effects were significantly reduced by modification of QDs


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with polyethylene glycol (PEG). Furthermore, a home-built single molecule FCS system was developed to in situ study the progression of the drug-induced apoptosis. We observed the significant change in the diffusion coefficients of QDs on cells during the progress of cell apoptosis. EXPERIMENTAL SECTION Materials and Chemicals. QdotITK™ amino-polyethylene glycol CdSe/ZnS QDs with 525/655 nm emission wavelength (QD-PEG-NH2), carboxyl quantum dots with 655 nm emission wavelength (QD-COOH) and Alexa Fluor® 488-AV/PI kit were purchased from Life technologies (USA). The 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDC) was from Fluka (Japan), and N-hydroxysulfo-succinimide (Sulfo-NHS) was from Shanghai Medpep Co., Ltd. (China). Suberate Bis-sulfosuccinimidyl (BS3) was purchased from Thermo Fisher Scientific (USA). Unlabeled AV was a product of Abcam ab (UK). Sephacryl S200 was purchased from GE Healthcare (Sweden). The empty gel columns (8 × 300 mm) were obtained from Shanghai Heqi Glassware (China). PANC-1 cells were provided by the Committee on Type Culture Collection of Chinese Academy of Sciences (China). Other reagents were obtained from Shanghai Chemical Reagent Company (China). Ultrapure water was used to prepare all solutions, which was provided by Milli-Q Plus System (Millipore Corporation, Bedford, MA, U.S.A.). Conjugation of QDs to AV protein. The procedures for conjugation of QDs to AV protein are shown in Figure S1. In this study, two kinds of QDs containing amino groups and carboxyl groups were used as labeling probes and two coupling reagents were used in conjugation. As shown in Figure S1a, QD-PEG-NH2 was efficiently conjugated to AV using BS3 as coupling reagent, and the two amino groups in proteins and QD-PEG-NH2 were linked with two-step


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procedure. Firstly, the molar ratio of QDs and BS3 was adjusted to 1:50 and reacted for 30 min at 25 oC. Then, ultra-filtration was applied to remove the excess of BS3. Finally, AV with 20fold excess was added to the activated QDs solution and reacted for another hour at 25 oC under gentle stirring, and then stored at 4 oC. The PBS with pH value 5.0 was used as reaction solution. As shown in Figure S1b, AV was successfully conjugated to QD-COOH by using EDC and Sulfo-NHS as coupling reagents. Firstly, QDs were activated by EDC and Sulfo-NHS with the molar ratio 1:2000:4000 for 15 min at 25 oC. Then, AV with 30-fold excess was added to the activated QDs solution and reacted for another hour at 25 oC under gentle stirring. Finally, the conjugated solution was stored at 4 oC. PBS solution with the pH value 7.0 was worked as reaction solution. Purification of QD-AV Conjugates. Each QD-AV conjugates were purified by home-built size-exclusion chromatographic column (SEC). The details of the experimental setup can be found elsewhere.36 The S-200 medium was used as the packing medium of the size-exclusion chromatographic column. The sample is visible by using a hand-held UV lamp to excit the fluorescence of QDs. Each drop was collected in separate tubes when the fluorescence appeared in the column tip. Characterization Techniques. Fluorescence of QDs and QD-AV conjugates were characterized by a home-built FCS setup system in study on the separation efficiency of QD-AV conjugates. The details of the experimental setup16 and the theory of FCS37 can be found elsewhere. The home-built FCS system mainly included an inverted Olympus IX 71 microscope (Japan) and an argon laser with 488 nm emission (Ion Laser Technology, Shanghai, China). The 505DRLP dichroic mirror and the 660DF50 and 530DF30 bandpass filters were from Omega Optical (USA). A digital correlator was used to correlate the fluorescence fluctuations (Flex02–


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12D/C, Correlator.com, USA). Microcal Origin 6.0 software was applied to nonlinearly fit the raw FCS data based on the Levenberg-Marquardt algorithm experiments. The diffusion coefficients of QDs in solutions and on cells were obtained by this FCS system. Capillary electrophoresis (CE) was applied to separate and characterize QDs and QD-AV conjugates using a P/ACE MDQ system under a normal polarity separation mode. The inner diameter of capillary was 75 µm and the effective length was 30 cm (40 cm total). The capillary was thermo-stated at 25 oC. The introduction mode of samples was electrokinetic injection. The capillary was rinsed with 10 mM NaOH for 5 min, and then with the electrophoresis buffer for 3 min between each run. Cell Experiments. PANC-1 cells were incubated in DMEM high glucose medium (Gibco, California, USA) including 10% (v/v) fetal bovine serum (FBS) (Gibco). LDM was used as a model drug to induce cell apoptosis and cells were treated with different concentrations of LDM for 20 h. Cells that were not treated by LDM were worked as control group. The normal and apoptotic cells were incubated with each 10 nM QD and QD-AV solution in 300 µl PBS (pH 7.2) with additional CaCl2 (1.0 mM), MgCl2 (0.5 mM) at 37 oC, 5% CO2 for 30 min. After QD and QD-AV incubation, cells were washed twice with PBS, and then incubated in PBS solution. Cell Imaging and Image Processing. Inverted fluorescent microscopy (Olympus IX 71, Olympus Optical Co., Japan) was used for fluorescence imaging of the QDs and QD-AV, and a mercury lamp was used as the excitation light source. An EMCCD camera (Cascade 128+, Roper Scientific, USA) was attached to acquire the bright field and fluorescence images of cells. The Image-Pro-Plus 6 software was used to perform the pseudo color processing.


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The Assay of Apoptosis by Flow Cytometer. LDM was used to induce PANC-1 cell apoptosis. Apoptotic cells were firstly trypsinized, and then washed with PBS, and finally centrifuged at 2000 rpm for 5 min to get cell pellets. The cell pellets were incubated with QD525PEG-NH2-AV (or Alexa Fluor® 488-AV) and PI staining solution at 25 °C for 15 min in the dark. Flow cytometer (Becton, Dickinson and Company, US) was used to analyze the apoptotic status of cells. FL1 channel with 488 nm excitation and 530 nm emission was used for the detection of QD525-PEG-NH2-AV or Alexa Fluor® 488-AV. FL3 channel with a band-pass filter (> 600 nm) was used for PI detection. Events falling in the QD525-PEG-NH2-AV or Alexa Fluor® 488 (+)/PI (-) region of the lower right quadrant are counted as apoptotic cells. The Study on Diffusion Behaviors of QDs on Membrane of Apoptotic Cells. FCS was coupled with confocal laser scanning microscopy (FCS-CLSM) and was used for imaging of cells and real-time study the diffusion behavior of QDs on cell membrane. FCS-CLSM system was shown in Figure S2 in Supporting Information. Cells used in FCS-CLSM analysis were cultured in ϕ 20 mm glass bottom cell culture dishes (NEST Biotechnology Co. LTD. USA), and other steps are same as fluorescence detection experiments. RESULTS AND DISCUSSIONS The Strategy of Cell Apoptosis Assay. The principle of apoptosis assay is based on the detection of PS exposed on the plasma membrane during the drug-induced apoptosis using QDslabeled AV as fluorescent probe. AV protein possesses a strong affinity to PS and is commonly used to recognize PS on apoptotic cells. In this assay, QDs-labeled AV as fluorescent probe is used to target PS on the plasma membrane, and PS is detected by fluorescent imaging and flow cytometer methods. The diffusion behaviors of QDs on apoptotic cell membrane can be investigated by single molecule FCS. The procedure of this method is shown in Figure 1. Firstly,


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the conjugation of QDs to AV is carried out using suitable coupling reagents, and QDs-AV conjugates were purified by SEC technique. Finally, the assay of the drug-induced apoptosis is performed by fluorescent detection methods using QDs-AV conjugates as target probes. In this strategy, LDM is used as an induction drug, and fluorescent imaging technique, flow cytometer and single molecule FCS are used in the assay of the drug-induced apoptosis, respectively.

Figure 1. The strategy for the assay of the drug-induced apoptosis using QDs-AV conjugates as fluorescent probes. Characterization of QDs and QD-AV Conjugates. The conjugation of QDs to AV and the purification of their conjugates were described in the experimental section. The QD-AV conjugates were characterized by three methods including CE, FCS and zeta potential analyzer. Figure S3 shows the electropherograms of QDs, purified QD-AV conjugates and raw QD-AV conjugates by using CE with laser-induced fluorescence (LIF) detector.38 As shown in Figure S3, QD-AV conjugates were well separated with free QDs within 14 min in the alkaline buffer. This is because of the significant difference in the mobility of free QDs and QD-AV conjugates in solution. This result demonstrated that AV proteins were linked to the QD surface and QDsAV conjugates were efficiently purified by SEC technique.


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Furthermore, the characterizations of QDs and QD-AV conjugates were performed by FCS technique. FCS is a single molecule method, and it is used to detect the signal fluctuations of single fluorescent molecule in a highly focused detection volume (less than 1 fL) due to Brownian motion. This method is widely used to study molecular diffusion, reaction kinetics, and molecular interaction in vitro37 and in vivo.39,40 Certain characteristic kinetic parameters can be obtained using the appropriate dynamic model. Figure 2 displays the autocorrelation curves of QD-PEG-NH2, QD-COOH, and their AV conjugates. As seen in Figure 2a, 2b and 2c, FCS curves of six compounds are well fitted with the single-component model equation (1) with correlation coefficients (R2) of 0.982~0.995, and the fitting residuals are less than 0.08. The obtained diffusion coefficients were 8.0 ± 0.14 µm2/s to 17.1 ± 0.85 µm2/s for QDs and were 6.9 ± 0.12 µm2/s to 13.7 ± 0.30 µm2/s for QD-AV conjugates. The characteristic diffusion times of QD-AV increased significantly when compared to free QDs. The brightness per particle (BPP), characteristic diffusion times and hydrodynamic radii of QDs and their AV conjugates measured by FCS are shown in Figure S4. As shown in Figure S4c, the diameters were 7.7 ± 0.4 nm and 9.6 ± 0.4 nm for the QD525-NH2 and obtained AV-QD conjugates, respectively. The diameters of the QD655-NH2 and their AV-QD conjugates were 16.5 ± 0.3 nm and 18.8 ± 1.0 nm, respectively. And the diameters of the QD655-COOH and their AV-QD conjugates were 12.7 ± 0.3 nm and 16.0 ± 1.4 nm, respectively. These data demonstrated that the hydrodynamic radii of QDs become larger after QDs were conjugated to AV proteins. The results were consistent with the CE analysis results, and further illustrated that AV proteins were successfully linked to the QD surface.


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Figure 2. The characterization results of QDs and purified QD-AV conjugates. Normalized autocorrelation curves of QD525-PEG NH2 (a), QD655-PEG-NH2 (b), QD655-COOH (c) and their purified QD-AV conjugates, the fitting residuals are shown in the below. Their Zeta potentials are shown in (d). The FCS measurement time was 60 s. The error bars represent the standard deviation of 3 time measurements. Finally, the zeta potentials of QDs are also used to characterize the surface states and conjugation of QDs with AV protein. In this study, Zeta potentials of QDs and QD-AV conjugates were measured using Zeta sizer (Malvern, UK). As shown in Figure 2d, QD525-PEGNH2, QD655-PEG NH2 and QD655-COOH show dramatic differences in their zeta potentials due to their different modified surfaces. Amino-modified nanoparticle often shows positively charged surface. Interestingly, QD525-PEG-NH2 and QD655-PEG-NH2 were negative charged in PBS (pH 7.4) in our experiments, which is similar to that of some references.41,42 The main reason is that the zeta potential of nanoparticles is impacted by the pH, the ionic strength of the


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buffer solution and the concentration of nanoparticles. The zeta potentials of PEG-modified QDs slightly increased after conjugation of QDs to AV protein, but the zeta potentials of QD655COOH significantly decreased after conjugation of QDs to AV protein. These results further confirmed that AV proteins were efficiently conjugated to the QD surface. Study of Non-Specific Adsorption of QDs. QDs are semiconductor nanoparticles, and show good photo stability and high brightness of single particle when compared to widely-used fluorescent dyes. However, QDs also have serious non-specific adsorption to biomolecules and cells in some applications. This is one of main reasons why the applications of QDs as labeling probes are not as wide as organic fluorescent dyes to date. In order to eliminate (or reduce) the non-specific adsorption of QDs, the efficiency and specificity of different surface modified QDs and their AV conjugates were investigated using fluorescent microscopy. The normal and apoptotic cell samples were used in this study. We investigated the levels of cellular internalization of various commercially available QDs. Figure 3 shows the images of the tested cells using different QDs and their AV conjugates as probes. Figure 3a and 3b reflect the levels of cellular internalization of QDs-COOH and their AV conjugates. As shown in Figure 3a and 3b, both normal and apoptotic cells seriously adsorbed QDs-COOH and their AV conjugates, and in this case, they were not distinguished using QD-AV conjugates as a target probes. We tried to add PEG or bovine serum albumin (BSA) to cell culture solution to reduce non-specific adsorption of QDs, but these attempts were failed to decrease non-specific adsorption of QD-AV to normal cells (Figure S5). Figure 3c shows representative images of normal and apoptotic cells using QD-PEG-NH2 as a probe. Interestingly, as shown in Figure 3c, no QDs were observed on normal and apoptotic cells. This preliminary result illustrated that QD-PEG-NH2 did not possess non-specific adsorption to normal and apoptotic cells. Figure 3d shows typical


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images of normal and apoptotic cells using QD-PEG-NH2-AV conjugates as probes. As shown in Figure 3d, nearly no QDs were observed on normal cells, but QDs were distributed on apoptotic cells. These results demonstrated that QD-PEG-NH2-AV conjugates selectively recognized apoptotic cells. The results above reflected that the non-specific adsorption of QDs was significantly associated with the surface states of QDs. When compared to QDs-COOH, the surface on QD-PEG-NH2 was coated with PEG polymers, and the PEG-layer reduced the internalization of QDs. That was mainly because the PEG-layer steric hindrance prevented the direct docking of QDs to the cellular membrane.43 Thus, QD-PEG-NH2-AV as a labeling probe can be used for detection of apoptotic cells.

Figure 3. Fluorescence images of cells using free QDs and QD-AV conjugates as probes. The PANC-1 cells were normal cultured and induced 20 h to apoptosis by 2.5 µg/mL LDM, respectively.


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The Assay of Drug-Induced Apoptosis by Flow Cytometry. The flow cytometry is a very important method for the assay of apoptotic cell. This method is based on the detection of PS exposed on the plasma membrane during cell apoptosis, which is similar to the fluorescent imaging method. In current assay, the apoptotic cells were labeled with fluorescent dye-AV (such as FITC and Alexa Fluor® 488), and death cells were labeled with PI. Two detection channels in flow cytometry are used to recognize apoptotic cells and death cells, respectively. PI dye is excluded by viable cells with intact membranes, however it is permeable to the membranes of dead and damaged cells. For example, viable cells show the negative results in AV and PI staining, cells in early apoptosis show the positive results in AV staining and the negative results in PI staining, and cells in late apoptosis show the positive results in both AV and PI staining. In order to further confirm the reliability of QD-AV as targeting probes, flow cytometry was used to analyze the same samples used in fluorescent microscopy. Amino-PEG modified QDs with 525 nm emission wavelength (QD525-PEG-NH2) were used to as labeling probe to detect apoptotic cells in flow cytometer. This is because that the emission wavelength of QD525 is nearly same as Alexa Fluor® 488, and also matches with the emission wavelength of PI commonly-used in commercial assay kit of cell apoptosis. Figure 4 shows the distribution of the total cell culture population. The fluorescence level of QDs labeled AV (FL-1) and PI (FL-3) was shown in Figure 4a as a dot plot of four quadrants scaled with logarithm, respectively. Control group cells showed the negative results in AV and PI staining, which indicated that the cells were viable with intact membranes. In contrast, the cells treated with 0.25 µg/mL LDM showed positive results in AV staining, which indicated partial PS externalization and early apoptosis. PS externalization increased significantly when the concentrations of LDM increased from 0.25 µg/mL to 25 µg/mL. When LDM concentration was 25 µg/mL, the cells showed high


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PS externalization and PI uptake, which means the loss of plasma membrane integrity and late apoptosis stage or necrosis. Figure 4b compares the results of two fluorescent labels for the assay of apoptosis. The results of flow cytometer using QD-AV as probes were similar to the results of Alexa Fluor® 488-AV/PI test kit assay. And the detection errors using QD-AV were smaller than Alexa Fluor® 488-AV/PI test kit. These results indicated that the QD-AV labeling method can specific detect apoptotic cells by using flow cytometry.

Figure 4. The assay results of flow cytometer (a), and comparison between QD-AV/PI and Alexa Fluor® 488-AV/PI are shown in (b, n = 3). Cells including the normal, primary apoptotic, late apoptotic, and necrotic cells were distinguished by the cytogram of four quadrants with the criteria of AV−/PI−, AV+/PI−, AV+/PI+, and AV−/PI+, respectively.


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The Study on Diffusion Behaviors of QDs on Apoptotic Cell Membrane. Next, we want to explore the possibility for study on the diffusion behaviors of QDs on apoptotic cell membrane by FCS method. In this study, a home-built FCS-CLSM system is established for confocal scanning imaging of cells and real-time study the diffusion behavior of QDs on cell membrane. This system is shown in Figure S2 of Supporting Information.

Figure 5. Confocal scanning image of apoptosis PANC-1 cells (A) and typical FCS curves (B) of QD-AV on the membrane of PANC-1 cells. QD655-PEG-NH2 was used in this study. QD655-PEG-NH2 was chose for fluorescence images of cells and the further tests because of its higher brightness per particle (BPP) than that of QD525-PEG-NH2 (as shown in Figure S4a). In


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order to study the diffusion behavior of QD-AV (QD655-PEG-NH2) probe on apoptotic cells, the images of the labeled cells were firstly obtained by confocal scanning imaging system, and then the diffusion behaviors of the probe in different positions on cell were investigated by FCS. The cells were cultured in a glass culture dish, and induced with drug to apoptosis. And then, the apoptotic cells were cultured for 30 min after addition of QD-AV probe. Finally, FCS-CLSM system was used for study on the diffusion behavior of QDs on apoptotic cell membrane after discarding the remaining probe solution and washing apoptotic cells. Figure 5a shows the fluorescent image of apoptotic PANC-1 cells induced with 2.5 µg/mL of LDM for 20 h. As shown in Figure 5a, the image of apoptotic cells is clear and high resolution which obtained by FCS-CLSM system. This image is used for locations in FCS measurements. FCS curves are shown in Figure 5b and they were obtained at different locations of the cell membrane shown in Figure 5a. FCS curves were fitted with the following equation (1).

G(τ ) =

1 1 1 ⋅ ⋅ 2 N  τ    1 +  1 +  ω0  ⋅ τ  τD   z0  τ D

(1)

Where, ω0 and z0 are the lateral and axial radii of the confocal volume, respectively. τD is the characteristic time of QDs through the detection volume. N is the average number of fluorescent molecules or particles in the volume. FCS curves of five different positions on the cell membrane were fitted, and the diffusion coefficients of QDs obtained were from 0.032 µm2/s to 0.115 µm2/s. These results displayed that the diffusion coefficients of QDs on the cell membrane are significantly different. These differences may be related to the change in membrane structure and the fluidity difference of the cell membrane after drug-induced apoptosis. In this drug induction, the concentration of LDM was 2.5 µg/mL, and in this case, a majority of cells were in the early


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stage of apoptosis and still maintained a more complete cell membrane structure. However, certain position (such as position 3) was at the later stage of apoptosis, the cell membrane had loss of integrity, and in this position the diffusion coefficient (D = 0.115 µm2/s) was significantly larger than others of restricted diffusion in the complete cell membrane (position 4, D = 0.032 µm2/s). These results show that the diffusions of QDs on the cells membrane significantly change with the apoptotic process. Furthermore, we studied the diffusion behaviors of QDs on cells membrane in 0.25, 2.5 and 25 µg/mL of LDM, respectively. We observed that the diffusion coefficients of QDs on the cells membrane increased when higher concentration of drug was used (data are shown in Table S1). The obtained diffusion coefficients of QDs on the cell membrane were determined as 0.026 ± 0.004 µm2/s to 0.116 ± 0.011 µm2/s when LDM concentration was 0.25 µg/mL. When LDM concentration was 25 µg/mL, the diffusion coefficients were increased to 0.047 ± 0.006 µm2/s to 0.18 ± 0.008 µm2/s. The results demonstrated that the diffusion coefficients increased significantly when the concentrations of induced drug were increased. These preliminary results show that the home-built FCS-CLSM system was used to real-time and in situ study the diffusion behaviors of QDs on the cells membrane during the process of drug-induced apoptosis.

CONCLUSIONS In this paper, we developed an in situ assay of single cell apoptosis using QDs as labeling probes. In this assay, QD-AV conjugates probes were firstly prepared to target PS on the membrane of apoptotic cells, and then were detected by fluorescent imaging, flow cytometer and single molecule FCS. We studied the nonspecific adsorption of cells to different surfaced modified QDs, and found that the nonspecific adsorption effects were efficiently eliminated by modification of QDs with PEG. The developed fluorescence methods were successfully used for


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assay of single cell apoptosis induced with a drug (LDM). The results of cell imaging and flow cytometric analysis were consistent with apoptosis detection test kit. Furthermore, we tried to study the diffusion behaviors of QDs on the membrane of apoptotic cells using single molecule FCS system, and observed the significant change in the diffusion coefficients of QDs on cells during the progress of cell apoptosis. Compared with the traditional methods, the fluorescent methods described here have high sensitivity for the use of high photo stability and brightness QDs as labeling probes. More importantly, the single molecule FCS method was used to realtime and in situ study the diffusion behaviors of QDs on the cell membrane, and it can provide the temp-spatial information on a single apoptotic cell. Our preliminary results demonstrate that PEG-modified QDs are sensitive fluorescent labeling probes for the assay of cell apoptosis, and the developed methods for apoptosis assay can be not only extended to other cell toxicity studies with interest for clinics, but also to the detection of certain disease-biomarkers expressed in the plasma membrane and screening of apoptosis-based anticancer drugs.

ASSOCIATED CONTENT

Supporting Information. The following files are available free of charge. Additional table and figures, including the scheme of conjugation of QDs to AV protein and Home-built FCS system, QDs and their AV conjugates measured by CE and FCS, details of QDAV on the membrane of five batch PANC-1 cell samples detected by FCS and the effects of BSA and PEG on normal cell nonspecific adsorption of QD655-COOH. (PDF) AUTHOR INFORMATION


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Corresponding Author * Prof. Jicun Ren and Prof. Xiangyi Huang, Tel: +86-21-54746001 Fax: +86-21-54741297 Email: [email protected], [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported financially by the NSFC (Grant 21675111), the Shanghai Natural Science Foundation (14ZR1423400), Key Project of Basic Research of Shanghai (18JC1413400), and the Innovation Fund (IFPM2016B007) from Joint Research Center for Precision Medicine Set Up by Shanghai Jiao Tong University & Affiliated Sixth People's Hospital South Campus (Fengxian Central Hospital).

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Assay of Single Cell Apoptosis by Ensemble and Single Molecule Fluorescence Methods: Annexin-V/PEG Functionalized Quantum Dots as Probes Lingao Ruan, Mei Ge, Xiangyi Huang*, Jicun Ren* In our paper, the QDs-labeled Annexin V (AV) were prepared to specifically target phosphatidylserine (PS) on the membrane of apoptotic cells, and PS was detected by fluorescent imaging, flow cytometer and single molecule fluorescence correlation spectroscopy (FCS). When compared to current methods, the fluorescent methods described here possess high sensitivity due to the use of high photostability and brightness QDs as labeling probes, and provide the temp-spatial information on a single apoptotic cell.


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Figure 1. Strategy for assay of the drug-induced apoptosis using QDs-AV conjugates as fluorescent probes. 118x64mm (300 x 300 DPI)


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Figure 2. The characterization results of QDs and purified QD-AV conjugates. Normalized autocorrelation curves of QD525-PEG NH2 (a), QD655-PEG-NH2 (b), QD655-COOH (c) and their purified QD-AV conjugates, the fitting residuals are shown in the below. Their Zeta potentials are shown in (d). The FCS measurement time was 60 s. The error bars represent the standard deviation of 3 time measurements. 183x164mm (150 x 150 DPI)


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Figure 3. Fluorescence images of cells using free QDs and QD-AV conjugates as probes. The PANC-1 cells were normal cultured and induced 20 h to apoptosis by 2.5 µg/mL LDM, respectively. 145x109mm (150 x 150 DPI)


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Figure 4. The assay results of flow cytometer (a), and comparison between QD-AV/PI and Alexa Fluor® 488AV/PI are shown in (b). The error bars represent the standard deviation of 3 time measurements. The cytogram of four quadrants was used to distinguish the normal, primary apoptotic, late apoptotic, and necrotic cells by the criteria of AV−/PI−, AV+/PI−, AV+/PI+, and AV−/PI+, respectively. 108x180mm (300 x 300 DPI)


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Figure 5. Confocal scanning image of apoptosis PANC-1 cells (A) and typical FCS curves (B) of QD-AV on the membrane of PANC-1 cells. QD655-PEG-NH2 was used in this study. 100x166mm (150 x 150 DPI)


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Assay of Single-Cell Apoptosis by Ensemble and Single-Molecule

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