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Real-time in situ Visualizing the Sequential Activation of Caspase Cascade Using a Multi-color Au-Se Bonding Fluorescent Nanoprobe Xiaojun Liu, Xiaoxiao Song, Dongrui Luan, Bo Hu, Kehua Xu, and Bo Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00452 • Publication Date (Web): 03 Apr 2019 Downloaded from http://pubs.acs.org on April 3, 2019

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Analytical Chemistry

Real-time in situ Visualizing the Sequential Activation of Caspase Cascade Using a Multi-color Au-Se Bonding Fluorescent Nanoprobe Xiaojun Liu†, Xiaoxiao Song†, Dongrui Luan, Bo Hu, Kehua Xu*, and Bo Tang* College of Chemistry, Chemical Engineering and Materials Science, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Institute of Molecular and Nano Science, Shandong Normal University, Jinan 250014, P. R. China * Email: [email protected]; [email protected]. Tel.: +86 0531-86180010. Fax: +86 0531-86180017

ABSTRACT: The caspase cascade is an ensemble of very important signaling molecules that plays a critical role in cell apoptosis. Real-time monitoring the upstream and downstream activation relationships of the caspases in the signal pathway is of great significance for understanding the regulatory mechanisms of these signaling molecules in the development of various diseases. Herein, a multi-color fluorescent nanoprobe, GNP-Se-Casp, has been developed based on Au-Se bonding for real-time in situ monitoring caspase- (casp-) 3, 8 and 9 during cell apoptosis. In the real-time fluorescence imaging of apoptotic HeLa cells induced by staurosporine (STS) using GNP-Se-Casp, the fluorescence signals corresponding to casp-8 and casp-9 sequentially turn on, followed by the appearance of the fluorescence of casp-3, which visualizes the upstream and downstream relationship of casp-3, 8 and 9. Thus, GNP-Se-Casp is an effective tool for real time in situ monitoring caspase cascade activation in the apoptosis process of tumor cells. This design strategy is easily adaptable to in situ detection of other signal molecules, especially those with upstream and downstream activation relationships.

INTRODUCTION Cell apoptosis is a type of programmed cell death that occurs by extrinsic and intrinsic apoptotic pathways.1-3 The caspase cascade, composed of initiator caspases (e.g., caspase(casp-) 2, 8, 9 and 10) and effector caspases (e.g., casp-3, 6 and 7), is an ensemble of cysteine protease which plays a critical role in the initiation and execution of the cell apoptosis process.4-7 In the caspase cascade, casp-3, 8 and 9 are in a close upstream and downstream relationship and are identified as attractive targets for the monitoring of cell apoptosis. In the extrinsic apoptotic pathway, casp-8 is first activated and then initiates casp-3; and in the intrinsic pathway, casp-9 is the initiator which activates casp-3.8-10 Real-time detection of the sequential activation of casp-3, 8 and 9 and elucidation of the upstream and downstream relationship of these caspases are essential to understand the apoptotic process and to evaluate the therapeutic efficacy of cancer treatments. Caspase activity is usually measured based on the reaction of caspases with the specific peptide substrates that are usually attached to signal molecules.11-12 As a caspase generated in the apoptotic cell cleaves the specific peptide, the attached signal molecule is released. Many fluorescent probes based on this principle have been developed, including molecular probes13-19, gold nanoparticles (GNPs)–peptide conjugate nanoprobes20-28, graphene oxide–peptide conjugate29,30 and quantum dots31-33. Among these probes, GNPs–peptide conjugates nanoprobes have been the favored ones, in which GNPs act as strong fluorescence quenchers which are linked to fluorophores by the thiol-terminal peptides via Au-S bonds. However, the Au-S bonds in the GNPs nanoprobes are vulnerable to be cleaved by the biological thiols, including glutathione (GSH) and cysteine which are present in relatively high con-

centrations and inevitably cause a false-positive result. To solve this problem, Wang et al. designed nanoflares labeled with dual-fluorophores and indirectly amended the false positive signals via ratiometric measurement.34 Recently, an innovative Au-Se nanoplatform has been developed by our group, utilizing Au-Se bond instead of Au-S bond, which effectively eliminates the interference by biological thiols.35 And a nanoprobe based on the Au-Se nanoplatform was developed for the detection of matrix metalloproteinases 2 (MMP-2).36 Undoubtedly, it is a sound strategy to reconstruct the conventional Au-S-bonding nanoprobe with the Au-Se bond for detection of the caspase cascade and reliable visualization of the activation sequence of casp-3, 8 and 9. Herein, we report a multi-color GNPs florescent nanoprobe (GNP-Se-Casp) by attaching three different fluorophoreslabelled caspase peptide substrates onto GNPs through Au-Se bonds to achieve in-situ visualization of the sequential activations of casp-3, 8 and 9. The design strategy is displayed in Scheme 1. In the nanoprobe, the caspase-specific peptides with the selenocysteine terminals were labelled with 5carboxytetramethylrhodamine (5-TAMRA), cyanine-5 (Cy5) and fluorescein isothiocyanate (FITC), respectively specific to casp-3, casp-8 and casp-9. In the absence of targeted caspases, GNP-Se-Casp showed no fluorescence due to the efficient quenching effect of GNPs. In the presence of casp-3 or casp-8 and casp-9, the specific peptides were cleaved at the specific sites, and the fluorophores were released from GNPs with fluorescence recovery. The results show that GNP-Se-Casp could detect casp-3, 8 and 9 with high sensitivity, and with negligible interference from GSH and some important proteins. Benefitting from the advantage of the Au-Se bond, GNP-SeCasp was successfully used for real-time in situ monitoring of

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the sequential activations of casp-3, 8 and 9 during the apoptosis of HeLa cells as induced by staurosporine (STS). The blue and green fluorescence, corresponding to casp-8 and casp-9 sequentially turned on, followed by appearance of the red fluorescence of casp-3. These events provided a visualization and demonstration of the upstream and downstream relationships of casp-3, 8 and 9. Together, GNPSe-Casp has been shown to be an effective tool for real-time monitoring of caspase cascade activation in the apoptosis process of tumor cells and serve as the foundation for better understanding of the cell apoptotic mechanism.

Scheme 1. Design of the nanoprobe GNP-Se-Casp. EXPERIMENTAL Synthesis of GNP-Se-Casp. The preparation procedure for GNPs is given in the Supporting Information (SI). 100 μL of sodium dodecyl sulfate solution (10%, wt%) was mixed with GNPs to achieve a solution with a final concentration of 0.1%. Then, the GNPs solution was mixed with the three peptide chains solution and then added dropwise to the serum bottle. The optimal concentration ratio of the three peptide chains was 1:1:1 according to the in vivo concentrations of casp-3, 8 and 9, 37-40 and the concentration ratio of the GNPs to each peptide chain was 1:300. The mixture was then stirred in the dark for 48 h to ensure that the peptide chains bind sufficiently to the GNPs surfaces. The product was then purified with H2O by centrifugation (14,000 rpm, 25 min, 4 °C) three times. The final product (the nanoprobe GNP-Se-Casp) was dispersed in Tris buffer solution (10 mM, pH = 7.4) and stored away from light. The concentration of the GNPs was calculated by the intensity of absorption at the extinction wavelength of 524 nm (ε = 2.7 ×108 L·mol-1·cm-1). Fluorescence Response of GNP-Se-Casp to casp-3, 8 and 9. GNP-Se-Casp (1 nM) and human casp-3, 8 and 9 recombinant proteins (final concentration of 300 ng·mL-1) were mixed at 37 °C and the reaction time were optimized first. The fluorescence intensity was measured at different times (0, 15, 20, 30, 60, 90, 120 and 150 min). The collection range of fluorescence spectra for casp-3, 8 and 9 was 555-650 nm, 650750 nm and 500-650 nm at excitations of 553, 641 and 494 nm, respectively. Caspase enzymatic kinetics assay of casp-3 (300 ng·mL-1), casp-9 (300 ng·mL-1) and casp-8 (300 ng·mL-1) with increasing substrate (GNP-Se-Casp) concentration (0, 5, 10, 15, 20, 30, 50 and 80 µM). For casp-3 and casp-8, fluorescence intensity was detected at 30 min and for casp-9 at 60 min. The collection range of fluorescence spectra for casp-3, 8 and 9 was 555-650 nm, 650-750 nm and 500-650 nm at excitations of 553, 641 and 494 nm, respectively. Fitting the data with the Michaelis-Menton equation (Equation 1) allows us to estimate the corresponding Michaelis constants Km and kinetic constants kcat. V=

d[P] 𝑉𝑚𝑎𝑥 [S] = d𝑡 𝐾𝑚 + [S]

(Equation 1)

𝑘cat=

Page 2 of 11 V 𝑚𝑎𝑥 [Enz ]

(Equation 2) Where [S] and [P] are the concentrations of the substrate and product (cleaved peptide) and t is the reaction time. Michaelis constants Km and maximal velocities Vmax were calculated by direct fitting the data to Equation 1 using a non-linear regression via origin software. Kinetic constants kcat is calculated according to Equation 2. [Enz] is the concentration of casp-3, casp-8 or casp-9. To study the effect of the inhibitors, GNP-Se-Casp (1 nM) was mixed with the caspase inhibitor (10 µM, Z-DEVD-FMK, Z-IETD-FMK and Z-LEHD-FMK for casp-3, casp-8, and casp-9, respectively) and incubated with casp-3, 8 and 9 recombinant proteins (300 ng·mL-1) at 37 °C for 1.5 h. The corresponding fluorescence was collected. To study the interference of GSH and various proteins, GNP-Se-Casp (1nM) were incubated with GSH (300 ng·mL-1) and different proteins (MMP-2, bovine serum albumin (BSA), human serum albumin (HSA), trypsin, urokinase plasminogen activator (uPA) and lysozyme; 300 ng·mL-1) at 37 °C for 90 minutes. Then the corresponding fluorescence was collected. The experiments were performed three times. Confocal Laser Scanning Microscope (CLSM) Imaging of intracellular Casp-3, 8 and 9 during Cell Apoptosis. Firstly, HeLa cells were cultured in a confocal dish for 24 h and at 37 °C. After 80% cover, the adherent cells were washed with PBS buffer three time. GNP-Se-Casp (1nM) was then added to the dishes. After a 4h incubation at 37 °C, the cells were washed twice with PBS buffer, then treated with STS (5 μM) at 37 °C for 2 h. For the inhibitor studies, cells were incubated with 100 μM corresponding caspase inhibitors 2 h before STS (5 μM) treatment. Subsequently, the CLSM imaging of the cells was carried out. For the real-time imaging test, HeLa cells were cultured in the dishes and incubated for 24 h. Then, STS (5 μM) was added and the dishes were then immediately put in the CLSM. The microscope was focused on a collection of cells and imaging was carried out. The fluorescence images at the Hoechst 33342 channel were acquired in the meantime. For colocalization with active casp3, 8 and 9 antibodies, the cells were in combination with 3.7% paraformaldehyde for 15 min at 37 °C, washed twice with cold PBS, and incubated with 0.1% Triton X-100 in PBS for 10 min. The cells were then blocked with 2% BSA in PBS for 30 min and washed twice with PBS. The cells were incubated with a mixture of anti-casp-3, 8 and 9 antibody and PBS (v/v =1:99) for 1 h at room temperature, washed once with PBS buffer, and then incubated with secondary antibody (a Donkey AntiRabbit IgG H&L, Alexa Flour 405) (2 μg·mL−1) in PBS for 1 h, followed by another wash with PBS. Then CLSM imaging was carried out. RESULTS AND DISCUSSION Synthesis and Characterization of GNP-Se-Casp. GNPs were synthesized by a classic method and functionalized with three different selenocysteine-terminated peptide linkers via the Au–Se bond, generating the nanoprobe GNP-Se-Casp. Each peptide linker was labelled with 5-TAMRA, cyanine-5 and FITC at the caspase specific cleavage site, i.e., DEVD (casp-3), IETD (casp-8), and LEHD (casp-9), respectively. The as-synthesized GNPs and GNP-Se-Casp were characterized using transmission electron microscopy (TEM). As the TEM results shown, the GNPs and GNP-Se-Casp were


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Figure 1. (A) UV/vis absorption spectra of GNPs and GNP-Se-Casp. (B) Fluorescence spectra of GNP-Se-Casp at excitation wavelengths of 494, 553 and 641 nm, respectively.

Figure 2. Kinetic responses of GNP-Se-Casp (1 nM) to casp-3 (A), casp-8 (B) and casp-9 (C). The concentrations of casp-3, 8 and 9 were 300 ng·mL-1.

of pseudo-spherical morphology with similar size, indicating that the surface profile did not change after functionalization (Figure S1A-C). The zeta potential values of the GNPs and GNP-Se-Casp determined by DLS were -6.07 and 5.67 eV, further verifying the good stability of the nanoprobe. Besides, the X-ray photoelectron spectroscopy (XPS) characterization (Figure S1D) verified the formation of Au–Se bond in GNPSe-Casp. Then, the UV-vis absorption spectra and fluorescent spectra of the GNPs and GNP-Se-Casp were collected. As Figure 1A shows, the distinct surface plasmon resonance (SPR) peak shifted from 520 nm (GNPs) to 524 nm (GNP-Se-Casp). After functionalization, a slight redshift of the characteristic SPR absorption peak occurred in the UV-vis absorption spectra, which confirmed that the peptide linkers were successfully attached to GNPs. As shown in Figures 1B, the distinct fluorescence emission spectra of GNP-Se-Casp were 520, 580 and 662 nm excited at 494, 553 and 641 nm, respectively. There was no overlapping of the excitation and emission spectra. Furthermore, the quenching effect of GNPs on the fluorescence of each dye-labeled was characterized (Figure S2A-C) and quantification of the peptides loaded on GNP-Se-Casp was determined according to the previously established method (Figure S2D-F).41 There were approximately 98 5-TAMRA labeled chains, 64 cyanine-5 labeled chains and 59 FITC labelled chains conjugated to each single GNP. These results indicate that GNP-Se-Casp was synthesized successfully and exhibited excellent fluorescent properties for detection and cell imaging. Fluorescence of GNP-Se-Casp Response to Caspases. Given that the pH value has a significant effect on the

performance of fluorescent probe, the fluorescent intensities of GNP-Se-Casp toward caspases in Tris buffers with different pH values were then tested. The fluorescence intensities were found to be relatively steady in the pH region of 4.0 to 10.0 (Figure S3B). Thus, GNP-Se-Casp could function properly at physiological pH, which is the basis for the application in living cells. Then the response time of GNP-Se-Casp with caspases was optimized. 1 nM GNP-Se-Casp was incubated with a mixture of casp-3, 8 and 9 (300 ng·mL-1) at 37 °C in Tris (pH 7.4). Figure S3C shows that GNP-Se-Casp could respond quite rapidly to caspases. As the reaction time lapsed, the fluorescence intensities corresponding to casp-3, 8 and 9 all increased gradually and then reached plateaus at approximately 60 min. It could be seen that the optimized response time of GNP-Se-Casp toward casp-3, 8 and 9 matched well, which made GNP-Se-Casp a good candidate for simultaneously monitoring casp-3, 8 and 9 in living cells. The kinetic analysis of the three enzymatic reaction was carried out by incubating casp-3, 8 or 9 with different concentrations of GNP-Se-Casp (Figure 2). The Michaelis constants (KM) of casp-3,8 and 9 were calculated to be 9.36± 0.2, 9.84 ± 0.3 and 8.53 ± 0.1µM; and the corresponding kinetic constants (kcat) were 6.3, 7.35 and 5.86 s-1, respectively. These KM and kcat values are comparable to those reported in the previous study or ever better than those of a commercial substrate,13,42,43 indicating the better affinity between GNP-SeCasp and casp-3, 8 and 9.



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Figure 3. (A-C) Fluorescence intensities of GNP-Se-Casp (1 nM) treated with increasing concentrations of casp-3, 8 and 9 in Tris (pH 7.4). The concentration range of casp-3, 8 and 9 were 0-300 ng·mL-1. Slit widths: 5 nm. (D-F) Linear correlation between fluorescence intensities and concentrations of casp-3, 8 and 9 in the range of 0-300 ng·mL-1.

Figure 4. (A-C) Fluorescence spectra of GNP-Se-Casp (1 nM) and its mixture with casp-3, 8 and 9 (0-300 ng·mL-1) in the absence and presence of the corresponding caspase inhibitor (10 µM). (E-F) Fluorescence responses toward different interferents. The concentrations of casp-3, 8, 9 and interferents were 300 ng·mL−1. Slit widths: 5 nm.

To test the validity of GNP-Se-Casp to targeted caspases, the in vitro proteolytic activities were investigated with recombinant caspases. GNP-Se-Casp was incubated with the mixture of recombinant casp-3, 8 and 9 in caspase assay buffer at 37 °C. At the optimized response time of 90 min, a concentration dependent activity for the probe was demonstrated by varying caspase concentrations at a fixed concentration of GNP-Se-Casp (1 nM). A gradual increase in fluorescence intensity was observed with the increase in concentrations of targeted caspase in range of 0-300 ng·mL-1. (Figure 3A-C).

There is good linearity between the fluorescence emissions of 5-TAMRA and casp-3 concentrations (Figure 3D). The regression equation is F=9236.89[casp-3] + 701.44 with a linear coefficient of 0.9970. The limit of detection (3S/m) was determined to be 0.073 ng·mL-1. Figure 3E depicts the linear relation between the fluorescence intensities of Cy5 and the concentrations of casp-8 from 0 to 300 ng·mL-1.


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Figure 5. (A) Fluorescence imaging of apoptotic HeLa cells induced by STS. In control group, HeLa cells were incubated with GNP-SeCasp (1 nM) for 4 h followed by treatment with STS (5 µM) for 2 h. In other groups, HeLa cells were incubated with GNP-Se-Casp (1 nM) for 4 h followed by treatment with caspase-specific inhibitor (100 µM) for 1 h and then treated with STS (5 µM) for 2 h. Red fluorescence (casp-3) at λex/em of 561/570–700 nm, blue fluorescence (casp-8) at λex/em of 633/650–750 nm, green fluorescence (casp-9) at λex/em of 488/500–600 nm, Hoechst at λex/em of 405/430–480 nm. Scale bar=50 μm. (B) Quantitative fluorescence intensities in apoptotic HeLa cells in (A).

The regression equation is F = 4356.65[casp-8] +460.28 and the line-ar coefficient is 0.9946. The limit of detection was 0.326 ng·mL-1. Likewise, Figure 3F shows good proportional relationships between the concentrations of casp-9 and the recovered fluorescence signals of FITC, where the linear

response range was from 0 to 300 ng·mL-1 with a detection limit of 0.218 ng·mL-1. The reported endogenous concentrations of casp-3, 8 and 9 are approximately 100, 50 and 20 nM37-40 and the above calculated values of limit of



detection in molarity are all below 0.1 nM, which are enough for the detection of target caspase in living cells and in vivo. In addition, to further verify the precision of the response of GNP-Se-Casp toward caspases, the inhibitors of the targeted caspases were introduced to the mixture of the nanoprobe and caspases. The corresponding results are shown in Figure 4A-C. The recovered fluorescence signals of fluorescent dyes in GNP-Se-Casp is weakened dramatically by the specific inhibitors of caspases, confirming the excellent sensitivity and specificity of GNP-Se-Casp toward targeted caspases. Then, to assess the selectivity of the nanoprobe, GNP-Se-Casp was treated with several important proteins, including BSA, HSA, pepsin, MMP-2, trypsin, uPA and lysozyme, under identical conditions. As shown in Figure 4D to 4F, GNP-Se-Casp produced the highest fluorescence response toward the targeted caspase among the analytes. In contrast, other interfering proteins or enzymes did not cause obvious fluorescence recovery. Moreover, considering that GSH is a potential interfering species, the fluorescence response of GNP-Se-Casp to GSH was studied. The results indicated that GSH did not induce remarkable fluorescence change in the detection system. The presence of GSH on the detection of fluorescence signals was further examined. Under simulated physiological conditions, the mixture of recombinant casp-3, 8 and 9 protein (300 ng·mL-1) and GSH was added to the GNPSe-Casp solution. In Figure S4, the background signal of GNP-Se-Casp was stable in the presence of GSH (5 mM), and the changes in the fluorescent intensity of the GNP-Se-Casp solution triggered by targeted caspase was similar to that in the absence of GSH. The results showed that physiological levels of GSH could not affect the detection sensitivity of GNP-SeCasp. Collectively, GNP-Se-Casp was ideal for the detection of the caspase cascade with high selectivity and suitability for application in complicated biological systems. Fluorescence Imaging of Caspase Cascade Activation in Cancer Cells. The potential application of the probe for imaging caspase cascade activation in living and apoptotic cells was further explored. Prior to intracellular usage, the cell cytotoxicity of GNP-Se-Casp was first evaluated by measuring cell viability with the widely used MTT assay in HeLa cells and HepG2 cells. As shown in Figure S5, the cell viabilities were over 90% after incubation with GNP-Se-Casp (1 nM) for up to 24 h, indicative of the low cytotoxicity and the good biocompatibility of the nanoprobe, supporting the promising use of the nanoprobe for imaging in living and apoptotic cells. Then, fluorescence microscopy was used to image the apoptotic cancer cells with GNP-Se-Casp. HeLa cells were seeded in a confocal dish for 24 h, and then incubated with GNP-Se-Casp in DMEM for 2 h at 37 °C followed by treatment with STS, a widely used apoptosis inducer. The HeLa cells were then studied with confocal fluorescence imaging. The optimized concentration of STS was 5 μM with an incubation time of 4 h (Figure S6). As shown in Figure 5, after the cells were treated with STS, strong fluorescence signals in the red, blue and green channels were observed. The increase in fluorescence signals was attributed to the activation of casp-3, 8 and 9 in apoptotic cells induced by STS. Similar fluorescence enhancement phenomena were also observed in HepG2 cells and murine B16-F10 melanoma cells cells (Figure S7). The initiator role of target caspase in the activation of the caspase cascade was confirmed by inhibitor treatment. The HeLa cells were

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pretreated with target caspase inhibitors before STS-treatment. After the HeLa cells were treated with casp-3 inhibitor, the blue fluorescence of Cy5 and the green fluorescence of FITC were visible, while the red fluorescence of 5-TAMRA disappeared, indicating that the inhibitory effect of Z-DEVDFMK on casp-3 did not affect the activation of casp-8 and casp-9. In contrast, after the HeLa cells were pretreated with Z-IETD-FMK (casp-8 inhibitor) or Z-LEHD-FMK (casp-9 inhibitor), the red fluorescence of 5-TAMRA corresponding to casp-3 decreased by a certain extent. As casp-8 and casp-9 were inhibited at the same time, the fluorescence intensity of casp-3 decreased notably. Thus, the activation of casp-3 depended on casp-8 and casp-9, which demonstrated that the apoptotic process in the HeLa cells stimulated with STS involved both the extrinsic apoptotic pathway and the intrinsic apoptotic pathway. Furthermore, immunofluorescence imaging was carried out by using anti-caspase antibodies to verify the specificity of GNP-Se-Casp (Figure S8). Excellent overlap was observed between the fluorescence signals and the immunofluorescence signals generated from a corresponding anti-caspase primary antibody and secondary antibody. Thus, these results validate the high selectivity and precision of GNP-Se-Casp for imaging cell apoptosis. Real-time Monitoring the Evolution of the Caspase Cascade in HeLa cells. Inspired by the excellent performance of GNP-Se-Casp in the fluorescence imaging of the caspase cascade activation in cancer cells, the use of GNP-Se-Casp for real-time monitoring the evolution of the caspase cascade was investigated. First, nanoprobes for the single detection of casp9 based on Au-Se and Au-S bonds developed previously in our group35 were used to inspect the interference of intracellular thiols on real-time fluorescence imaging. The HeLa cells were pretreated with N-ethylmaleimide (NEM, an irreversible thiol scavenger) before STS-treatment, followed by incubation with the Au-Se bonding nanoprobe and the Au-S bonding nanoprobe for casp-9. The CLSM results are shown in Figure S9. In the HeLa cells incubated with the Au-Se bonding nanoprobe, fluorescence signals with almost the same intensity were observed regardless of whether cells were pre-treated with NEM. By comparison, in the HeLa cells incubated with the Au-S bonding nanoprobe, due to the scavenging of the intracellular thiols, the fluorescence intensity sharply weakened after the pretreatment with NEM. As a result, the high-level biothiols in cancer cells showed little interference when the Au-Se bonding nanoprobe was used for the specific detection of casp-9. Thus, the Au-Se bonding nanoprobe was further used to real-time monitor the activation of casp-9 during the apoptosis process in HeLa cells. As the apoptotic pathway was induced by treatment with STS, a quite weak fluorescence signal was observed until 45 min and then gradually increased as time lapsed (Figures 6A and 6C). In contrast, when the Au-S bonding nanoprobe was used in the control group, fluorescence intensity was strong at beginning, and the gradual change in the fluorescence signal over time was not obvious (Figure 6B and 6D). As a result, the use of the Au-S bonding nanoprobe would cause a high fluorescence background and a false positive result, whereas the Au-Se bonding nanoprobe brought out a high-fidelity fluorescent signal in favor of the real-time application.


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Figure 6. Real-time monitoring of casp-9 in HeLa cells incubated with the Au-Se bonding nanoprobe (A) and the Au-S bonding nanoprobe (B) for 4 h and followed by treatment with STS (5 μM). The concentration of nanoprobe was 1 nM. Green fluorescence at λex/em of 488/500–600 nm, Hoechst at λex/em of 405/430–480 nm. Scale bar=50 μm. (C) Quantitative fluorescence intensities in Hela cells incubated with the Au-Se bonding nanoprobe in (A). (D) Quantitative fluorescence intensities in Hela cells incubated with the Au-S bonding nanoprobe in (B).

Based on the above results, GNP-Se-Casp was used to real time monitor the activation of the caspase cascade during HeLa cell apoptosis, and the evolution of casp-3, 8 and 9 was observed. HeLa cells were first incubated with GNP-Se-Casp at 37 °C. After 4 h of incubation, the cells were treated with STS and then were observed with CLSM (Figure 7A) and the quantitative data on fluorescent intensities were shown in Figure 7B-7D. No fluorescence in the background was observed in the cells before stimulation with STS. After incubation for 30 min, the fluorescence of Cy5 (blue, casp-8) in HeLa cells first appeared, and increased gradually with the progression of cell apoptosis, reaching the maximum intensity after 90 min. As the incubation time elapsed, FITC fluorescence (green, casp-9) was observed after incubation for 45 min, reaching a maximum at 120 min. The time-dependent fluorescence signal of 5-TAMRA associated with the activation of casp-3 was not detectable until 60 min of STS treatment, and then the signal gradually increased along with the cellular apoptotic progress. In accordance with the inhibitor treatment results, these observations indicated that casp-8 and casp-9, initiators in the caspase cascade, were first activated in turn in the initial stage of cell apoptosis and initiated the apoptotic progress both in the extrinsic apoptotic pathway and the intrinsic apoptotic pathway. As the effector of apoptosis, casp-3 was finally activated to show the fluorescence of 5-TAMRA as the degree of cell apoptosis deepened. Notably, as the bright field images showed, apoptotic HeLa cells showed obvious shrinkage after incubation for 60 min, revealing the progress of apoptosis. These results clearly demonstrate that GNP-Se-Casp not only could be used for sensing the activation of the caspase cascade

but also be applied to real time monitor the evolution of the caspase cascade in cancer cells. CONCLUSIONS In conclusion, a multi-color nanoprobe GNP-Se-Casp was developed based on Au-Se bond to monitor the sequential activation of casp-3, 8 and 9 real time. GNP-Se-Casp exhibited high sensitivity and precision, free from the interference of biothiol and successfully used for visualizing the upstream and downstream activation of casp-3, 8 and 9 in apoptotic tumor cells. During the apoptosis process of HeLa cells, the upstream casp-8 and casp-9 could activate downstream casp-3, indicating that the apoptosis process was carried out by both the extrinsic and the intrinsic apoptotic pathways. Collectively, GNP-Se-Casp is a promising tool for the study of cell apoptosis and offers a new strategy for the real-time investigation of the complicated upstream and downstream relationship about other signal molecules in signalling pathways.



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Figure 7.(A) Real-time fluorescence imaging of the cell apoptosis process in HeLa cells incubated with GNP-Se-Casp (1 nM) for 4 h and followed by treatment with STS (5 μM). Scale bar=50 μm. Red fluorescence (casp-3) at λex/em of 561/570–700 nm, blue fluorescence (casp8) at λex/em of 633/650–750 nm, green fluorescence (casp-9) at λex/em of 488/500–600 nm, Hoechst at λex/em of 405/430–480 nm. Scale bar=50 μm. (B) Quantitative fluorescence intensities of casp-3 in Hela cells as time lapsed in (A). (C) Quantitative fluorescence intensities of casp-8 in Hela cells as time lapsed in (A). (D) Quantitative fluorescence intensities of casp-9 in Hela cells as time lapsed in (A).

ASSOCIATED CONTENT

* Email: [email protected]; [email protected]

Supporting Information. Experimental procedures and additional data (Materials and instruments, TEM image, Quantitation of three kinds of peptide chains loaded on GNP-SeCasp, MTT assay, Quantification results of fluorescence intensities in real-time CLSM imaging). The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

Author Contributions

AUTHOR INFORMATION

ACKNOWLEDGMENT

Corresponding Author

† These authors contributed equally. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

This work was supported by the National Natural Science Foundation of China (21575081, 21535004, 91753111 and


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21775091) and the Key Research and Development Program of Shandong Province (2018YFJH0502)

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As for Table of Contents Only


Real-time in situ Visualizing the

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