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Phospholipid-Tailored Titanium Carbide Nanosheets as a Novel Fluorescent Nanoprobe for Activity Assay and Imaging of Phospholipase D Xiaohua Zhu, Lin Fan, Shigong Wang, Chunyang Lei, Yan Huang, Zhou Nie, and Shouzhuo Yao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00581 • Publication Date (Web): 08 May 2018 Downloaded from http://pubs.acs.org on May 8, 2018

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Analytical Chemistry

Phospholipid-Tailored Titanium Carbide Nanosheets as a Novel Fluorescent Nanoprobe for Activity Assay and Imaging of Phospholipase D Xiaohua Zhu, Lin Fan, Shigong Wang, Chunyang Lei*, Yan Huang, Zhou Nie*, Shouzhuo Yao

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China

*to whom corresponding should be addressed. Tel: +86-731-88821626; Fax: +86-731-88821848 Email: [email protected]; [email protected].

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ABSTRACT: As one of emerging inorganic graphene analogues, two-dimensional titanium carbide (Ti3C2) nanosheets have attracted extensive attention in recent years due to their remarkably structural and electronic properties. Herein, a sensitive and selective nanoprobe for fluorescent probing the phospholipase D activity was developed based on ultrathin Ti3C2 nanosheets-mediated fluorescence quenching effect. Ultrathin Ti3C2 nanosheets with ~1.3 nm in thickness were synthesized from bulk Ti3AlC2 powder by a two-step exfoliation procedure, and further modified by natural

phospholipid

that

doped

with

rhodamine

B-labeled

phospholipid

(RhB-PL-Ti3C2). The close proximity between RhB and Ti3C2 leads to efficient fluorescence quenching (> 95%) of RhB by energy transfer. Phospholipase D-catalyzed lipolysis of the phosphodiester bond in RhB-PL results in RhB moving away from the surface of Ti3C2 nanosheets and subsequent fluorescence recovery of RhB, providing a fluorescent “switch-on” assay for the phospholipase D activity. The proposed nanoprobe was successfully applied to quantitative determine phospholipase D activity with a low limit of detection (0.10 U L-1), and for its inhibition measurement. Moreover, in situ monitoring and imaging the activity of phospholipase D in living cells were achieved using this biocompatible nanoprobe. These results reveal that Ti3C2 nanosheets-based probes exhibit great potential in fluorometric assay and clinical diagnostic applications.

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INTRODUCTION Two-dimension (2D) nanomaterials have received increasing interest on the development of biosensing devices in recent years because of their uniquely physical and chemical properties.1,2 Up to now, a variety of 2D nanomaterials, such as graphene,3 graphene oxide (GO),4 transition metal dichalcogenides,5,6 and metal oxides7,8 have been investigated. More recently, transition metal carbides (MXenes), a new family of multifunctional 2D nanomaterials with exceptionally optical and electric properties, have been developed by Gogotsi and colleague.9,10 Compared to traditional 2D nanomaterials, MXenes are structurally diverse, and have shown more improved properties with superior conductivity (2400 S cm−1) and high surface area (98 m2 g−1).11,12 The superior properties of 2D MXenes make them promising candidates for energy storage12 and environmental applications.13 In addition, the MXenes nanosheets generally show relatively high sensitivity and environmentally friendly characteristics in applications such as highly selective sensors.14 For instance, the ultrathin Ti3C2 nanosheets can be utilized to fabricate field-effect transistors for sensitive detection of dopamine, and to monitor spiking activity in hippocampal neurons.15 Particularly, the ultrathin Ti3C2 nanosheets show a forceful and broad absorption from 400 to 1200 nm, which enables the material to be an excellent quencher of electronic excited states of dyes through fluorescence resonance energy transfer (FRET). Therefore, it is possible to realize sensitively fluorescent sensing using Ti3C2 nanosheets as fluorescence quenchers. However, there has been little research reported on Ti3C2 nanosheets-based fluorescent sensors. 3



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On the other hand, the development of highly sensitive and robust assays for detecting enzyme activity is of critical importance for clinical diagnostics and drug screening in biomedicine.16,17 Phospholipase D belongs to the lipolysis enzyme subclass that degrades phospholipids into phosphatidic acid and choline.18 Phospholipase D-mediated synthesis of phosphatidic acid can cause modifications to membrane curvature, vesicle trafficking, activation of protein kinases. These changes ultimately lead to diversely cytological behaviors, including cell growth, division, and migration.19 Moreover, the abnormalities in phospholipase D expression and activity have been considered as an indicator of many diseases, such as several cancers, thrombotic and neurodegenerative diseases.20 Furthermore, phospholipase D has been regarded as a potentially diagnostic biomarker of cancers, and a potential target for drug development.21 As a result, rapid and sensitive assessment of phospholipase D activity and screening of its inhibitors will benefit biochemical research and pharmaceutical development. Currently, conventional assays of phospholipase D activity rely on radiolabeled phospholipids methods,22 making it versatile and appropriate for both cellular and acellular uses. However, the radioactive wastes, multistep procedures, and intricate labeled substrates restrict their widespread applications. To overcome this deficiency, several approaches have been exploited for the determination of phospholipase D activity,

such

as

chromatography,23,24

mass

spectrometry,25,26

infrared

spectroscopy,27,28 conductimetry,29 electrophoretic-electroosmotic focusing,30 and fluorescence spectroscopy.31,32 Among them, fluorometric method possesses the 4


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advantages of high sensitivity, fast analysis and good reproducibility, and has attracted extensive attentions.33,34 For example, Ferguson et al35 reported the synthesis and validation of two fluorogenic phospholipid substrates for activity assay. Abousalham and colleague36 reported a fluorescent method for the detection of phospholipase D activity in plant and bacterial origin. However, this method con not image phospholipase D activity in living cells. Bumpus and Baskin37,38 established an approach for imaging of phospholipase D-mediated phosphatidic acid synthesis based on click chemistry, but multiple washing and incubating cannot avoid. Chu’s group39 developed a phospholipid-coated upconversion nanoparticles (UCNPs)-based probe for ratiometric fluorescent sensing and bioimaging of phospholipase D activity. Nevertheless, the potential toxicity of UCNPs is still an issue for both in vivo and in vitro applications.40 Accordingly, the development of a new sensing platform for enzyme-coupled assays is highly desirable to facilitate routine analysis. Herein, we presented a proof-of-concept demonstrating the application of Ti3C2 nanosheets as a novel fluorescence-based enzyme activity detection platform. The nanoprobe was prepared by a one-step self-assembly of rhodamine B-labeled phospholipids onto the Ti3C2 surface. The Ti3C2 nanosheets as energy acceptor can quench the fluorescence of rhodamine B (RhB)-labeled phospholipid via FRET. Phospholipase D-catalyzed cleavage of the phosphodiester bonds in RhB-labeled phospholipids leads to the RhB moving away from the surface of Ti3C2 nanosheets, and subsequent the prohibition of FRET process. As a result, the fluorescence intensity of RhB is restored and provides a facile “turn-on” assay for the detection of 5



phospholipase D activity. In addition, the determination and imaging of phospholipase D activity using this nanoprobes in cell lysates and living cells were also achieved. This work demonstrates that Ti3C2 nanosheets can be used as a novel biosensing platform, which will inspire the exploration of more widespread applications of engineered nanomaterials in bioanalytical chemistry. EXPERIMENTAL SECTION Chemicals. The Ti3AlC2 starting material was purchased from Beijing Forsman Scientific. Hydrofluoric acid (HF) and tetramethylammonium hydroxide (TMAOH) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Phospholipids

with

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine

rhodamine B sulfonyl) (PE-RhB) was obtained from Avanti Polar Lipids. GO was purchased from XF Nano (Nanjing, China). Phospholipase D from Streptomyces chromofuscus was obtained from Merck (Darmstadt, Germany). 5-Fluoro-2-indolyl des-chlorohalo-pemide (FIPI) was from MedChem Express (New Jersey, USA). Human serum albumin (HSA), L-glutathione (GSH), hemoglobin (Hb), trypsin, lysozyme, glucose, and all the amino acids were purchased from Aladdin Chemistry Co. Ltd. (Shanghai, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), Acetylcholinesterase (AChE, from Electrophorus electricus), matrix metalloproteinase-2 (MMP-2), and thrombin were purchased from Sigma-Aldrich (Shanghai, China). Alkaline phosphatase (ALP) were purchased from Sangon Biotechnology Inc. (Shanghai, China). Cell Counting Kit-8 (CCK-8) was obtained from Beyotime Biotechnology (Shanghai, China). All chemicals are analytical grade. 6


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All solutions were prepared using ultrapure water (18.2 MΩ·cm) from Millipore (Milli-Q) system. Preparation of Ti3C2 nanosheets. Ti3C2 nanosheets were synthesized according to a previous report with little modification.41 Briefly, Ti3AlC2 (0.5 g) powder was pretreated by immersing in diluted HF (20%) at a concentration of 0.1 g mL-1for 30 min to clean surfaces oxidant. The obtained powder was then rinsed three times with ultrapure water. Then, the cleaned powder was dispersed in 25% aqueous TMAOH to deintercalate the gallery Al and simultaneously intercalate Al(OH)4- along with TMA+. The TMA- intercalated powder was finally re-dispersed in 300 mL H2O, and shaken repeatedly for 10 min to achieve complete delamination. Then, the Ti3C2 nanosheets were collected by centrifugation. After being washed by distilled water for more than 5 times, ultrapure water was added to obtain the stock solution of Ti3C2 nanosheets. Atomic force microscope (AFM) images were performed on a Multimode 8 AFM (Bioscope system, Brucker) with tapping mode. X-ray photoelectron spectroscopy (XPS) was performed on an ESCALAB 250 photoelectron spectrometer (Thermo Fisher Scientific, USA) with Al Kα (1486.6 eV) as the X-ray source. Fluorescence experiments were carried out using a QuantaMaster fluorescence spectrophotometer (PTI, Canada). Preparation of phospholipid modified Ti2C3 (PL-Ti2C3). Red blood cells are the most abundant type of blood cells, lacking a nucleus and most organelles, and their plasma membranes are highly biocompatible.42 Therefore, phospholipid (PL) 7



derived from red blood cells was used to stabilize Ti2C3 based on previously described protocols.43 Detailed procedures are shown in Supporting Information. PE-RhB was dissolved in chloroform (300 µL, 0.1 mg mL-1) and subsequently evaporated in a glass vial to form a thin film. Solution containing PL was then added to the vial and stirred at 37 °C for 50 min. After that, the free dye was washed away by centrifuging the natural phospholipid at 21000 × g for 15 min four times. A mixture of Ti2C3 nanosheets and natural phospholipid (2:1, w/w) was then sonicated for 10 min to form the PL modified Ti2C3 (denoted as RhB-PL-Ti2C3). The excess PL was removed by centrifugation, and RhB-PL-Ti2C3 was re-dispersed in water for future use. Determination of phospholipase D activity in vitro. In a typical in virto assay, RhB-PL-Ti2C3 solution (20 µL, 0.1 mg mL−1) was added into 20 µL of reaction buffer (25 mM Tris-HCl, 12.5 mM CaCl2 and pH 8.0). Then, 10 µL of phospholipase D solution at different final concentrations ranging from 0 to 1000 U L−1was added and incubated at 37 °C for 1 h. After that, 50 µL of ultrapure water was added to a final volume of 100 µL. For the selectivity of phospholipase D detection, the potential interferents were used in place of phospholipase D under the same experimental condition. Subsequently, the fluorescence spectra of the mixture were recorded (excited at 560 nm). Fluorescence imaging of phospholipase D activity in living cells. Human breast adenocarcinoma (MDA-MB-231, high aggressive human breast cancer; and MCF-7, low aggressive human breast cancer) cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium ) supplemented with fetal bovine serum(10%, v/v), 100 U 8


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mL−1 penicillin, and 100 µg mL−1 streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C. These cells were used in fluorescence imaging experiments. In detailed procedures, the RhB-PL-Ti2C3 (0.1 mg mL−1) were added to the cell culture, and the cells were incubated for another 4 h at 37 °C. The cells were washed with PBS three times before observation under a fluorescence microscope in PBS. Stained cells were examined using a confocal laser scanning microscope (CLSM, C1-Si, Nikon, Japan) and the images of the maximum intensity z-projection are shown. The mean gray value of each single cell was determined by Image J (NIH) and used as a data point. RESULTS AND DISCUSSION Preparation and Characterization of PL-Ti3C2. The ultrathin Ti3C2 nanosheets were synthesized from the bulk Ti3AlC2 powder by a two-step exfoliation procedure, including initial HF etching and subsequent TMAOH intercalation (Figure 1A). Then the as prepared material was analyzed by TEM, and well-defined ultrathin sheet-like structures were observed (Figure S1, Supporting Information). To confirm the single layer formation through exfoliation, the nanosheets were characterized by AFM on mica. As shown in Figure 1B, the thickness of Ti3C2 nanosheets is about 1.3 nm. The real thickness of a monolayer Ti3C2 nanosheets is slightly larger than its theoretical one, which is caused by the surface absorption of O or OH after exfoliation.41,44 The formation of Ti3C2 nanosheets was also confirmed by X-ray diffraction (XRD). After TMAOH intercalation, the most intense XRD peak of Ti3AlC2 (2θ ≈ 38°) disappeared, and the characteristic periodic peaks originated from 9



stacked Ti3C2 layers appeared instead45 (Figure S2, Supporting Information). In X-ray photoelectron spectroscopy (XPS), Ti−C (2p3) and Ti−O (2p3) doublets at 455.9 eV and 458.7 eV were identified, confirming the chemical composition of Ti3C2 nanosheets (Figure S3, Supporting Information). Taken together, the above results demonstrated the successful synthesis of Ti3C2 nanosheets. Interestingly, the Ti3C2 nanosheets showed a broad absorption ranging from 400 to 1000 nm with a peak absorption at approximately 815 nm (Figure S4, Supporting Information), implying that Ti3C2 nanosheets can be employed as a versatile quencher for various fluorophores. The prepared Ti3C2 nanosheets can be well dispersed in water for up to several months without apparent aggregation, but they tend to aggregate in saline solution (Figure S5, Supporting Information). Therefore, surface modification of Ti3C2 nanosheets is required prior to be exploited in bioanalytical applications. Previous studies have suggested that the surface of Ti3C2 nanosheets can bind phospholipid molecules.44,46 To further improve the stability and biocompatibility, the surface of Ti3C2 nanosheets was modified by natural phospholipids (PL) derived from human red blood cells. The PL modified Ti3C2 nanosheets (designated as PL-Ti3C2) exhibited good dispersity in saline solution (Figure S5, Supporting Information) and kept the sheet-based morphology (Figure S6, Supporting Information). After surface modifying, the PL-Ti2C3 nanosheets gave a topological height over 9 nm. Since the thickness of red blood cell membrane was 3~5 nm,47 the assembly of PL was supposed to form a bilayer structure on each surface of Ti3C2 nanosheets (Figure 1C). 10


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Moreover, after the PL modification, the surface zeta potential of Ti3C2 nanosheets increased from -11.2 to -16.7 mV, which is comparable to that of PL (Figure S7, Supporting Information). In addition, as shown in Figure 1D, the new P 2p peak in the XPS spectra of PL-Ti2C3 nanosheets at binding energy 133.2 ev is characteristic of the phosphate moiety of the phosphor-choline head group, in agreement with previously reported values.48 These results further confirm the successful modification of Ti3C2 nanosheets with PL. FRET between RhB-labeled phospholipid and Ti3C2 nanosheets. Owing to the broad-spectrum absorption, ultrathin 2D nanosheets (e. g. graphene,49 MoS2,50 and MnO2 nanosheets,51) can efficiently quench the fluorescence emission of nearby dye molecules through energy transfer, and have been extensively exploited to fabricate fluorescent sensors for biomolecules assay. Herein, the fluorescence quenching effect of Ti2C3 nanosheets was evaluated. To test the fluorescence quenching efficiency, the PL was doped with an RhB fluorescent dye labelled PL (RhB-PL, Figure S8, Supporting Information), and a nanoassembly of RhB doped PL modified Ti2C3 nanosheets (RhB-PL-Ti2C3) was prepared (Figure 2A). The fluorescence quenching capacity of Ti2C3 nanosheets was evaluated by fluorescence spectra. As shown in Figure 2B, the fluorescence intensity of RhB decreased obviously after the Ti3C2 nanosheets were added into the RhB-PL (~ 1.0 mg mL-1). About 90% fluorescence was quenched by 0.1 mg mL-1 of Ti3C2 nanosheets. This phenomenon indicated that the Ti2C3 nanosheets can be used as an excellent quencher for constructing 11



nanoprobes. The interactions of PL with Ti3C2 nanosheets enable the close proximity of RhB with Ti3C2 nanosheets and the efficient fluorescence quenching of the dyes through efficient FRET. Moreover, the fluorescence quenching of RhB-PL by GO (the typical 2D nanosheets quencher) was performed under the sample experimental conditions. As shown in Figure 2C, the fluorescence quenching efficiency of GO at the concentration of 0.1 mg mL-1 was 67.3%, and more than 0.2 mg mL-1 of GO were required to reach the high quenching efficiency of 90%. Therefore, compared to GO, Ti3C2 nanosheets possess a much stronger fluorescence quenching capacity, which can be partially attributed the fact that the mass extinction coefficients of Ti3C2 nanosheets are greater than these of GO in the wavelength range of 400-1000 nm (Figure S9, Supporting Information). Moreover, the RhB-PL failed to form supported bilayer on GO surface, and was adsorbed as intact liposomes might also lead to the lower quenching efficiency of GO.52,53 Having confirmed the fluorescence intensity of RhB can be effectively quenched by Ti3C2 nanosheets, the RhB-PL-Ti2C3 was separated by centrifugation and re-dispersed in water for future usage. After removing the excess RhB-PL, the quenching efficiency of Ti3C2 nanosheets toward RhB-PL reached more than 95% (Figures S10, Supporting Information). In addition, two absorption bands at 408 nm and 578 nm were observed in the UV-vis spectra of the final RhB-PL-Ti2C3, which corresponded well to that of PL54 and RhB, respectively, suggesting the successful RhB-PL membrane modification (Figures S11, Supporting Information). 12


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Fluorescence recovery of RhB-PL-Ti2C3 induced by phospholipase D. Considering the phospholipase D can catalyze the hydrolysis of phosphatidylcholine into choline and phosphatidic acid, a “turn-on” fluorescent method for phospholipase D activity assay was proposed. Figure 3A illustrated the analytical principle of our nanoprobe for the activity assay of phospholipase D. In the presence of active phospholipase D, the phosphodiester bonds were catalytically cleaved, which released the RhB labels apart from the Ti3C2 nanosheets surface, accompanied by the recovery of their fluorescence emission. To test the feasibility of the proposed assay, RhB-PL-Ti2C3 were incubated with phospholipase D (500 U L-1) for 1 h. In comparison to the control sample (RhB-PL-Ti2C3 nanoprobes), phospholipase D-catalyzed reaction induced a significant fluorescence enhancement, which is 20.4 folds as high as that of the control (Figure 3B, curve b). In contrast, a high concentration of human serum albumin (HSA, 5 mg mL-1) caused no obvious fluorescence changes (Figure 3B, curve c). In addition, when the probe was incubated with both phospholipase D (500 U L-1) and its inhibitor (FIPI, 0.1 mg mL-1),55 a much weaker fluorescence response was obtained (Figure 3B, curve d). In addition, the fluorescence anisotropy was measured to verify whether the activated fluorescence was due to the release of the RhB labels. Anisotropy value of RhB was decreased from 0.18 to 0.05 after the phospholipase D-catalyzed reaction (Figure S12, Supporting Information), indicating the release of labelled RhB from nanoprobes to solution.33 These results indicate that the ﬂuorescence response of the probe can be attributed to the cleavage reaction catalyzed by phospholipase D. 13



Moreover, time-dependent fluorescence responses at 583 nm were recorded at different concentrations of phospholipase D for real time monitoring phospholipase D-catalyzed hydrolysis reaction. As shown in Figure 3C, the reaction rate continuously increased as an increase in the concentration of phospholipase D, which could offer insight into the kinetics of the hydrolysis reactions through real-time monitoring of the fluorescence activation responses. Detection of Phospholipase D activity using RhB-PL-Ti2C3 nanoprobes. The ability of the RhB-PL-Ti2C3 for quantitatively determining the activity of phospholipase D was evaluated. Figure 4A displayed typical fluorescence responses of the RhB-PL-Ti2C3 toward phospholipase D at different concentrations. Fluorescence emission at 583 nm was continually increasing as the concentration of phospholipase D increased from 0.5 to 1000 U L−1. The plot of fluorescence response (F/F0) versus the concentration of phospholipase D was shown in Figure 4B, and the fluorescence response reached a plateau point at 600 U L−1 of phospholipase D. A good linear range of 0.5−50 U L−1 was obtained with a limit of detection of 0.1 U L−1 (S/N = 3). The comparison of the analytical performance for phospholipase D activity assay between the proposed method and several previously reported methods is summarized in Table S2, Supporting Information. These results implied that the proposed nanoprobe provided a desirably sensitive and robust method for activity assay of phospholipase D.

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Furthermore, the specificity of the nanoprobe toward phospholipase D was investigated. Aliquots of RhB-PL-Ti2C3 solution was incubated with potential interferents including glucose, amino acids, glutathione (GSH), human serum albumin (HSA) trypsin, , acetylcholinesterase (AChE), lysozyme, matrix metalloproteinase-2 (MMP-2), thrombin, alkaline phosphatase (ALP), and fetal bovine serum (FBS). As shown in Figure 5A, fluorescence of the nanoprobe showed negligible changes for these interferents, while a signiﬁcant fluorescence enhancement was observed for phospholipase D. The values of F/F0 of these interferences were almost the same as the blank, which clearly suggests that they did not interfere with our assay. And the feasibility of the proposed nanoprobe for enzyme-inhibitor assay was tested. We incubated RhB-PL-Ti2C3 with phospholipase D (400 U L−1) in the presence of FIPI at different concentrations (from 10−6 to 1 mg mL−1) to obtain the dose-dependent inhibition curve (Figure 5B). From the inhibition curve, IC50 value of FIPI toward phospholipase D (400 U L−1) was calculated to be 1.58 µg mL-1 (3.75 µM). These results suggest that the RhB-PL-Ti2C3 nanoprobes hold great potential in the application of screening inhibitors for phospholipase D. Application of RhB-PL-Ti2C3 for cell lysates detection and living cell imaging. Combined with the good properties of excellent selectivity, high sensitivity, and low detection limit of the proposed sensing system, the application of the RhB-PL-Ti2C3 nanoprobes for phospholipase D detection in biological fluids was evaluated. MDA-MB-231 cells that have high phospholipase D activity, and MCF-7 cell with low phospholipase D activity were chosen as the model cells.56 Cell lysates of the two 15



cell lines containing 3.0 mg mL-1 total protein were tested by RhB-PL-Ti2C3 nanoprobes. As depicted in Figure S13, Supporting Information, fluorescence enhancements induced by MDA-MB-231 cells and MCF-7 cells were 3.4 and 1.3 folds, respectively. Phospholipase D activities in MDA-MB-231 and MCF-7 cell lysates were calculated to be 6.9 ± 0.3 and 0.8 ± 0.2 U L−1, respectively, which are consist well with the different phospholipase D expression levels in the two cell lines.56 Real-time imaging of target enzymes in living cells is crucial to better understand their performances in both physiological and pathological processes. Therefore, the intracellular imaging of phospholipase D using the RhB-PL-Ti2C3 nanoprobes was also explored. MDA-MB-231 cells was further used for living cell bioimaging because it is a specific cell line with high expression of phospholipase D. Before the experiments, the cytotoxicity of the RhB-PL-Ti2C3 nanoprobes were studied by the MTT and CCK-8 assays. RhB-PL-Ti2C3 nanoprobes were proven to show no significant apparent cytotoxicity to MDA-MB-231 cells at a high concentration of 200 µg mL−1 (Figure S14, Supporting Information). Then, MDA-MB-231 cells and MCF-7 cells were incubated with RhB-PL-Ti2C3 (50 µg mL−1) at 37 °C for 4 h, and analyzed by CSLM. As shown in Figure 6A, strong red fluorescence can be detected in the cytoplasm. This phenomenon could be attributed that the nanoprobes delivered into cytosol were hydrolyzed by intracellular phospholipase D of MDA-MB-231 cells. Phospholipase D-mediated hydrolysis results in the release of RhB from the nanoprobes, along with the dramatic enhancement of red fluorescence in the 16


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cytoplasm. In contrast, only slightly red fluorescence was observed in the cytosol of MCF-7 cells. As the control, a remarkable decrease (decreased 79.2%) in fluorescence intensity was observed in the same cells treated by phospholipase D inhibitor (FIPI, 0.1 mg mL-1,). The average fluorescence intensity in MDA-MB-231 cells is 10.2 times as that in MCF-7 cells (Figure 6B), which is roughly consistent with the quantification data of phospholipase D mRNA by real-time quantitative reverse transcription PCR (Figure 6C), as well as the previously reported difference in phospholipase D activity of the two cell lines.56 Such promising imaging results demonstrate that the RhB-PL-Ti2C3 probe could employed as an effective tracer to report the phospholipase D activity in living cells. CONCLUSIONS In summary, we have demonstrated that a novel two-dimension Ti3C2 nanosheets can be used as a platform for fast, sensitive, and selective sensing and imaging of phospholipase D activity. The proposed nanoprobe was prepared by self-assembly of natural phospholipids and RhB-labeled phospholipids onto the surface of Ti3C2 nanosheets. Through inhibition of the FRET between RhB and Ti3C2 by phospholipase D-catalyzed phosphodiester bond cleavage, the RhB-PL-Ti2C3 nanoprobes could be used for fluorogenic detection of phospholipase D activity. The developed nanoprobe can be readily applied for the rapid and sensitive detection of phospholipase D activity in cell lysates, as well as in situ activity imaging in living cells, exhibiting great opportunities for practical applications in fundamental biological research and clinical diagnosis fields. On the basis of their excellent 17



performance, this novel 2D Ti3C2 nanomaterial also provides a promising platform for the optical sensing applications. Acknowledgments This research was financially supported by National Natural Science Foundation of China (Nos. 21725503, 21575038, 21505120 and 21475038), the Foundation for Innovative Research Groups of NSFC (Grant 21521063), the Young Top-notch Talent for Ten Thousand Talent Program, the Fundamental Research Funds for the Central Universities and the Hunan Provincial Innovation Foundation for Postgraduate (CX2016B117). Notes The authors declare no competing financial interest. Supporting Information Available Additional supplementary material, including the description of extensive method and figures. This material is available free of charge on the ACS Publications website.

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FIGURE CAPTIONS Figure 1. (A) Schematic illustration of the intercalation and delamination processes. AFM images and depth profiles of Ti3C2 nanosheets (B) and PL-Ti3C2 (C). (D) XPS P2p spectra of the Ti3C2 nanosheets and PL-Ti3C2.

Figure 2. (A) Structure of assembled RhB-PL on Ti3C2 nanosheets surfaces. (B) Fluorescence spectra of RhB-PL solution in the absence and presence of Ti3C2 nanosheets (1.0 mg mL-1). (C) Fluorescence spectra of RhB-PL in the presence of various concentrations of GO.

Figure 3. (A) Analytical principle of the biosensor for activity assay of phospholipase D. (B) Fluorescence spectra of RhB-PL-Ti2C3 before (curve a) and after incubation with 500 U L−1 phospholipase D (curve b), 50 mg mL−1 HSA (curve c), and denatured phospholipase D (curve d). (C)Time-dependent fluorescence recovery curves at 580 nm obtained for phospholipase D of 0, 10, 30, 100, and 500 U L−1 at 37 °C.

Figure 4. (A) Fluorescence response of RhB-PL-Ti2C3 upon addition of various concentrations of phospholipase D (from bottom to top: 0, 0.5, 1.0, 2.0, 3.0, 5.0, 10.0, 20.0, 30.0, 50.0, 100.0, 200.0, 400.0, 600.0, 800.0 and 1000.0 U L−1). (B) Plot of F/F0 of RhB-PL-Ti2C3 vs. the concentration of phospholipase D. F0 and F represent the fluorescence intensity of RhB-PL-Ti2C3 probes in the absence and presence of

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phospholipase D, respectively. Error bars represent the standard deviation from three repetitive experiments.

Figure 5. (A) Fluorescence responses of RhB-PL-Ti2C3 toward several interferences. Relative fluorescence intensity of the RhB-PL-Ti2C3 incubated in blank buffer; glucose (20 mM), 20 mM for various amino acids, 20 mM for GSH, 50 mg mL−1 for BSA, Hb, 20% for FBS, 100 nM for lysozyme, trypsin, AchE, MMP-2, thrombin and ALP, respectively. F0 and F represent the fluorescence intensity of RhB-PL-Ti2C3 probes

in

the

absence

and

presence

of

biomolecules,

respectively.

(B)

Dose-dependent inhibition curve of phospholipase D using the RhB-PL-Ti3C2 in the presence of 400 U L-1 phospholipase D and FIPI of varying concentrations (10-6, 10-5, 10-4, 10-3, 10-2, 10-1, 1 mg mL-1). Error bars represent the standard deviation from three repetitive experiments.

Figure 6. (A) Typical CSLM images of MDA-MB-231 cells incubated with 50 µg mL−1 RhB-PL-Ti2C3 for 4 h at 37 °C in the absence (I) and presence of 1.0 mg mL-1 FIPI (II), and CSLM images of MCF-7 cells (III) incubated with 50 µg mL−1 RhB-PL-Ti2C3 for 4 h at 37 °C. Blue: nuclei stained with Hoechst. Scale bar, 50 µm. (B) Quantitative analysis of individual MDA-MB-231 cells in the absence and presence of 0.1 mg mL-1 FIPI, and MCF-7 cells. (C) Quantitative RT-PCR results for endogenous gene expression in breast cancer cells MCF-7 and MDA-MB-231. For each gene, results have been normalized 22


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(to 1-fold) to MCF-7 levels. Results are relative to housekeeping genes GAPDH.

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4

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Figure 5.

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Figure 6

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For TOC only

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Phospholipid-Tailored Titanium Carbide ... - ACS Publications

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