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A novel cell-penetrating Janus nanoprobe for ratiometric fluorescence detection of pH in living cells
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A novel cell-penetrating Janus nanoprobe for ratiometric fluorescence detection of pH in living cells Lei Wanga, Ying Zhoua,∗∗, Yan Zhanga, Guomei Zhanga, Caihong Zhanga, Yujian Hec,∗∗∗, Chuan Dongb, Shaomin Shuanga,∗ a
School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan, 030006, China Institute of Environmental Science, Shanxi University, Taiyuan, 030006, China c College of Chemistry and Chemical Engineering, University of the Chinese Academy of Sciences, Beijing, 100049, China b
ARTICLE INFO
ABSTRACT
Keywords: Janus nanoprobe Ratiometric detection Intracelluar pH
pH regulates the function of many organelles and plays a pivotal role in requiring multitud cellular behaviors. Compared with single fluorescent probes, ratio fluorescent probes have higher sensitivity and immunity to interference. Herein, a novel Janus ratio nanoprobe was developed for intracellular pH detection. Modified rhodamine B probe and fluorescein isothiocyanate (FITC) were individually encapsulated in the independent hemispheres of Janus microparticles fabricated via Pickering emulsion. Moreover, it exhibits a satasified ratiometric detection of pH compared to the previous core-shell structure and organic small molecule probe. Accordingly, the Janus nanoprobe possesses many important features as an attractive sensor, including high antijamming capability, excellent stability, good reversibility and low cytotoxicity. Variations of the two fluorescence intensities (Fgreen/Fred) resulted in a ratiometric pH fluorescent sensor, which can respond to wide range of pH values from 3 to 8. To be more specific, with a single excitation wavelength of 488 nm, there are dual emission bands centered at 538 nm and 590 nm. Also the Janus nanoprobe displays a excellent linear relationship in the physiologically relevant pH range of 4.0–6.0. Consequently, detecting of pH and imaging was successfully achieved in living cells, which provides a simple and reliable method for detecting intracelluar pH and other similar substances.
1. Introduction Many important physiological processes of cells and organelles are closely related to pH when performing or completing a chemical reaction [1,2]. It is necessary to control the acidity and alkalinity of the reaction solutions, because many chemical reactions need to be carried out at a specific pH condition; otherwise, the desired product will not be obtained [3–5]. Additionally, from a medical point of view, abnormal cellular pH values are known to be associated with inappropriate cellular functions, which are linked with many diseases including Alzheimers disease [6], cancer [7–9], and others. Therefore, accurate measurement of pH is important for chemical biology research. So far, many approaches have been developed to construct pH sensors [10–13], such as electrochemical, nuclear magnetic resonance (NMR) and absorption spectroscopy. Glass electrodes are generally used to determine pH, but not suitable for live pH monitoring due to defects
such as electrochemical interference, possible mechanical damage, and cannot applied for extreme pH measurements [14]. Among other measuring methods, fluorescence was considered as an optimal choice because of its excellent sensitivity, selectivity and minimal damage to samples [15]. It is known that the fluorescence or absorption properties of certain organic compounds vary with pH and can be used to indicate changes in acidity and alkalinity in the target medium [16–18]. The pH measurement method based on the change of the optical signal can compensate for the above disadvantages of the glass electrode. Fluorescent dye-based changes in fluorescence with pH often depend on the fact that a single functional group is protonated or deprotonated [19]. Among many options, fluorescent probes with single excitation and dual-emission wavelengths fluorescent probes with single excitation and dual-emission wavelengths can eliminate the effects of their own concentration, excitation intensity, and photobleaching as well as external environmental changes, providing more accurate analysis in
Corresponding author. Corresponding author. ∗∗∗ Corresponding author. E-mail addresses: [email protected] (Y. Zhou), [email protected] (Y. He), [email protected] (S. Shuang). ∗
∗∗
https://doi.org/10.1016/j.talanta.2019.120436 Received 28 June 2019; Received in revised form 29 September 2019; Accepted 3 October 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Lei Wang, et al., Talanta, https://doi.org/10.1016/j.talanta.2019.120436
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complex samples [20]. In addition, the fluorescence resonance energy transfer between Rhodamine B and fluorescein reported in the previous literature does not allow for effective ratio detection. Lei et al. [21] constructed a ratiometric fluorescent probe which fluorescein isothiocyanate (FITC) rhodamine B isothiocyanate was co-located in mesoporous silica pores. However, the detection result was not quite well due to fluorescence resonance energy transfer. A core-shell ratio fluorescent probe has also been reported in the literature [22]. A probe of a core-shell structure was constructed to avoid fluorescence resonance energy transfer by changing the thickness of the shell. However, the fluorescence of rhodamine B can only be used as a reference signal due to the protection of the silica shell and does not exhibit a significant ratio of fluorescence changes, and is not suitable for the measurement of acidic environments with pka 6.86. In 1991, Pierre-Gilles de Gennes firstly presented the concept of Janus particles in a speech at the Nobel Prizes in Physics [23]. Janus granules are particles of a double nature and are structurally assembled from two hemispheres with different physicochemical properties. Janus composite nanoparticles are a kind of nano-particles with asymmetric structure which have been developed in the past ten years. Due to their asymmetry in shape and performance and along with the deepening of research, the special properties and attractive applications of Janus particles are constantly emerging, involving in many fields such as catalyst [24], surfactants [25] and biological carriers [26]. The application of Janus particles has also become a research focus [27–29]. Janus particles with two different kinds of groups have amphipathic properties. Because of this property, Janus particles can well combine probes with different acid or base sensitivity [30,31]. After the modified Rhodamine B and FITC to form a Janus probe, the thickness of the silica shell layer can effectively avoid the occurrence of fluorescence resonance energy transfer, and can achieve a good ratio detection of pH. Considering the inherent complexity and constant evolvement of cells, ratiometric fluorescent assay with pH probe is one of effective methods, which is able to cancel out most effects of probes concentration, external environment variations, photobleaching and excitation intensity. Therefore, ratiometric fluorescent assay offer a more reliable detection in complex samples. In this study, we selected silica with excellent biocompatibility as the raw material. Firstly, Pickering emulsion was taken in wax-water phase, one side of SiO2–NH2 NPs were partially “locked” in the wax and modified with FITC, then exposed half of Janus nanoparticles can react with modified Rhodamine B probe in order to complete a pH ratio detection. Herein, as shown in Scheme 1 and Scheme 2, we designed a novel Janus ratio fluorescent probe.
studies(including size and zeta potential)measured by Zatesizer Nano ZS90 dynamic light scattering particle size analyzer. Thin-layer chromatography (TLC) was conducted on silica gel 60F254 plates (Merck KGaA). 1H NMR and 13C NMR spectra were recorded on a Bruker Avance 600 spectrometer, The pH measurements were performed on a PHS-3C digital pH-meter (YouKe, Shanghai, China). JEOL-JSM-7701F scanning electron microscope (SEM) was used for characterizing the morphological features of the nanoparticles. IR spectra were recorded with an fourier infrared spectrometer (TENSOR 27 Bruke Germany), Cell imaging is finished by confocal laser scanning microscopy (CLSM). 2.3. Synthesis of modified probe 2.3.1. Synthesis of intermediate 1 As shown in Scheme 1, compounds 1 and 2 were obtained according to the reported literature [32,33]. The synthetic methods of compounds 1 and 2 are shown in the supplementary information (Figs. S1–S2). 2.3.2. Synthesis of probe Compound 3 was synthesized via a method similar to the literature [34]. The ethanol solution of intermediate 1 (1.071g, 2.34 mmol) and compound 2 (414 mg, 2.3 mmol) was heated for 2 h under reflux and cooled to room temperature. The solvent was removed under reduced pressure and the crude product was purified by column chromatography using CH2Cl2/CH3OH (15/1, v/v) as an eluent. The synthetic methods and data characterization of probe are shown in the supplementary information(Figs. S3–S6). 2.4. Preparation of SiO2 NPs and its amino functionalization Followed by the reported literature [35], first, 5 ml of TEOS was dissolved in 50 mL of absolute ethanol at room temperature, and then this solution was quickly added to a solution mixed with 49.5 mL of deionized water, 88.5 mL of absolute ethanol, and 7.0 mL of ammonia water, and stir at room temperature for 2 h. The samples were centrifuged, washed, dried overnight and analyzed for further analysis. Disperse 1g of SiO2 NPs in a solution of 3-aminopropyltriethoxysilane (APTES, 1 mL) and anhydrous methanol (total amount 50 mL). The reaction was refluxed at 70 °C for 8 h, and after the reaction was completed, it was centrifuged to remove unreacted APTES. The resulting solid sample was dried overnight at 50 °C [36]. 2.5. Preparation of Janus particles After the amination of silica, 0.25g of modified silica nanoparticles were homogeneously dispersed in 25 mL of the distilled water and heated to 65 °C. Since aminated silica nanoparticles are inherently hydrophilic, it is essential to partially hydrophobize their surfaces for facilitate their adsorption at the oil-water interface. Therefore, 1 ml of SDS was added into the nanoparticles suspension (0.325 g/L) to neutralize surface charge and to obtain stable wax-in-water emulsions. In a second step, 5 g of wax is then deposited at the top of the particles suspension. After the wax melting, the mixture is submitted to vigorous stirring by means of an Ultra-Turrax homogenizer (T25 JANKE & KUNKEL), operating at 2200 rpm for 2 h. Then washed with deionized water and collected as described above. Since the half of the aminated silica surface is wrapped in paraffin wax, the amino group on the surface can form a stable chemical bond with FITC [37], 400 mg of paraffin-coated silica is ultrasonically dispersed into 60 mL of absolute ethanol, and then quickly added 3.5 mg of FITC. Reflux for 6 h, after completion of the reaction, the product was washed by filtration, dissolved in chloroform, and dried. Weigh 4 mg of the probe, add 2 equivalents of NHS, DCC for 6 h [38], then add ice-cold acetonitrile to precipitate, filter and dry to dissolve in 20 ml DCM, add 20 mg of SIO2-FITC, react overnight, decompress distillation to obtain Janus probe. The exact ratio of two
2. Materials and methods 2.1. Materials and equipments Aminopropyl)triethoxysilane(APTES, 99%; Aladdin), Tetraethyl orthosilicate (TEOS, 98%; Aladdin), Rhodamine B (RB; Aladdin). methanol, absolute ethanol, sodium dodecyl sulfate (SDS) and chloroform were analytical grade and purchased from Damao Chemical Reagent Factory, Tianjin, China. Fluorescein isothiocyanate (FITC) and wax were purchased from Yuanye Bio-Technology Co., Ltd, Shanghai, China. Salicylaldehyde(98%) and Chloroacetic acid(98%) were purchased from Macklin. Britton-Robinson (B-R) buffer was mixed by 40 mM acetic acid, boric acid, phosphoric acid. Dilute hydrochloric acid or sodium hydroxide was used for adjusting pH values. The solvent for H+ detection was a mixed solvent of EtOH/Britton-Robinson buffer (1/ 9, v/v). 2.2. Apparatus Fluorescence spectra of the probe were recorded by F-4500 fluorescence spectrometer (Hitachi, Japan). Dynamic light scattering (DLS) 2
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Scheme 1. Synthesis of modified rhodamine B probe.
probe molecule is showed in Fig. S8.
7721 cells (a human hepatocellular carcinoma cells) were supplemented with 100 U/mL 1% penicillin and streptomycin (v/v) and 10% fetal bovine serum in a 5% CO2 incubator. After the cells were adherently grown to about 70%, they were separated by 0.25 trypsinethylene diaminetetraacetic acid (EDTA) growth medium in time to continue passage.
2.6. Cell cytotoxicity assay SMMC-7721 cells were harvested and seeded in 96-well microplates for The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Different concentrations of probes with the cells incubated in a 5% CO2 incubator for 24 h at 37 °C. Then, the MTT solution was removed and 100 μL of DMSO was added to each well to dissolve formed formazan. Set the wavelength number at 490 nm and calculated the cell viability. The result was shown in Fig. S7.
3. Results and discussion 3.1. Characterization of SiO2–NH2 NPs By measuring the Zeta potential, the value was changed from −34.8 mV to 17.3 mV, and it can be proved that the silicon surface is successfully modified with amino group. The surface morphology of the silica was investigated by scanning electron microscopy and the
2.7. Cell cultures A-549 cells (a human lung adenocarcinoma cells and SMMC-
Scheme 2. Preparation of Janus nanoprobe. 3
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Fig. 1. (A)Zeta potentialof SiO2 NPs and SiO2–NH2 NPs. (B)SEM images of SiO2–NH2 NPs.(C)The average diameter of SiO2–NH2 NPs.
the surface of colloidosomes was not smooth (Fig. 3B). As the magnification increases, we can clearly see SiO2–NH2 NPs was closely aligned on the surface of the paraffin(Fig. 3C), and the exposed half is just involved in the new chemical reaction. It can be seen from Fig. 3D that the nanoparticles were monolayer on the colloidosomes surface. 3.3. Spectroscopic properties of pH probe 3.3.1. Determination of fluorescence spectra and pKa value Britton-Robison buffer solution test system with different pH values was prepared, and a certain amount of probe mother liquid was added to obtain a concentration of 10 μM. The excitation wavelength was selected at 488 nm and the fluorescence spectrum was recorded with a fluorometer. It is clear from the fluorescence spectrum that the probe has strong fluorescence at wavelengths of 535 nm and 590 nm, and the fluorescence intensity at 535 nm significantly reduced starts to decrease from pH 8. The fluorescence intensity at 590 nm was significantly enhanced. The ratio between the two (I535/I590) changes from 1.630 to 0.381 (Fig. 4A). Under acidic conditions, modified Rhodamine B probe showed strong fluorescence due to the spiro ring open (Fig. 4B). This equation can be expressed as:
Fig. 2. FTIR spectra of SiO2 NPs (top) and SiO2–NH2 NPs(bottom).
pKa = pH
average diameter of SiO2–NH2 nanoparticles is 171.9 nm, which all presented in Fig. 1. As shown in Fig. 2, the amination of silica can be confirmed by infrared spectroscopy. By comparing the infrared spectra of the two sample, it can be known that both of them have the bending vibration and the symmetric stretching vibration of Si–O [39], corresponding to the absorption peaks near the wave numbers of 466 cm−1 and 793 cm−1. The absorption peaks near the wave numbers of 945 cm−1, 1643 cm−1, and 1082 cm−1 are the bending vibration peaks of Si–OH and –OH, respectively. In the infrared spectrum of SiO2–NH2, the absorption peak at 2856 cm−1 and 2976 cm−1 can be attributed to the stretching vibration of –CH, and the absorption peak at 3400 cm−1 wave number corresponds to the asymmetric and symmetric stretching vibration of the primary amine, which further indicates that the silican surface was successfully modified with amino groups.
log(Imax
I)/(log(I
Imin))
(1)
Where I is the observed fluorescence intensity at a fixed wavelength, and Imin and Imax are the corresponding minimum and maximum, respectively [40,41]. From this equation, the pKa value was determined to be 6.68 ± 0.04, which means it can be used for monitoring weak acid environment and for intracellular pH determination (Fig. 4C). Fig. 4C shows the fluorescence versus pH curve at ratio(I535/I590). The emission intensity showed good linearity in the range of pH 4.00–6.00, and its linear regression equation was F = 0.242*pH – 0.534 with a correlation coefficient of 0.9920 The Janus nanoprobe exhibit good linearity (pH from 4 to 6) (Fig. 4D). 3.3.2. Testing of the reversibility and selectivity There is a complicated environment inside the cell, which contains these kinds of metal ions such as Na+, Mg2+, Zn2+, Ca2+ and so on. The rhodamine-like compounds themselves can easily complex with these metal ions, making their own spiro ring structure undergoes a certain degree of ring opening, which greatly affects the selectivity and sensitivity of the probe of the rhodamine B derivative to acidic conditions, thereby interfering with the response of the probe to the acidic environment. In order to confirm whether the probe still has high selectivity and sensitivity to acidic conditions in the presence of various metal ions, this experiment is followed by adding 10 equivalents of various metal ions in the probe solution of pH 7 and pH 4. These common metal ions include Na+, K+, Ca2+, Mg2+, Cu2+, Fe3+, Ba2+
3.2. Characterization of SiO2–NH2/wax colloidosomes The modified silicon spheres are dispersed in a certain concentration of SDS solution to neutralize the charge, and the paraffin is added at a high rotation speed to stir 1 h to form a stable emulsion to be suspended above, and filtered, dried and observed by a scanning electron microscope. As shown in Fig. 3A, morphology of the colloidosomes were confirmed to be spherical. Amplification of a single colloidosomes, due to the distribution of the nanoparticles on the surface of the wax, 4
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Fig. 3. SiO2–NH2 NPs are arranged on the surface of the wax with monolayer to form SiO2–NH2/wax colloidosomes. SEM images illustrating that the colloidosomes at magnifications of (A) 100×, (B) 800×,(C) 30000× and (D) 35000×.
Fig. 4. (A) Fluorescence spectra of Janus probes with pH (3–8). (λex = 488 nm). (B)Fluorescence spectra of the modified Rhodamine B probe with pH (4.10–7.96). (C) Ratiometric calibration curve showing peak sensor emission intensity (538 nm) divided by peak reference emission intensity (590 nm) versus pH value. (D) The Linear fitting of the probe between pH 4.0 and 6.0. 5
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Fig. 5. (A) Selectivity of the Janus nanoparticles with different metal ions (all metal ions concentrations were 1.0 mM).(B) Selectivity of the modified rhodamine B probe with different metal ions (all metal ions concentrations were 1.0 mM).(C) The reversibility of the Janus nanoparticles between pH 4.0 and 7.(D) The reversibility of the modified rhodamine B probe between pH 4.0 and 7.0.
and Sn2+ to explore whether this probe can resist the interference of different metal ions on its ability to detect strong acid. The final concentration of the probe is 10 μM. As shown in Fig. 5A and B, the probe's response to pH does not change significantly due to the presence of other types of metal ions, so we have reason to believe that this probe has higher sensitivity and selectivity for acidic environments. A candidate for pH probes that can be used to explore the acidic environment within cells. Moreover, since the intracellular pH tends to be oscillating and non-uniformly distributed, it is important that the pH probe has reversible responsiveness. As shown in Fig. 5C, Janus nanoprobe was subjected to four cycles of change at the same pH and shown good reversible response to pH. In Fig. 5D, modified rhodamine B probe was also yielded to four cycles, the result showed good reversible response to pH.
The Janus probe was added to the medium with MC7721 cells at a final concentration of 5 μM. After incubation for 4 h, it was washed three times with PBS balanced salt buffer solution. The laser was used to scan the microscope and the excitation wavelength was 488 nm. At pH 4.0, probe-stained SMMC-7721 cells showed little fluorescence in the green channel and bright fluorescence in the red channel. Fig. 7 shows as the pH increases to 8.0, the red fluorescence intensity decreases significantly while the green fluorescence intensity increases. Bright field transmission images confirmed the viability of SMMC-7721 cells after incubation with probes. In addition, we obtained a Merge image (third row) superimposed on the red-green channel, which shows a characteristic pH-dependent signal with a pH 4.0 to 8.0. To further verify whether the probe can be used for quantitative detection of viable cells, SMMC-7721 cells and BEAS-2B cells were incubated with 1 mL Janus nanoparticles and 1 mL medium for 4 h at 37 °C. (The final concentration of the Janus nanoparticles is 10 μM). The cells were washed with PBS (pH 7.4) to remove nanoparticles that did not enter the cells. Then treated with 1.0 mL of growth medium and analyzed by laser confocal microscopy. The pH is calculated by the calibration curve of the intracellular pH and the different regions of the calibration (Fig. 8) indicating that the pH of different regions in the living cells can be accurately determined. In addition, when the Janus nanoparticles were used to determine the pH in different regions of normal cells, from Fig. 9 it can be shown that the Janus nanoparticles have good dispersibility throughout the BEAS 2B cells rather than specific organelles. This suggests that Janus nanoparticles have the potential to monitor pH in living cells.
3.3.3. Testing light stability of the probe at different pH values The photostability of the probe is critical for subsequent detection, and the stability of the probe (10 μM) is determined by recording the fluorescence response within 0.5 h. Fig. 6 shows the fluorescence intensity of the probe at pH 4, 5.5 and 7.4 as a function of time at room temperature. The results demonstrate that the probe can be stably present at different hydrogen ion concentrations and exhibits good stability. 3.4. Cellular imaging The Janus probe has good pH response characteristics. Therefore, it is necessary to further verify whether it can be imaged intracellularly. 6
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Fig. 6. (A)The ratio strength (I590/I538) of Janus nanoparticles changes with time.(B)Fluorescence intensity of modified rhodamine B probe changes with time.Both were placed in B–F buffer solution (5% EtOH) at pH 4.0 (▲), 5.5 (●) and 7.4(▉).
Fig. 7. CLSM images of probes-labeled SMMC-7721 cells at pH 8.0, 7.0, 6.0, 5.0, and 4.0, respectively. Channel 1 and 2 were collected with red channel and green channel, respectively. Channel 3 was the merged image of green channel and red channel. Channel 4 was the bright field image. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 8. (A) Probe-labeled SMMC-7721 cells pH titration curve. The pseudocolored ratiometric image of probe-labeled SMMC-7721 cells (B) and single SMMC7721 cells (C).
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Fig. 9. (A) Probe-labeled BEAS-2B cells. (B) The pseudocolored ratiometricimage of probe-labeled BEAS-2B cells. (C) The estimated pH values at the region of interest indicated by black circle in (A).
4. Conclusions
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In summary, we constructed a novel ratio of Janus nanoparticle probes based on the pickering emulsion method, and the modified rhodamine B probe and FITC simultaneously attached to the surface of the silica. This new type of nanoparticle not only avoids fluorescence resonance energy transfer between fluorescent dyes, but also achieves true ratio detection. The nanoparticles exhibit excellent linearity (pH from 4 to 6), high light stability, good reversibility, and low cytotoxicity. The Janus nanoparticle probe has a pKa value of 6.86, and the fluorescence intensity at 538 nm and 5 90 nm produces a significant opposite change with pH. It is worth noting that the Janus nanoparticle probe can be used for quantitative measurement of normal cells and cancer cells. Notes We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work. Acknowledgements This work was financially supported by National Key Research and Development Plan (2016YFF0203700), the National Natural Science Foundation of China (No. 51772289 and 21575084), Shanxi Scholarship Council of China (No. 201701D121017 and 201701D221029), Shanxi Scholarship Council of China (2017-Key1), and the Shanxi Province Hundred Talents Project. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2019.120436. References [1] E.R. Chin, D.G. Allen, The contribution of pH-dependent mechanisms to fatigue at different intensifies in mammalian single muscle fibres, J. Physiol.-London 512 (3) (1998) 831–840. [2] R. Tsuboi, I. Ko, K. Takamori, H. Ogawa, Isolation of a keratinolytic proteinase from Trichophyton mentagrophytes with enzymatic activity at acidic pH, Infect. Immun. 57 (11) (1989) 3479–3483. [3] J. Li, X. Li, J. Jia, X. Chen, Y. Lv, Y. Guo, J. Li, A ratiometric near-infrared fluorescence strategy based on spiropyran in situ switching for tracking dynamic changes of live-cell lysosomal pH, Dyes Pigments 166 (2019) 433–442. [4] S. Rabiee, S. Tavakol, M. Barati, M.T. Joghataei, Autophagic, apoptotic, and necrotic cancer cell fates triggered by acidic pH microenvironment, J. Cell. Physiol. 234 (7) (2019) 12061–12069. [5] Y. Yue, F. Huo, S. Lee, C. Yin, J. Yoon, A review: the trend of progress about pH probes in cell application in recent years, Analyst 142 (1) (2016) 30–41. [6] T.A. Davies, R.E. Fine, R.J. Johnson, C.A. Levesque, W.H. Rathbun, K.F. Seetoo, S.J. Smith, G. Strohmeier, L. Volicer, L. Delva, E.R. Simons, Non-age related differences in thrombin responses by platelets from male-patients with advanced alzheimers-disease, Biochem. Biophys. Res. Commun. 194 (1) (1993) 537–543.
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L. Wang, et al. switchable oil/water separation, ACS Appl. Mater. Interfaces (2019) 22408–22418. [28] T.Y. Yang, L.J. Wei, L.Y. Jing, J.F. Liang, X.M. Zhang, M. Tang, M.J. Monteiro, Y. Chen, Y. Wang, S. Gu, D.Y. Zhao, H.Q. Yang, J. Liu, G.Q.M. Lu, Dumbbell-shaped Bi-component mesoporous Janus solid nanoparticles for biphasic interface Catalysis, Angew. Chem. Int. Ed. 56 (29) (2017) 8459–8463. [29] T.C. Zhao, X.H. Zhu, C.T. Hung, P.Y. Wang, A. Elzatahry, A.A. Al-Khalaf, W.N. Hozzein, F. Zhang, X.M. Li, D.Y. Zhao, Spatial isolation of Carbon and silica in a single Janus mesoporous nanoparticle with tunable amphiphilicity, J. Am. Chem. Soc. 140 (31) (2018) 10009–10015. [30] J.M. Alvarez-Pez, L. Ballesteros, E. Talavera, J. Yguerabide, Fluorescein excitedstate proton exchange reactions: nanosecond emission kinetics and correlation with steady-state fluorescence intensity, J. Phys. Chem. A 105 (26) (2001) 6320–6332. [31] V. Dujols, F. Ford, A.W. Czarnik, A long-wavelength fluorescent chemodosimeter selective for Cu(II) ion in water, J. Am. Chem. Soc. 119 (31) (1997) 7386–7387. [32] S. Davaran, A.A. Entezami, Synthesis and hydrolysis of modified polyvinyl alcohols containing ibuprofen pendant groups, Iran. Polym. J. (Engl. Ed.) 5 (3) (1996) 183–191. [33] Y. Shiraishi, R. Miyamoto, X. Zhang, T. Hirai, Rhodamine-based fluorescent thermometer exhibiting selective emission enhancement at a specific temperature range, Org. Lett. 9 (20) (2007) 3921–3924. [34] H.S. Lv, S.Y. Huang, Y. Xu, X. Dai, J.Y. Miao, B.X. Zhao, A new fluorescent pH probe for imaging lysosomes in living cells, Bioorg. Med. Chem. Lett. 24 (2) (2014)
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Talanta journal homepage: www.elsevier.com/locate/talanta
A novel cell-penetrating Janus nanoprobe for ratiometric fluorescence detection of pH in living cells Lei Wanga, Ying Zhoua,∗∗, Yan Zhanga, Guomei Zhanga, Caihong Zhanga, Yujian Hec,∗∗∗, Chuan Dongb, Shaomin Shuanga,∗ a
School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan, 030006, China Institute of Environmental Science, Shanxi University, Taiyuan, 030006, China c College of Chemistry and Chemical Engineering, University of the Chinese Academy of Sciences, Beijing, 100049, China b
ARTICLE INFO
ABSTRACT
Keywords: Janus nanoprobe Ratiometric detection Intracelluar pH
pH regulates the function of many organelles and plays a pivotal role in requiring multitud cellular behaviors. Compared with single fluorescent probes, ratio fluorescent probes have higher sensitivity and immunity to interference. Herein, a novel Janus ratio nanoprobe was developed for intracellular pH detection. Modified rhodamine B probe and fluorescein isothiocyanate (FITC) were individually encapsulated in the independent hemispheres of Janus microparticles fabricated via Pickering emulsion. Moreover, it exhibits a satasified ratiometric detection of pH compared to the previous core-shell structure and organic small molecule probe. Accordingly, the Janus nanoprobe possesses many important features as an attractive sensor, including high antijamming capability, excellent stability, good reversibility and low cytotoxicity. Variations of the two fluorescence intensities (Fgreen/Fred) resulted in a ratiometric pH fluorescent sensor, which can respond to wide range of pH values from 3 to 8. To be more specific, with a single excitation wavelength of 488 nm, there are dual emission bands centered at 538 nm and 590 nm. Also the Janus nanoprobe displays a excellent linear relationship in the physiologically relevant pH range of 4.0–6.0. Consequently, detecting of pH and imaging was successfully achieved in living cells, which provides a simple and reliable method for detecting intracelluar pH and other similar substances.
1. Introduction Many important physiological processes of cells and organelles are closely related to pH when performing or completing a chemical reaction [1,2]. It is necessary to control the acidity and alkalinity of the reaction solutions, because many chemical reactions need to be carried out at a specific pH condition; otherwise, the desired product will not be obtained [3–5]. Additionally, from a medical point of view, abnormal cellular pH values are known to be associated with inappropriate cellular functions, which are linked with many diseases including Alzheimers disease [6], cancer [7–9], and others. Therefore, accurate measurement of pH is important for chemical biology research. So far, many approaches have been developed to construct pH sensors [10–13], such as electrochemical, nuclear magnetic resonance (NMR) and absorption spectroscopy. Glass electrodes are generally used to determine pH, but not suitable for live pH monitoring due to defects
such as electrochemical interference, possible mechanical damage, and cannot applied for extreme pH measurements [14]. Among other measuring methods, fluorescence was considered as an optimal choice because of its excellent sensitivity, selectivity and minimal damage to samples [15]. It is known that the fluorescence or absorption properties of certain organic compounds vary with pH and can be used to indicate changes in acidity and alkalinity in the target medium [16–18]. The pH measurement method based on the change of the optical signal can compensate for the above disadvantages of the glass electrode. Fluorescent dye-based changes in fluorescence with pH often depend on the fact that a single functional group is protonated or deprotonated [19]. Among many options, fluorescent probes with single excitation and dual-emission wavelengths fluorescent probes with single excitation and dual-emission wavelengths can eliminate the effects of their own concentration, excitation intensity, and photobleaching as well as external environmental changes, providing more accurate analysis in
Corresponding author. Corresponding author. ∗∗∗ Corresponding author. E-mail addresses: [email protected] (Y. Zhou), [email protected] (Y. He), [email protected] (S. Shuang). ∗
∗∗
https://doi.org/10.1016/j.talanta.2019.120436 Received 28 June 2019; Received in revised form 29 September 2019; Accepted 3 October 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Lei Wang, et al., Talanta, https://doi.org/10.1016/j.talanta.2019.120436
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complex samples [20]. In addition, the fluorescence resonance energy transfer between Rhodamine B and fluorescein reported in the previous literature does not allow for effective ratio detection. Lei et al. [21] constructed a ratiometric fluorescent probe which fluorescein isothiocyanate (FITC) rhodamine B isothiocyanate was co-located in mesoporous silica pores. However, the detection result was not quite well due to fluorescence resonance energy transfer. A core-shell ratio fluorescent probe has also been reported in the literature [22]. A probe of a core-shell structure was constructed to avoid fluorescence resonance energy transfer by changing the thickness of the shell. However, the fluorescence of rhodamine B can only be used as a reference signal due to the protection of the silica shell and does not exhibit a significant ratio of fluorescence changes, and is not suitable for the measurement of acidic environments with pka 6.86. In 1991, Pierre-Gilles de Gennes firstly presented the concept of Janus particles in a speech at the Nobel Prizes in Physics [23]. Janus granules are particles of a double nature and are structurally assembled from two hemispheres with different physicochemical properties. Janus composite nanoparticles are a kind of nano-particles with asymmetric structure which have been developed in the past ten years. Due to their asymmetry in shape and performance and along with the deepening of research, the special properties and attractive applications of Janus particles are constantly emerging, involving in many fields such as catalyst [24], surfactants [25] and biological carriers [26]. The application of Janus particles has also become a research focus [27–29]. Janus particles with two different kinds of groups have amphipathic properties. Because of this property, Janus particles can well combine probes with different acid or base sensitivity [30,31]. After the modified Rhodamine B and FITC to form a Janus probe, the thickness of the silica shell layer can effectively avoid the occurrence of fluorescence resonance energy transfer, and can achieve a good ratio detection of pH. Considering the inherent complexity and constant evolvement of cells, ratiometric fluorescent assay with pH probe is one of effective methods, which is able to cancel out most effects of probes concentration, external environment variations, photobleaching and excitation intensity. Therefore, ratiometric fluorescent assay offer a more reliable detection in complex samples. In this study, we selected silica with excellent biocompatibility as the raw material. Firstly, Pickering emulsion was taken in wax-water phase, one side of SiO2–NH2 NPs were partially “locked” in the wax and modified with FITC, then exposed half of Janus nanoparticles can react with modified Rhodamine B probe in order to complete a pH ratio detection. Herein, as shown in Scheme 1 and Scheme 2, we designed a novel Janus ratio fluorescent probe.
studies(including size and zeta potential)measured by Zatesizer Nano ZS90 dynamic light scattering particle size analyzer. Thin-layer chromatography (TLC) was conducted on silica gel 60F254 plates (Merck KGaA). 1H NMR and 13C NMR spectra were recorded on a Bruker Avance 600 spectrometer, The pH measurements were performed on a PHS-3C digital pH-meter (YouKe, Shanghai, China). JEOL-JSM-7701F scanning electron microscope (SEM) was used for characterizing the morphological features of the nanoparticles. IR spectra were recorded with an fourier infrared spectrometer (TENSOR 27 Bruke Germany), Cell imaging is finished by confocal laser scanning microscopy (CLSM). 2.3. Synthesis of modified probe 2.3.1. Synthesis of intermediate 1 As shown in Scheme 1, compounds 1 and 2 were obtained according to the reported literature [32,33]. The synthetic methods of compounds 1 and 2 are shown in the supplementary information (Figs. S1–S2). 2.3.2. Synthesis of probe Compound 3 was synthesized via a method similar to the literature [34]. The ethanol solution of intermediate 1 (1.071g, 2.34 mmol) and compound 2 (414 mg, 2.3 mmol) was heated for 2 h under reflux and cooled to room temperature. The solvent was removed under reduced pressure and the crude product was purified by column chromatography using CH2Cl2/CH3OH (15/1, v/v) as an eluent. The synthetic methods and data characterization of probe are shown in the supplementary information(Figs. S3–S6). 2.4. Preparation of SiO2 NPs and its amino functionalization Followed by the reported literature [35], first, 5 ml of TEOS was dissolved in 50 mL of absolute ethanol at room temperature, and then this solution was quickly added to a solution mixed with 49.5 mL of deionized water, 88.5 mL of absolute ethanol, and 7.0 mL of ammonia water, and stir at room temperature for 2 h. The samples were centrifuged, washed, dried overnight and analyzed for further analysis. Disperse 1g of SiO2 NPs in a solution of 3-aminopropyltriethoxysilane (APTES, 1 mL) and anhydrous methanol (total amount 50 mL). The reaction was refluxed at 70 °C for 8 h, and after the reaction was completed, it was centrifuged to remove unreacted APTES. The resulting solid sample was dried overnight at 50 °C [36]. 2.5. Preparation of Janus particles After the amination of silica, 0.25g of modified silica nanoparticles were homogeneously dispersed in 25 mL of the distilled water and heated to 65 °C. Since aminated silica nanoparticles are inherently hydrophilic, it is essential to partially hydrophobize their surfaces for facilitate their adsorption at the oil-water interface. Therefore, 1 ml of SDS was added into the nanoparticles suspension (0.325 g/L) to neutralize surface charge and to obtain stable wax-in-water emulsions. In a second step, 5 g of wax is then deposited at the top of the particles suspension. After the wax melting, the mixture is submitted to vigorous stirring by means of an Ultra-Turrax homogenizer (T25 JANKE & KUNKEL), operating at 2200 rpm for 2 h. Then washed with deionized water and collected as described above. Since the half of the aminated silica surface is wrapped in paraffin wax, the amino group on the surface can form a stable chemical bond with FITC [37], 400 mg of paraffin-coated silica is ultrasonically dispersed into 60 mL of absolute ethanol, and then quickly added 3.5 mg of FITC. Reflux for 6 h, after completion of the reaction, the product was washed by filtration, dissolved in chloroform, and dried. Weigh 4 mg of the probe, add 2 equivalents of NHS, DCC for 6 h [38], then add ice-cold acetonitrile to precipitate, filter and dry to dissolve in 20 ml DCM, add 20 mg of SIO2-FITC, react overnight, decompress distillation to obtain Janus probe. The exact ratio of two
2. Materials and methods 2.1. Materials and equipments Aminopropyl)triethoxysilane(APTES, 99%; Aladdin), Tetraethyl orthosilicate (TEOS, 98%; Aladdin), Rhodamine B (RB; Aladdin). methanol, absolute ethanol, sodium dodecyl sulfate (SDS) and chloroform were analytical grade and purchased from Damao Chemical Reagent Factory, Tianjin, China. Fluorescein isothiocyanate (FITC) and wax were purchased from Yuanye Bio-Technology Co., Ltd, Shanghai, China. Salicylaldehyde(98%) and Chloroacetic acid(98%) were purchased from Macklin. Britton-Robinson (B-R) buffer was mixed by 40 mM acetic acid, boric acid, phosphoric acid. Dilute hydrochloric acid or sodium hydroxide was used for adjusting pH values. The solvent for H+ detection was a mixed solvent of EtOH/Britton-Robinson buffer (1/ 9, v/v). 2.2. Apparatus Fluorescence spectra of the probe were recorded by F-4500 fluorescence spectrometer (Hitachi, Japan). Dynamic light scattering (DLS) 2
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Scheme 1. Synthesis of modified rhodamine B probe.
probe molecule is showed in Fig. S8.
7721 cells (a human hepatocellular carcinoma cells) were supplemented with 100 U/mL 1% penicillin and streptomycin (v/v) and 10% fetal bovine serum in a 5% CO2 incubator. After the cells were adherently grown to about 70%, they were separated by 0.25 trypsinethylene diaminetetraacetic acid (EDTA) growth medium in time to continue passage.
2.6. Cell cytotoxicity assay SMMC-7721 cells were harvested and seeded in 96-well microplates for The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Different concentrations of probes with the cells incubated in a 5% CO2 incubator for 24 h at 37 °C. Then, the MTT solution was removed and 100 μL of DMSO was added to each well to dissolve formed formazan. Set the wavelength number at 490 nm and calculated the cell viability. The result was shown in Fig. S7.
3. Results and discussion 3.1. Characterization of SiO2–NH2 NPs By measuring the Zeta potential, the value was changed from −34.8 mV to 17.3 mV, and it can be proved that the silicon surface is successfully modified with amino group. The surface morphology of the silica was investigated by scanning electron microscopy and the
2.7. Cell cultures A-549 cells (a human lung adenocarcinoma cells and SMMC-
Scheme 2. Preparation of Janus nanoprobe. 3
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Fig. 1. (A)Zeta potentialof SiO2 NPs and SiO2–NH2 NPs. (B)SEM images of SiO2–NH2 NPs.(C)The average diameter of SiO2–NH2 NPs.
the surface of colloidosomes was not smooth (Fig. 3B). As the magnification increases, we can clearly see SiO2–NH2 NPs was closely aligned on the surface of the paraffin(Fig. 3C), and the exposed half is just involved in the new chemical reaction. It can be seen from Fig. 3D that the nanoparticles were monolayer on the colloidosomes surface. 3.3. Spectroscopic properties of pH probe 3.3.1. Determination of fluorescence spectra and pKa value Britton-Robison buffer solution test system with different pH values was prepared, and a certain amount of probe mother liquid was added to obtain a concentration of 10 μM. The excitation wavelength was selected at 488 nm and the fluorescence spectrum was recorded with a fluorometer. It is clear from the fluorescence spectrum that the probe has strong fluorescence at wavelengths of 535 nm and 590 nm, and the fluorescence intensity at 535 nm significantly reduced starts to decrease from pH 8. The fluorescence intensity at 590 nm was significantly enhanced. The ratio between the two (I535/I590) changes from 1.630 to 0.381 (Fig. 4A). Under acidic conditions, modified Rhodamine B probe showed strong fluorescence due to the spiro ring open (Fig. 4B). This equation can be expressed as:
Fig. 2. FTIR spectra of SiO2 NPs (top) and SiO2–NH2 NPs(bottom).
pKa = pH
average diameter of SiO2–NH2 nanoparticles is 171.9 nm, which all presented in Fig. 1. As shown in Fig. 2, the amination of silica can be confirmed by infrared spectroscopy. By comparing the infrared spectra of the two sample, it can be known that both of them have the bending vibration and the symmetric stretching vibration of Si–O [39], corresponding to the absorption peaks near the wave numbers of 466 cm−1 and 793 cm−1. The absorption peaks near the wave numbers of 945 cm−1, 1643 cm−1, and 1082 cm−1 are the bending vibration peaks of Si–OH and –OH, respectively. In the infrared spectrum of SiO2–NH2, the absorption peak at 2856 cm−1 and 2976 cm−1 can be attributed to the stretching vibration of –CH, and the absorption peak at 3400 cm−1 wave number corresponds to the asymmetric and symmetric stretching vibration of the primary amine, which further indicates that the silican surface was successfully modified with amino groups.
log(Imax
I)/(log(I
Imin))
(1)
Where I is the observed fluorescence intensity at a fixed wavelength, and Imin and Imax are the corresponding minimum and maximum, respectively [40,41]. From this equation, the pKa value was determined to be 6.68 ± 0.04, which means it can be used for monitoring weak acid environment and for intracellular pH determination (Fig. 4C). Fig. 4C shows the fluorescence versus pH curve at ratio(I535/I590). The emission intensity showed good linearity in the range of pH 4.00–6.00, and its linear regression equation was F = 0.242*pH – 0.534 with a correlation coefficient of 0.9920 The Janus nanoprobe exhibit good linearity (pH from 4 to 6) (Fig. 4D). 3.3.2. Testing of the reversibility and selectivity There is a complicated environment inside the cell, which contains these kinds of metal ions such as Na+, Mg2+, Zn2+, Ca2+ and so on. The rhodamine-like compounds themselves can easily complex with these metal ions, making their own spiro ring structure undergoes a certain degree of ring opening, which greatly affects the selectivity and sensitivity of the probe of the rhodamine B derivative to acidic conditions, thereby interfering with the response of the probe to the acidic environment. In order to confirm whether the probe still has high selectivity and sensitivity to acidic conditions in the presence of various metal ions, this experiment is followed by adding 10 equivalents of various metal ions in the probe solution of pH 7 and pH 4. These common metal ions include Na+, K+, Ca2+, Mg2+, Cu2+, Fe3+, Ba2+
3.2. Characterization of SiO2–NH2/wax colloidosomes The modified silicon spheres are dispersed in a certain concentration of SDS solution to neutralize the charge, and the paraffin is added at a high rotation speed to stir 1 h to form a stable emulsion to be suspended above, and filtered, dried and observed by a scanning electron microscope. As shown in Fig. 3A, morphology of the colloidosomes were confirmed to be spherical. Amplification of a single colloidosomes, due to the distribution of the nanoparticles on the surface of the wax, 4
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Fig. 3. SiO2–NH2 NPs are arranged on the surface of the wax with monolayer to form SiO2–NH2/wax colloidosomes. SEM images illustrating that the colloidosomes at magnifications of (A) 100×, (B) 800×,(C) 30000× and (D) 35000×.
Fig. 4. (A) Fluorescence spectra of Janus probes with pH (3–8). (λex = 488 nm). (B)Fluorescence spectra of the modified Rhodamine B probe with pH (4.10–7.96). (C) Ratiometric calibration curve showing peak sensor emission intensity (538 nm) divided by peak reference emission intensity (590 nm) versus pH value. (D) The Linear fitting of the probe between pH 4.0 and 6.0. 5
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Fig. 5. (A) Selectivity of the Janus nanoparticles with different metal ions (all metal ions concentrations were 1.0 mM).(B) Selectivity of the modified rhodamine B probe with different metal ions (all metal ions concentrations were 1.0 mM).(C) The reversibility of the Janus nanoparticles between pH 4.0 and 7.(D) The reversibility of the modified rhodamine B probe between pH 4.0 and 7.0.
and Sn2+ to explore whether this probe can resist the interference of different metal ions on its ability to detect strong acid. The final concentration of the probe is 10 μM. As shown in Fig. 5A and B, the probe's response to pH does not change significantly due to the presence of other types of metal ions, so we have reason to believe that this probe has higher sensitivity and selectivity for acidic environments. A candidate for pH probes that can be used to explore the acidic environment within cells. Moreover, since the intracellular pH tends to be oscillating and non-uniformly distributed, it is important that the pH probe has reversible responsiveness. As shown in Fig. 5C, Janus nanoprobe was subjected to four cycles of change at the same pH and shown good reversible response to pH. In Fig. 5D, modified rhodamine B probe was also yielded to four cycles, the result showed good reversible response to pH.
The Janus probe was added to the medium with MC7721 cells at a final concentration of 5 μM. After incubation for 4 h, it was washed three times with PBS balanced salt buffer solution. The laser was used to scan the microscope and the excitation wavelength was 488 nm. At pH 4.0, probe-stained SMMC-7721 cells showed little fluorescence in the green channel and bright fluorescence in the red channel. Fig. 7 shows as the pH increases to 8.0, the red fluorescence intensity decreases significantly while the green fluorescence intensity increases. Bright field transmission images confirmed the viability of SMMC-7721 cells after incubation with probes. In addition, we obtained a Merge image (third row) superimposed on the red-green channel, which shows a characteristic pH-dependent signal with a pH 4.0 to 8.0. To further verify whether the probe can be used for quantitative detection of viable cells, SMMC-7721 cells and BEAS-2B cells were incubated with 1 mL Janus nanoparticles and 1 mL medium for 4 h at 37 °C. (The final concentration of the Janus nanoparticles is 10 μM). The cells were washed with PBS (pH 7.4) to remove nanoparticles that did not enter the cells. Then treated with 1.0 mL of growth medium and analyzed by laser confocal microscopy. The pH is calculated by the calibration curve of the intracellular pH and the different regions of the calibration (Fig. 8) indicating that the pH of different regions in the living cells can be accurately determined. In addition, when the Janus nanoparticles were used to determine the pH in different regions of normal cells, from Fig. 9 it can be shown that the Janus nanoparticles have good dispersibility throughout the BEAS 2B cells rather than specific organelles. This suggests that Janus nanoparticles have the potential to monitor pH in living cells.
3.3.3. Testing light stability of the probe at different pH values The photostability of the probe is critical for subsequent detection, and the stability of the probe (10 μM) is determined by recording the fluorescence response within 0.5 h. Fig. 6 shows the fluorescence intensity of the probe at pH 4, 5.5 and 7.4 as a function of time at room temperature. The results demonstrate that the probe can be stably present at different hydrogen ion concentrations and exhibits good stability. 3.4. Cellular imaging The Janus probe has good pH response characteristics. Therefore, it is necessary to further verify whether it can be imaged intracellularly. 6
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Fig. 6. (A)The ratio strength (I590/I538) of Janus nanoparticles changes with time.(B)Fluorescence intensity of modified rhodamine B probe changes with time.Both were placed in B–F buffer solution (5% EtOH) at pH 4.0 (▲), 5.5 (●) and 7.4(▉).
Fig. 7. CLSM images of probes-labeled SMMC-7721 cells at pH 8.0, 7.0, 6.0, 5.0, and 4.0, respectively. Channel 1 and 2 were collected with red channel and green channel, respectively. Channel 3 was the merged image of green channel and red channel. Channel 4 was the bright field image. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 8. (A) Probe-labeled SMMC-7721 cells pH titration curve. The pseudocolored ratiometric image of probe-labeled SMMC-7721 cells (B) and single SMMC7721 cells (C).
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Fig. 9. (A) Probe-labeled BEAS-2B cells. (B) The pseudocolored ratiometricimage of probe-labeled BEAS-2B cells. (C) The estimated pH values at the region of interest indicated by black circle in (A).
4. Conclusions
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In summary, we constructed a novel ratio of Janus nanoparticle probes based on the pickering emulsion method, and the modified rhodamine B probe and FITC simultaneously attached to the surface of the silica. This new type of nanoparticle not only avoids fluorescence resonance energy transfer between fluorescent dyes, but also achieves true ratio detection. The nanoparticles exhibit excellent linearity (pH from 4 to 6), high light stability, good reversibility, and low cytotoxicity. The Janus nanoparticle probe has a pKa value of 6.86, and the fluorescence intensity at 538 nm and 5 90 nm produces a significant opposite change with pH. It is worth noting that the Janus nanoparticle probe can be used for quantitative measurement of normal cells and cancer cells. Notes We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work. Acknowledgements This work was financially supported by National Key Research and Development Plan (2016YFF0203700), the National Natural Science Foundation of China (No. 51772289 and 21575084), Shanxi Scholarship Council of China (No. 201701D121017 and 201701D221029), Shanxi Scholarship Council of China (2017-Key1), and the Shanxi Province Hundred Talents Project. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2019.120436. References [1] E.R. Chin, D.G. Allen, The contribution of pH-dependent mechanisms to fatigue at different intensifies in mammalian single muscle fibres, J. Physiol.-London 512 (3) (1998) 831–840. [2] R. Tsuboi, I. Ko, K. Takamori, H. Ogawa, Isolation of a keratinolytic proteinase from Trichophyton mentagrophytes with enzymatic activity at acidic pH, Infect. Immun. 57 (11) (1989) 3479–3483. [3] J. Li, X. Li, J. Jia, X. Chen, Y. Lv, Y. Guo, J. Li, A ratiometric near-infrared fluorescence strategy based on spiropyran in situ switching for tracking dynamic changes of live-cell lysosomal pH, Dyes Pigments 166 (2019) 433–442. [4] S. Rabiee, S. Tavakol, M. Barati, M.T. Joghataei, Autophagic, apoptotic, and necrotic cancer cell fates triggered by acidic pH microenvironment, J. Cell. Physiol. 234 (7) (2019) 12061–12069. [5] Y. Yue, F. Huo, S. Lee, C. Yin, J. Yoon, A review: the trend of progress about pH probes in cell application in recent years, Analyst 142 (1) (2016) 30–41. [6] T.A. Davies, R.E. Fine, R.J. Johnson, C.A. Levesque, W.H. Rathbun, K.F. Seetoo, S.J. Smith, G. Strohmeier, L. Volicer, L. Delva, E.R. Simons, Non-age related differences in thrombin responses by platelets from male-patients with advanced alzheimers-disease, Biochem. Biophys. Res. Commun. 194 (1) (1993) 537–543.
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