FITC and Ru(phen)3 2+ co-doped silica particles as visualized ratiometric pH indicator
© Xu et al; licensee Springer. 2011
Received: 14 June 2011
Accepted: 25 October 2011
Published: 25 October 2011
The performance of fluorescein isothiocyanate (FITC) and tris(1, 10-phenanathroline) ruthenium ion (Ru(phen)3 2+) co-doped silica particles as pH indicator was evaluated. The emission intensity ratios of the pH sensitive dye (FITC) and the reference dye (Ru(phen)3 2+) in the particles were dependent on pH of the environment. The changes in emission intensity ratios of the two dyes under different pH could be measured under single excitation wavelength and readily visualized by naked eye under a 365-nm UV lamp. In particular, such FITC and Ru(phen)3 2+ co-doped silica particles were identified to show high sensitivity to pH around the pKa of FITC (6.4), making them be potential useful as visualized pH indicator for detection of intracellular pH micro-circumstance.
KeywordspH indicator visualized silica particles ratiometric fluorescein ruthenium complex
In recent years, ratiometric fluorescent pH indicators had been developed for sensitive detection of pH of an analyte [1–6]. To fabricate a ratiometric pH indicator, usually two dyes, one pH sensitive and one reference dyes, were incorporated into a silica or polymer matrix. In this approach, a core/shell architecture in which the reference dye was mainly located in the core and the pH-sensitive dye located primarily in the shell was preferred [2, 7]. The ratios in emission intensity of the two dyes were correlated to pH of the analyte. Compared to pH indicator containing only the pH-sensitive dye [8–13], such ratiometric pH indicator was more reliable since the ratios in emission intensity were less sensitive to the fluctuations in excitation light source intensity and variations in other experimental conditions except pH [3, 4, 14–16]. However, most of the ratiometric pH indicators reported required the measurements of the emission intensity of the two dyes under two different excitation wavelengths, which made the analysis process be complicated and difficult to be visualized by naked eye [2, 4, 6, 7, 17].
In our previous work, we developed a kind of multicolor silica particles co-doped by fluorescent (fluorescein isothiocyanate - FITC) and phosphorescent (Ru(phen)3 2+) dyes. The green FITC and red Ru(phen)3 2+ dyes could be synchronously excited by a single excitation wavelength since there was large overlapping region in their absorption spectra. Color of the dye-doped silica particles was tunable by simply the ratios of the two dyes, which was readily visualized under a 365-nm UV lamp by naked eye . In this work, we explored the feasibility of such FITC and Ru(phen)3 2+ co-doped silica particles as visualized pH indicator, in which the green FITC was used as the pH sensitive dye and the red Ru(phen)3 2+ was employed as reference dye. It is expected that the particles may present different colors under different pH since the emission intensity of FITC was sensitive to pH. Experimental results revealed that the particles showed visualized color changes from red to yellowish-green distinguishable under a 365-nm UV lamp when pH of the buffer solutions increased from 2 to 8. Specially, such ratiometric pH indicator was very sensitive to pH around the pKa of FITC (6.4), making it potential useful for detection of intracellular pH micro-circumstance.
FITC, 3-aminopropyltriethoxysilane (APS), and dichloro tris (1,10-phenanathroline) ruthenium (II) hydrate (Ru(phen)3 2+) were purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Tetraethoxysilane (TEOS, Tiantai Chemical Int., Tianjin, China) was distilled under reduced pressure before use. Analytical grade ethanol, ammonia hydroxide (25%), NaOH (98%), H3PO4 (85%), H3BO3 (99%), and CH3COOH (36%) were purchased from Beijing Chemical Int. (Beijing, China) and used without further purification. Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were purchased from Invitrogen Gibco Corp. (Carlsbad, CA, USA). The human hepatoma cell line SMMC-7721 was purchased from Cell Resource Center of Shanghai Institutes for Biological Sciences (Shanghai, China). Britton-Robinson buffer solutions (denoted as BR buffer solution hereafter) in the pH range of 2.0-10 were prepared from a solution containing H3BO3, H3PO4, and CH3COOH with the same concentration of 0.04 mol L-1, and the desired pH value were acquired by adding different volume of 0.2 mol L-1 of NaOH. High-purity water with a resistivity of 18.2 MΩ cm (Pall Purelab Plus) was used in all experiments.
Synthesis of Ru(phen)3 2+-doped silica particles
Ru(phen)3 2+-doped silica particles were prepared by a modified Stöber method. In a typical reaction, 3 mL TEOS was added to ethanol solution (60 mL) containing ammonia (2.4 mL), Ru(phen)3 2+ (0.6 mg, dissolved in 1 mL ethanol), and water (1.2 mL). The reaction mixture was kept at 40°C for 6 h, then another 0.8 ml TEOS was added for the growth of an additional silica layer, and then the reaction was continued for another 6 h. The reaction solution was centrifuged at 10,000 rpm for 15 min to collect the silica particles. The particles were further washed with ethanol for three times to remove the unreacted chemicals and then dispersed in 60 mL ethanol.
Synthesis of FITC and Ru(phen)3 2+ co-doped silica particles
Ammonia (2.4 mL) and water (1.2 mL) were added into the ethanol dispersion of the Ru(phen)3 2+-doped silica particles (60 mL) and then 80 μL APS was added into the mixture. After being kept at 40°C under magnetic stirring for 8 h, the reaction solution was centrifuged at 10,000 rpm for 15 min to collect the aminated silica particles. After being washed three times with ethanol to remove the unreacted chemicals, the particles were dispersed to 60 mL ethanol and then 1 mg FITC dissolved in 1 mL ethanol was added. The mixture was allowed to stand at 40°C under magnetic stirring for 12 h. After the reaction, the particles were centrifuged at 10,000 rpm for 15 min to remove the unreacted dyes. The particles were washed by water until no fluorescence was detectable in the supernatant.
Cell handing process
SMMC-7721 cells were cultured in DMEM containing 10% FBS (fetal bovine serum) with 100 U/ml penicillin and 100 μg/ml streptomycin and incubated at 37°C under a humidified atmosphere containing 5% CO2. The cells were seeded in culture plates at a density of 1 × 105 cell/mL. After 24-h culturing, the cells were treated with the as-prepared silica particles which dispersed in serum-free DMEM at a concentration of 100 μg/mL for 4 h. After treatment, the cells were isolated by trypsin and washed with PBS for three times, and then the cells after endocytosis of the silica particles were observed by a fluorescence microscopy.
Transmission electron microscopic (TEM) observations were carried out on a JEOL-2010 electron microscope (JEOL, Tokyo, Japan) operating at 200 kV for determining the sizes of silica particles. The samples were prepared by depositing a drop of the dispersion of the particles onto carbon grids (200 mesh) and allowing evaporation of the solvent in air at room temperature. Emission spectra were measured on an Edinburgh FS900 steady-state fluorescence spectrometer (Edinburgh Instruments Ltd., Livingston, UK) with a 450-W xenon lamp as excitation source. Absorption spectra were collected with a Varian Cary-100 scan UV-vis spectrophotometer. Fluorescence images were taken under a 400 times OLYMPUS IX71 fluorescence microscope excited at 450 nm.
Results and discussion
Figure 2A shows the emission spectra of the co-doped silica particles dispersed in BR buffer solutions with different pH. The excitation wavelength was set at 450 nm under which both FITC and Ru(phen)3 2+ present reasonable extinction coefficients higher than 104 M-1 cm-1 (see Figure S3 of Additional file 1) [23, 24]. At pH = 2, the emission of FITC around 520 nm was quenched greatly. With the increased pH, the emission intensity of FITC increased gradually and then kept almost unchanged at pH ≥8, which was consistent with the behaviors of free FITC in aqueous solutions (see Figure S4 of Additional file 1). At the same time, the emission intensity of the reference dye located in the core part of the particles kept almost constant under the different pH. After being dispersed in BR buffers with pH of 2 to 8, the particles showed tunable emission color from red to yellowish-green which could be readily distinguished by naked eye under a 365-nm UV lamp (see insert of Figure 2). It is known that FITC may exist in dianionic, monoanionic, cationic, or neutral form dependent on pH of the solution (see Figure 2B). The monoanionic and neutral forms could be transformed into the non-luminous ester-type structure [25, 26]. The pH-sensitive emission of the co-doped silica particles was primarily related to the equilibrium of FITC between the low quantum yield monoanionic form (φ = 0.36) and high quantum yield dianionic one (φ = 0.93). When pH of the solution was lowered, the emission intensity of FITC decreased greatly mainly attributed to formation of the non-luminous ester-type structure since there was no great difference in molar extinction coefficients of the momoanionic and dianionic forms (see Figure S4 of Additional file 1). Therefore, the particles showed a yellowish-green color at high pH and red color at low pH since the emission intensity of the red reference dye was almost insensitive to the changes in pH of the buffers.
In summary, visualized ratiometric pH indicator was fabricated by using a fluorescent dye (FITC) and a phosphorescent dye (Ru(phen)3 2+). The two dyes were introduced into silica particles in a core/shell architecture to maximize the contact of the pH sensitive dye FITC with analyte while protecting the reference dye Ru(phen)3 2+ from the environment. Such ratiometric pH indicator could be excited simultaneously by using single wavelength due to the large overlapping in absorption features of the two dyes. The co-doped silica particles were sensitive to pH in the range of 2 to 8 distinguishable either by the emission spectra or in color observable by naked eye. The pH indicator showed good sensitivity around physiological pH, making it potential useful as a simple visualization pH indicator from detection of intracellular micro-environment.
- Ru(phen)3 2+ :
tris(1, 10-phenanathroline) ruthenium ion
Dulbecco's Modified Eagle Medium
fetal bovine serum
transmission electron microscopic.
This work was supported by the National Basic Research Program of China (no. 2009CB939701, no. 2011CB935800), the National Nature Science Foundation of China (50825202), and Graduate Innovation Fund of Jilin University (10201044)
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