Article pubs.acs.org/ac
Fluorescence Resonance Energy Transfer-Based Ratiometric Fluorescent Probe for Detection of Zn2+ Using a Dual-Emission SilicaCoated Quantum Dots Mixture Liang Wu, Qing-Sheng Guo, Yu-Qian Liu, and Qing-Jiang Sun* State Key Laboratory of Bioelectronics, School of Biological Science & Medical Engineering, Southeast University, Nanjing 210096, China S Supporting Information *
ABSTRACT: In this work, we report the design and application of a new ratiometric fluorescent probe, which contains different-colored quantum dots (QDs) as dual fluorophores, ultrathin silica shell as spacer, and meso-tetra(4-sulfonatophenyl)porphine dihydrochloride (TSPP) as receptor, for Zn2+ detection in aqueous solution and living cells. In the architecture of our designed probe, the silica shell plays the key roles in controlling the locations of QDs, TSPP, and Zn2+, preventing the direct contact between QDs and Zn2+ but affording fluorescence resonance energy transfer (FRET) from dual-color QDs to TSPP. In the presence of Zn2+, the analyte-receptor reaction changes the absorption in the range of the Q-band of TSPP and accordingly the efficiencies of two independent FRET processes from the dual-colored QDs to the acceptor, respectively, leading to fluorescence enhancement of green-emission QDs whereas fluorescence quenching of yellow-emission QDs. Benefiting from the well-resolved dual emissions from different-colored QDs and the large range of emission ratios, the probe solution displays continuous color changes from yellow to green, which can be clearly observed by the naked eye. Under physiological conditions, the probe exhibits a stable response for Zn2+ from 0.3 to 6 μM, with a detection limit of 60 nM in aqueous solutions. With respect to single-emission probes, this ratiometric probe has demonstrated to feature excellent selectivity for Zn2+ over other physiologically important cations such as Fe3+ and Cu2+. It has been preliminarily used for ratiometric imaging of Zn2+ in living cells with satisfying resolution. attracted significant attention.15−17 The ratiometric fluorescent probe exhibits improved sensitivity and meanwhile obtains a great built-in correction for environmental effects.18,19 Furthermore, the rapid visual identification can be achieved by the perceived color changes of the probe, which is particularly useful for applications in cellular imaging investigations. The mechanisms of internal charge transfer and intramolecular FRET are used most widely for sensing Zn2+ by the ratiometric fluorescent method.20−27 However, the development of molecular ratiometric probe for intracellular measurement of Zn2+ is still limited by the design of new organic fluorophores with well-resolved dual emissions. Quantum dots (QDs) are a recently developed class of inorganic fluorophores owing to their unique properties, such as high quantum yields, size-tunable narrow emission, and exceptional resistance to photobleaching.28−32 With the benefit of such optical properties, different colored QDs can be used to create a multicolor system for constructing a ratiometric fluorescent probe. In the past few years, researchers have made
Z
inc ions (Zn2+), which rank second in abundance among the transition metals in the human body, is believed to play critical roles in many biological processes, influencing the brain function, gene transcription, immune function, and mammalian reproduction.1−4 The disorder of Zn2+ metabolism is confirmed to be closely correlated with several diseases such as Alzheimer’s disease, diabetes, epilepsy, and cerebral ischemia.5−7 Therefore, the development of methods for spatially and temporally tracking intracellular Zn2+ is challenging and has become the subject of current chemical/biological research.8−10 Because Zn2+ is spectroscopically silent due to its d10 electron configuration, fluorescent probe methods for detecting Zn2+ have been developed intensely with the advantages of sensitivity, simplicity, cost-effective, and capability of affording high spatial resolution via fluorescence microscopy.11−14 The first-generation probes are single-emission molecular probes operated with a turn-on or turn-off mode, which fluorescence signal is easily disturbed by environmental factors such as pH, temperature, and solvent polarity. In recent years, the ratiometric fluorescent probe by utilizing the simultaneous measurement of two fluorescence signals at different wavelengths followed by calculation of their intensity ratio has © 2015 American Chemical Society
Received: February 8, 2015 Accepted: May 1, 2015 Published: May 1, 2015 5318
DOI: 10.1021/acs.analchem.5b00514 Anal. Chem. 2015, 87, 5318−5323
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Analytical Chemistry
Scheme 1. Schematic Illustration of the Preparation and Application of Dual-Colored QDs Based Ratiometric Fluorescent Probe for Detection of Zn2+
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EXPERIMENTAL SECTION Materials and Reagents. Tetraethyl orthosilicate (TEOS, 99.999%), TSPP (≥95.0%), igepal CO-520, cadmium oxide (99.99%), selenium powder (99.9%), zinc acetate (99.9%), sulfur powder (99.9%), trioctylphosphine oxide (90%), oleic acid (90%), and 1-octadecene (90%) were purchased from Sigma-Aldrich. (3-Aminopropyl)triethoxysilane (APS, 97%), octadecylamine (98%), and octadecylphosphonic acid (97%) were received from Alfa Aesar. Toluene (HPLC) was purchased from Mtedia. Acetone (AR), ethanol (AR), cyclohexane (AR), ZnCl2 (AR), and ammonia solution (AR, 28 wt %) were purchased from Aladdin. The pure water (18.2 MΩ cm) was obtained from a Pall Cascade AN synthesis system. Synthesis of 512QD@SiO2 and 572QD@SiO2. The oil soluble 512QDs and 572QDs were synthesized following the method in the literature.41,42 512QD@SiO2 and 572QD@SiO2 were synthesized according to a two-step microemulsion method published previously.43 For step 1, as-prepared QDs (512QDs or 572QDs) were redispersed in 0.3 mL of anhydrous toluene, and then added 2 μL of TEOS with stirring for 24 h to get silanized QDs. For step 2, 1 g of igepal CO-520 was dispersed in 10 mL of cyclohexane and stirred for 30 min to obtain the stock solution. A volume of 0.3 mL of silanized QDs and different amounts (4, 6, or 8 μL) of TEOS were then added to the stock solution, respectively. After stirring for 30 min, 0.3 mL of ammonia aqueous solution (7 wt %) was introduced to initiate the reaction. The silica growth was completed after 24 h of stirring in the dark at room temperature. Next, APS was added to introduce the amino groups onto the surface of QD@ SiO2 with 24 h polymerization. The products were isolated from the microemulsion using acetone and washed several times by ethanol to remove any surfactant. The resulting nanoparticles were redispersed in PBS buffer (pH 7.4, 10 mM) for further use. Characterization of QD@SiO2 and QD@SiO2/TSPP. The transmission electron microscopy (TEM) was used to determine the size and morphology of as-prepared nanoparticles. The samples were prepared by dropping diluted solutions of 512QD@SiO2 and 572QD@SiO2 onto carbon films supported by a Cu grid, respectively. To prepare the samples for Fourier transform-infrared (FT-IR) measurements, the
efforts toward the development of QDs-based ratiometric probes for detecting some metal ions. One successful sensing platform is the fabrication of QDs-based dual-emission nanohybrids for the ratiometric fluorescent detection of Cu2+ or Hg2+, in which the inside QDs provide an invariable background signal and the outside QDs (or carbon dots) are used as the sensing signal.33−35 To the best of our knowledge, no literature has reported QDs-based probes for ratiometric Zn2+ detection in aqueous solution and living cells, although a few of QDs-based single-emission probes for detecting Zn2+ are available.36−40 As a proof-of-concept, herein we have designed a new FRETbased ratiometric fluorescent probe for detection of Zn2+ with dual-colored QDs (green 512QDs and yellow 572QDs) as fluorophores, ultrathin silica shell as spacer, and TSPP as receptor, as shown in Scheme 1. The dual-colored QDs are chosen as the FRET donors since they exhibit size-dependent narrow emission, which can be easily engineered for precisely matching the discrete, narrow, and multiplex absorption bands of TSPP (acceptor). The silica shell is designed as the spacer, primarily for achieving the improved signal-to-background ratio of fluorescent probe by avoiding the direct contact between QDs and metal ions. In the constructed ratiometric sensing system, two independent FRET processes (that from 512QDs to TSPP is referred as FRET-1 and that from 572QDs to TSPP is referred as FRET-2) take place. Upon the chelating of TSPP with Zn2+, the FRET-1 efficiency decreases, accompanying with fluorescence enhancement of 512QDs, whereas the FRET-2 efficiency increases, leading to fluorescence quenching of 572 QDs. By measuring the emission intensity ratios between512QDs and 572QDs, the dual FRETs-based ratiometric sensing for Zn2+ is achieved. With respect to those probes based on single-emission QDs or dual-emission QDs nanohybrids, where only one FRET process is utilized, our proposed ratiometric probe features with improved sensitivity and selectivity for Zn2+, benefiting from the large range of intensity ratios induced by the two independent FRET processes. Together with the dual well-resolved narrow emissions of 512 QDs and 572QDs, this probe is practically useful for ratiometric imaging of Zn2+ in living cells with satisfying resolution. 5319
DOI: 10.1021/acs.analchem.5b00514 Anal. Chem. 2015, 87, 5318−5323
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Instruments. Fluorescence spectra were collected with an F-7000 fluorescence spectrophotometer (Hitachi, Japan), and UV−vis absorption spectra were obtained by a Hitachi U-4100 spectrophotometer. FT-IR spectra were acquired with Nicolet5700 instrument. Zeta potentials were measured using dynamic light scattering on a Malvern Zetasizer Nano-ZS particle analyzer. QD@SiO2 nanoparticles were photographed on a transmission electron microscope of the JEOL JEM-2100 instrument. The fluorescence images of living cells were observed under a TI confocal fluorescence microscope (Nikon, Japan).
solutions of QD@SiO2, amino-modified QD@SiO2, and QD@ SiO2/TSPP were centrifuged at 12 000 rpm for 15 min, respectively. The precipitates were extensively dried with the help of a hot air flux under vacuum. Then, the samples were characterized by FT-IR with the spectral resolution of 1.928 cm−1. The zeta potential measurements were conducted in aqueous solutions of 512QD@SiO2 or 512QD@SiO2 mixed with equal volume of TSPP solution with different concentrations at room temperature and pH 7.4. Zeta potentials were calculated from the measured electrophoretic mobility. Fluorescence Measurements. 512QD@SiO2/TSPP and 572 QD@SiO2/TSPP solutions were prepared by adding different amounts of TSPP with a volume of 300 μL into an equal volume of 512QD@SiO2 or 572QD@SiO2 solutions, respectively. The ratiometric probe solutions were prepared by mixing 512 QD@SiO2/TSPP and 572QD@SiO2/TSPP solutions under appropriate proportions. The ratio of emission intensity at 512 and 572 nm is defined as 1:1.5. For experiments of sensing Zn2+, aqueous solutions of Zn2+ with different concentrations were added into an equal volume of the ratiometric probe solution, and the final concentrations of Zn2+ ions were 0, 0.375, 0.75, 1.5, 3.0, 4.5, 6.0, 9.0, and 12 μM. For accelerating the fluorescent response, catalysts of imidazole and Cd2+ were added with the final concentrations of 60 mM and 5.4 μM, respectively.44 Fluorescence spectra data were collected in 30 min after each addition under ambient condition, and the excitation wavelength was fixed at 460 nm with the slit width of 5 nm. Selectivity and Interference Measurements. For the selectivity experiments, aqueous solutions with various metal ions were added into an equal volume of the ratiometric probe solution or a single-emission probe solution, with a PBS buffer (10 mM) at pH 7.4. For simulating the physiological conditions, the final concentrations were 6.0 μM for Zn2+, 140.0 mM for Na+, 5.0 mM for K+, 2.5 mM for Ca2+, 1.0 mM for Mg2+, 20.0 μM for Fe2+, 25.0 μM for Fe3+, 1.0 μM for Mn2+, Cd2+, Cr3+, and 0.5 μM for Ni2+, Co2+, Pb2+, Cu2+, Ag+, and Hg2+. For the interference experiments, 6.0 μM Zn2+ solutions coexisted with another metal ion with the concentration mentioned above were first prepared and were then mixed with the ratiometric fluorescent probe solution. In all solutions, catalysts of imidazole and Cd2+ were not added. After the incubation time of 12 h, the fluorescent responses of the ratiometric probe and single-emission probes were examined by a fluorescence spectrophotometer with the excitation wavelength of 460 nm. Ratiometric Fluorescent Imaging of Zn2+ in Living Cells. The living HCT116 cells were cultured in the mccoy5A medium to get a suitable density. For Zn2+-spiked cells, the cells were incubated with 6.0 μM ZnCl2 and 12 μM pyrithione for 2 h. Prior to the imaging experiments, the cells were washed with PBS three times. The controlled imaging experiments were carried out with HCT116 cells (with/without Zn2+), incubated with the mixture of 512QD@SiO2 and 572QD@SiO2 for 12 h at 37 °C. The ratiometric fluorescent imaging experiments for intracellular Zn2+ were carried out with HCT116 cells (with/ without Zn2+), incubated with the ratiometric probe for 12 h at 37 °C. The excitation wavelength of the laser was 488 nm and the emissions were centered at 520 ± 10 nm and 575 ± 10 nm of the double channels. The fluorescence for the single cell (totally 10 cells in each image) was analyzed by ImageJ (NIH, http://rsb.info.nih.gov/nih-image/index.html).
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RESULTS AND DISCUSSION The ratiometric fluorescent probe is prepared by first synthesis of amino modified 512QD@SiO2 and 572QD@SiO2 nanoparticles via a reverse microemulsion approach and subsequent deposition of TSPP on the surfaces of mixed nanoparticles by electrostatic adsorption, as shown in Scheme 1. In our sensing system, encapsulation of QDs by silica is designed to play the key roles in preventing the direct contact between QDs and Zn2+ but affording FRET from dual-color QDs to TSPP. The effect of Zn2+ on fluorescence of QD@SiO2 is examined. It is found that the fluorescence signals of 512QD@SiO2 and 572 QD@SiO2 are insensitive to Zn2+ in a wide range of concentration (Figure S1 in the Supporting Information).
Figure 1. (A) Fluorescence spectra of the mixture of 512QD@SiO2 and572QD@SiO2 in the absence (square)/presence (triangle) of 6.0 μM Zn2+. Inset: evolution of emission intensities of 512QD@SiO2 and572QD@SiO2 versus time in a mixture solution with 6.0 μM Zn2+. (B) Evolution of fluorescence spectra of 512QD@SiO2 and 572QD@ SiO2 with the TSPP concentration (0, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 12, and 20 μM).
Figure 1A shows the emission characteristic of a mixture of 512 QD@SiO2 and 572QD@SiO2 with fixed concentrations of nanopaticles under a single wavelength excitation of 460 nm, exhibiting two well-resolved emission peaks at 512 and 572 nm, respectively. It is found that, in the absence/presence of 6.0 μM Zn2+, emission intensities of 512QD@SiO2 and 572QD@SiO2 are kept constant in the mixture solutions. Furthermore, both emission intensities of 512QD@SiO2 and 572QD@SiO2 are invariable as a function of incubation time (the inset of Figure 1A). In contrast, by mixing 512QDs and 572QDs directly, the fluorescence of 512QDs or 572QDs fluctuates with the time in the absence of Zn2+. In the presence of Zn2+, both emission intensities of 512QDs and 572QDs slightly increase in 0.5 h due to the formation of ZnS passivation layer,28,29 but thereafter decrease gradually with the time (Figure S2 in the Supporting 5320
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Analytical Chemistry Information). These results indicate that the fluorescence signal of QDs is much improved by the encapsulation of silica shell. Figure 1B shows the evolution of emission intensities of 512 QD@SiO2 and 572QD@SiO2, respectively, upon addition of TSPP. It is found that both emission intensities of 512QD@SiO2 and 572QD@SiO2 decrease steadily with the increased TSPP concentration. At a TSPP concentration of 3.0 μM, the emission intensity decreases to 40% for 512QD@SiO2 and 50% for 572QD@SiO2, respectively. This result indicates that the FRET processes take place from different-colored QDs to TSPP, even with the encapsulation of a silica shell. The architecture of the ratiometric probe is investigated in detail. TEM images show that each QD (512QD or 572QD) is well embedded into a 7.0 nm-thick silica shell, and the ultrathin silica coated nanoparticles are monodisperse (Figure S3 in the Supporting Information). Zeta potential measurements show that Zeta potential of either 512QD@SiO2 or 572QD@SiO2 decreases gradually with the increased TSPP concentration, verifying the electrostatic adsorption of TSPP onto the surface of QD@SiO2 (Figure S4 in the Supporting Information). FTIR spectra show characteristic vibration bands at 1096 and 801 cm−1 for silica, band at 1558 cm−1 for amine groups, and bands at 738 and 636 cm−1 for TSPP on the surface of the silica shell, revealing clearly the architecture of our proposed probe (Figure S5 in the Supporting Information). The effect of pH (from 4.0 to 9.0) on the fluorescence of as prepared ratiometric probe is examined. It is found that the fluorescence signals from both 512QD@SiO2 and 572QD@SiO2 are stable in the range of pH 5.0−8.0 (Figure S6 in the Supporting Information), indicating that this probe is suitable for further usage under physiological conditions. To make this probe feasible and biocompatible for cell imaging studies, pH 7.4 is selected. In addition, the stability of the ratiometric probe against time is investigated. The result indicates that the ratiometric probe is stable at pH 7.4 for 20 h (Figure S7 in the Supporting Information). The response of the ratiometric fluorescent probe to Zn2+ is determined by the chelation of TSPP with Zn2+. The rate of native TSPP-Zn2+ reaction is fairly slow, but it can be accelerated by some catalysts.44 Imidazole and Cd2+ are added into the probe solution as the catalysts (Figure S8 in the Supporting Information). The catalysts do not affect the fluorescence signals of our probe but shorten the response time to be 30 min. As shown in Figure 2A, the probe exhibits wellresolved dual emissions from 512QDs and 572QDs. In the absence of Zn2+, the ratio of emission intensity of 512QDs to that of 572QDs (I512/I572) is 1:1.5. Upon addition of Zn2+, the emission intensity at 512 nm gradually increases, whereas that at 572 nm decreases continuously. At a Zn2+ concentration of 6.0 μM, the value of I512/I572 becomes 1.6:1. As can be seen in Figure 2B, the relative emission intensity ratio is closely related to the amount of Zn2+, ranging from 0.3 to 6.0 μM, which can be used for quantitative determination of Zn2+ with a correlation coefficient of 0.996. The limit of detection (LOD) is defined as 60 nM following the 3σ IUPAC criteria, which is better than those of previously reported QDs-based probes and comparable to those of some ratiometric molecular probes (Table S1 in the Supporting Information). For comparison, the fluorescent responses of single-emission probes (512QD@SiO2/ TSPP or 572QD@SiO2/TSPP) to Zn2+ are examined and the linear relationships are also presented in Figure 2B. Clearly, the ratiometric fluorescent probe possesses higher sensitivity to Zn2+ than the single-emission probes. The LOD for the
Figure 2. (A) Fluorescence spectra of the ratiometric probe treated by various amounts of Zn2+. (B) Evolution of relative emission intensities of I512 (square), I572 (triangle), and their ratio (diamond) with the Zn2+ concentration for the ratiometric probe. For comparison, Zn2+ induced evolutions of relative emission intensity for the 512QD@SiO2/ TSPP probe (circle) and the 572QD@SiO2/TSPP probe (hexagon) are also presented. Inset: photographs of aqueous solutions with (a) the 512 QD@SiO2/TSPP probe and (b) the ratiometric probe upon adding different amounts of Zn2+ under a UV lamp. The Zn2+ concentrations in the solutions from left to right are 0, 1.5, 3.0, 6.0, and 9.0 μM, respectively. 512
QD@SiO2/TSPP probe is 130 nM, and that for the 572QD@ SiO2/TSPP probe is 220 nM. Benefiting from the well-resolved dual emissions and the relatively large range of emission ratios of 512QDs and 572QDs, the ratiometric probe solutions display continuous color changes from yellow to green, which can be clearly distinguished by the naked eye under a UV lamp. Unlike the ratiometric probe, the color changes of single-emission turn-on probe (512QD@SiO2/TSPP) solutions upon addition of Zn2+ are hard to observe (the inset of Figure 2B). The comparison clearly shows that the ratiometric probe has higher reliability than a single-emission probe for visual detection. As aforementioned above, the mechanism for the ratiometric probe is based on dual independent FRET processes. For FRET systems, the efficiency is controlled by spectral overlap between the donor emission and the acceptor absorption. As can be seen in Figure 3, there is a sufficiently large spectral overlap between the 512QDs emission and the absorption band centered at 515 nm of TSPP, affording an efficient FRET-1. The spectral overlap between the 572QDs emission and the absorption bands centered at 555 and 592 nm of TSPP is small, resulting in an inefficient FRET-2. Upon addition of Zn2+, the
Figure 3. Fluorescence spectra of 512QDs and 572QDs and evolution of absorption spectra of TSPP by chelating with different amounts of Zn2+. 5321
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Ag+, or Hg2+ quenches the fluorescence of both single-emission probes due to the strong electronic interactions between such cations and 512QDs (or 572QDs) despite the silica encapsulation.47,48 The selective Zn2+ detection by the ratiometric probe, free of the Cu2+, Ag+, or Hg2+ interference, is thus ascribed to the built-in correction for environment effects. Furthermore, in considering the complexity of the intracellular system, the selectivity for Zn2+ coexisting with various metal ions is examined for this ratiometric probe (Figure 4B). Clearly, these potential metal ion interferences show negligible effects on the ratiometric fluorescent signal for Zn2+ detection, indicating that the ratiometric probe is advantageous in reliability over the single-emission probe for applications in cell imaging studies. The silica-encapsulated QDs can be considered to be low toxicity and biocompatible for living cells.29 Figure S10 in the Supporting Information shows the double-channel fluorescent images for HCT116 cells incubated with the mixture of 512 QD@SiO2 and 572QD@SiO2 for 12 h at 520 ± 10 nm and 575 ± 10 nm. The yellow/green emission ratio for the pristine mixture is 1.29:1 (Figure S11 in the Supporting Information). After the incubation, HCT116 cells show both bright yellow emission and green emission, with the calculated yellow/green emission ratios of 1.24:1 and 1.22:1, in the absence/presence of Zn2+, respectively. These results suggest the ratios of dual-color silica-encapsulated QDs uptaken by the cells are almost equal, and the fluorescence of the mixture maintains its insensitivity toward labile Zn2+ in a cell environment. The application of our probe for ratiometric fluorescent imaging of Zn2+ in living HCT116 cells is thus carried out. Figure 5 shows the double-
chelation of TSPP with Zn2+ induces the specific evolution of absorption in the range of the Q-band.45,46 The absorbance at 515 nm decreases continuously, while those at 555 and 592 nm increase gradually. The spectral overlap between the 512QDs emission and the absorption band centered at 515 nm of ZnTSPP becomes smaller, while that between the 572QDs emission and the absorption bands centered at 555 and 592 nm of ZnTSPP becomes larger. Accordingly, the FRET-1 efficiency is gradually decreased, accompanying with fluorescence enhancement of 512QDs, whereas the FRET-2 efficiency is increased, leading to fluorescence quenching of 572QDs. The independence of the dual FRETs is confirmed by the consistent fluorescent responses to Zn2+ between the single-emission probes and the ratiometric probe by monitoring the emission intensity at 512 and 572 nm, respectively (Figure 2B). The selectivity for Zn2+ of this ratiometric probe as well as single-emission probes (512QD@SiO2/TSPP and 572QD@ SiO2/TSPP) is investigated, and various metal ions such as abundant cellular cations (Na+, K+, Ca2+, Mg2+), trace cations in organisms (Fe2+, Fe3+, Zn2+, Cu2+, Ni2+, Co2+, Cr3+, Mn2+), and toxic heavy metal cations (Cd2+, Pb2+, Ag+, Hg2+) are tested under physiological conditions. As shown in Figure 4A, a
Figure 4. (A) Selectivity of single-emission probes and the ratiometric probe in 10 mM PBS buffer solution at pH 7.4 for various ions. (B) Effect of coexisting ions on the signal (I512/I572) of the ratiometric probe in the presence of Zn2+. Final concentrations of the ions: Zn2+, 6.0 μM; Na+, 140 mM; K+, 5.0 mM; Ca2+, 2.5 mM; Mg2+, 1.0 mM; Fe2+, 20 μM; Fe3+, 25 μM; Cr3+, Mn2+, and Cd2+, 1.0 μM; Ni2+, Co2+, Pb2+, Cu2+, Ag+, and Hg2+, 0.5 μM.
Figure 5. Fluorescent images of live HCT116 cells incubated with the ratiometric probe: top row, treatment without Zn2+; bottom row, treatment with 6 μM ZnCl2 and 12 μM pyrithione. (A, D) Images obtained through the yellow channel. (B, E) Images obtained through the green channel. (C, F) Overlay of fluorescent images from the two channels and the corresponding bright field image.
remarkable enhancement of I512/I572 is induced by Zn2+, and no obvious changes of I512/I572 are observed for the other metal ions, demonstrating the excellent selectivity of this ratiometric probe for Zn2+. In contrast, the single-emission probes suffer from the interference of Fe3+, Cu2+, Ag+, and Hg2+. Fe3+ enhances but Cu2+, Ag+, or Hg2+ quenches the fluorescence of both 512QD@SiO2/TSPP and 572QD@SiO2/TSPP. The Fe3+ induced fluorescence enhancement of single-emission probes is because that Fe(III)TSPP exhibits all decreased absorbance centered at 515, 555, and 592 nm, respectively, compared to TSPP, leading to simultaneously reduced efficiencies of FRET1 (from 512QDs to Fe(III)TSPP) and FRET-2 (from 572QDs to Fe(III)TSPP) (Figure S9 in the Supporting Information). Cu2+,
channel fluorescent images for HCT116 cells incubated with the probe for 12 h. In the absence of Zn2+, HCT116 cells show a strong yellow emission (Figure 5A) and a weak green emission (Figure 5B), with the yellow/green emission ratio of 2.61:1 (Figure S11 in the Supporting Information). After the cells are treated with 6.0 μM ZnCl2 and 12 μM pyrithione, a strong quenching of the yellow signal (Figure 5D) and a remarkable enhancement of the green signal (Figure 5E) can be clearly observed, and the yellow/green emission ratio becomes 0.73:1, which agrees well with the evolution of dual-emissions of this probe in an aqueous solution. These preliminary results demonstrate that this ratiometric fluorescent probe is practi5322
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Analytical Chemistry cally useful for tracking intracellular labile Zn2+ with satisfying resolution.
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CONCLUSION In summary, by preparing a mixture of dual-emission silicaencapsulated QDs, in conjugation with TSPP, we have developed a selective and sensitive strategy for the ratiometric fluorescent detection of Zn2+. Dual independent FRET processes are involved in the ratiometric detection of Zn2+, which is advantageous in specificity and sensitivity over singleemission probes. A perfect visual identification for Zn2+ by distinct color changes is achieved by this probe in aqueous solutions. This ratiometric probe exhibits well-resolved dual emissions and a large range of emission ratios and thus can be successfully applied for imaging and biosensing of Zn2+ in living cells. This work provides a method for designing QDs-based ratiometric fluorescent assays with high specificity and sensitivity for in vivo imaging and biosensing of metal ions, which can be eventually useful for metalloneurochemistry studies.
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ASSOCIATED CONTENT
S Supporting Information *
Additional fluorescence data and spectra, TEM images, zeta potential data, FT-IR spectra, comparison of principles and analytical parameters of different fluorescent Zn2+ probes, and absorbance spectra. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b00514.
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AUTHOR INFORMATION
Corresponding Author
*Fax: (86) 25-83792349. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is financially supported by NSFC and Jiangsu Province (Grants 21375015, 21005017, and BK20141334).
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REFERENCES
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DOI: 10.1021/acs.analchem.5b00514 Anal. Chem. 2015, 87, 5318−5323
Fluorescence Resonance Energy Transfer-Based Ratiometric Fluorescent Probe for Detection of Zn2+ Using a Dual-Emission SilicaCoated Quantum Dots Mixture Liang Wu, Qing-Sheng Guo, Yu-Qian Liu, and Qing-Jiang Sun* State Key Laboratory of Bioelectronics, School of Biological Science & Medical Engineering, Southeast University, Nanjing 210096, China S Supporting Information *
ABSTRACT: In this work, we report the design and application of a new ratiometric fluorescent probe, which contains different-colored quantum dots (QDs) as dual fluorophores, ultrathin silica shell as spacer, and meso-tetra(4-sulfonatophenyl)porphine dihydrochloride (TSPP) as receptor, for Zn2+ detection in aqueous solution and living cells. In the architecture of our designed probe, the silica shell plays the key roles in controlling the locations of QDs, TSPP, and Zn2+, preventing the direct contact between QDs and Zn2+ but affording fluorescence resonance energy transfer (FRET) from dual-color QDs to TSPP. In the presence of Zn2+, the analyte-receptor reaction changes the absorption in the range of the Q-band of TSPP and accordingly the efficiencies of two independent FRET processes from the dual-colored QDs to the acceptor, respectively, leading to fluorescence enhancement of green-emission QDs whereas fluorescence quenching of yellow-emission QDs. Benefiting from the well-resolved dual emissions from different-colored QDs and the large range of emission ratios, the probe solution displays continuous color changes from yellow to green, which can be clearly observed by the naked eye. Under physiological conditions, the probe exhibits a stable response for Zn2+ from 0.3 to 6 μM, with a detection limit of 60 nM in aqueous solutions. With respect to single-emission probes, this ratiometric probe has demonstrated to feature excellent selectivity for Zn2+ over other physiologically important cations such as Fe3+ and Cu2+. It has been preliminarily used for ratiometric imaging of Zn2+ in living cells with satisfying resolution. attracted significant attention.15−17 The ratiometric fluorescent probe exhibits improved sensitivity and meanwhile obtains a great built-in correction for environmental effects.18,19 Furthermore, the rapid visual identification can be achieved by the perceived color changes of the probe, which is particularly useful for applications in cellular imaging investigations. The mechanisms of internal charge transfer and intramolecular FRET are used most widely for sensing Zn2+ by the ratiometric fluorescent method.20−27 However, the development of molecular ratiometric probe for intracellular measurement of Zn2+ is still limited by the design of new organic fluorophores with well-resolved dual emissions. Quantum dots (QDs) are a recently developed class of inorganic fluorophores owing to their unique properties, such as high quantum yields, size-tunable narrow emission, and exceptional resistance to photobleaching.28−32 With the benefit of such optical properties, different colored QDs can be used to create a multicolor system for constructing a ratiometric fluorescent probe. In the past few years, researchers have made
Z
inc ions (Zn2+), which rank second in abundance among the transition metals in the human body, is believed to play critical roles in many biological processes, influencing the brain function, gene transcription, immune function, and mammalian reproduction.1−4 The disorder of Zn2+ metabolism is confirmed to be closely correlated with several diseases such as Alzheimer’s disease, diabetes, epilepsy, and cerebral ischemia.5−7 Therefore, the development of methods for spatially and temporally tracking intracellular Zn2+ is challenging and has become the subject of current chemical/biological research.8−10 Because Zn2+ is spectroscopically silent due to its d10 electron configuration, fluorescent probe methods for detecting Zn2+ have been developed intensely with the advantages of sensitivity, simplicity, cost-effective, and capability of affording high spatial resolution via fluorescence microscopy.11−14 The first-generation probes are single-emission molecular probes operated with a turn-on or turn-off mode, which fluorescence signal is easily disturbed by environmental factors such as pH, temperature, and solvent polarity. In recent years, the ratiometric fluorescent probe by utilizing the simultaneous measurement of two fluorescence signals at different wavelengths followed by calculation of their intensity ratio has © 2015 American Chemical Society
Received: February 8, 2015 Accepted: May 1, 2015 Published: May 1, 2015 5318
DOI: 10.1021/acs.analchem.5b00514 Anal. Chem. 2015, 87, 5318−5323
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Analytical Chemistry
Scheme 1. Schematic Illustration of the Preparation and Application of Dual-Colored QDs Based Ratiometric Fluorescent Probe for Detection of Zn2+
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EXPERIMENTAL SECTION Materials and Reagents. Tetraethyl orthosilicate (TEOS, 99.999%), TSPP (≥95.0%), igepal CO-520, cadmium oxide (99.99%), selenium powder (99.9%), zinc acetate (99.9%), sulfur powder (99.9%), trioctylphosphine oxide (90%), oleic acid (90%), and 1-octadecene (90%) were purchased from Sigma-Aldrich. (3-Aminopropyl)triethoxysilane (APS, 97%), octadecylamine (98%), and octadecylphosphonic acid (97%) were received from Alfa Aesar. Toluene (HPLC) was purchased from Mtedia. Acetone (AR), ethanol (AR), cyclohexane (AR), ZnCl2 (AR), and ammonia solution (AR, 28 wt %) were purchased from Aladdin. The pure water (18.2 MΩ cm) was obtained from a Pall Cascade AN synthesis system. Synthesis of 512QD@SiO2 and 572QD@SiO2. The oil soluble 512QDs and 572QDs were synthesized following the method in the literature.41,42 512QD@SiO2 and 572QD@SiO2 were synthesized according to a two-step microemulsion method published previously.43 For step 1, as-prepared QDs (512QDs or 572QDs) were redispersed in 0.3 mL of anhydrous toluene, and then added 2 μL of TEOS with stirring for 24 h to get silanized QDs. For step 2, 1 g of igepal CO-520 was dispersed in 10 mL of cyclohexane and stirred for 30 min to obtain the stock solution. A volume of 0.3 mL of silanized QDs and different amounts (4, 6, or 8 μL) of TEOS were then added to the stock solution, respectively. After stirring for 30 min, 0.3 mL of ammonia aqueous solution (7 wt %) was introduced to initiate the reaction. The silica growth was completed after 24 h of stirring in the dark at room temperature. Next, APS was added to introduce the amino groups onto the surface of QD@ SiO2 with 24 h polymerization. The products were isolated from the microemulsion using acetone and washed several times by ethanol to remove any surfactant. The resulting nanoparticles were redispersed in PBS buffer (pH 7.4, 10 mM) for further use. Characterization of QD@SiO2 and QD@SiO2/TSPP. The transmission electron microscopy (TEM) was used to determine the size and morphology of as-prepared nanoparticles. The samples were prepared by dropping diluted solutions of 512QD@SiO2 and 572QD@SiO2 onto carbon films supported by a Cu grid, respectively. To prepare the samples for Fourier transform-infrared (FT-IR) measurements, the
efforts toward the development of QDs-based ratiometric probes for detecting some metal ions. One successful sensing platform is the fabrication of QDs-based dual-emission nanohybrids for the ratiometric fluorescent detection of Cu2+ or Hg2+, in which the inside QDs provide an invariable background signal and the outside QDs (or carbon dots) are used as the sensing signal.33−35 To the best of our knowledge, no literature has reported QDs-based probes for ratiometric Zn2+ detection in aqueous solution and living cells, although a few of QDs-based single-emission probes for detecting Zn2+ are available.36−40 As a proof-of-concept, herein we have designed a new FRETbased ratiometric fluorescent probe for detection of Zn2+ with dual-colored QDs (green 512QDs and yellow 572QDs) as fluorophores, ultrathin silica shell as spacer, and TSPP as receptor, as shown in Scheme 1. The dual-colored QDs are chosen as the FRET donors since they exhibit size-dependent narrow emission, which can be easily engineered for precisely matching the discrete, narrow, and multiplex absorption bands of TSPP (acceptor). The silica shell is designed as the spacer, primarily for achieving the improved signal-to-background ratio of fluorescent probe by avoiding the direct contact between QDs and metal ions. In the constructed ratiometric sensing system, two independent FRET processes (that from 512QDs to TSPP is referred as FRET-1 and that from 572QDs to TSPP is referred as FRET-2) take place. Upon the chelating of TSPP with Zn2+, the FRET-1 efficiency decreases, accompanying with fluorescence enhancement of 512QDs, whereas the FRET-2 efficiency increases, leading to fluorescence quenching of 572 QDs. By measuring the emission intensity ratios between512QDs and 572QDs, the dual FRETs-based ratiometric sensing for Zn2+ is achieved. With respect to those probes based on single-emission QDs or dual-emission QDs nanohybrids, where only one FRET process is utilized, our proposed ratiometric probe features with improved sensitivity and selectivity for Zn2+, benefiting from the large range of intensity ratios induced by the two independent FRET processes. Together with the dual well-resolved narrow emissions of 512 QDs and 572QDs, this probe is practically useful for ratiometric imaging of Zn2+ in living cells with satisfying resolution. 5319
DOI: 10.1021/acs.analchem.5b00514 Anal. Chem. 2015, 87, 5318−5323
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Analytical Chemistry
Instruments. Fluorescence spectra were collected with an F-7000 fluorescence spectrophotometer (Hitachi, Japan), and UV−vis absorption spectra were obtained by a Hitachi U-4100 spectrophotometer. FT-IR spectra were acquired with Nicolet5700 instrument. Zeta potentials were measured using dynamic light scattering on a Malvern Zetasizer Nano-ZS particle analyzer. QD@SiO2 nanoparticles were photographed on a transmission electron microscope of the JEOL JEM-2100 instrument. The fluorescence images of living cells were observed under a TI confocal fluorescence microscope (Nikon, Japan).
solutions of QD@SiO2, amino-modified QD@SiO2, and QD@ SiO2/TSPP were centrifuged at 12 000 rpm for 15 min, respectively. The precipitates were extensively dried with the help of a hot air flux under vacuum. Then, the samples were characterized by FT-IR with the spectral resolution of 1.928 cm−1. The zeta potential measurements were conducted in aqueous solutions of 512QD@SiO2 or 512QD@SiO2 mixed with equal volume of TSPP solution with different concentrations at room temperature and pH 7.4. Zeta potentials were calculated from the measured electrophoretic mobility. Fluorescence Measurements. 512QD@SiO2/TSPP and 572 QD@SiO2/TSPP solutions were prepared by adding different amounts of TSPP with a volume of 300 μL into an equal volume of 512QD@SiO2 or 572QD@SiO2 solutions, respectively. The ratiometric probe solutions were prepared by mixing 512 QD@SiO2/TSPP and 572QD@SiO2/TSPP solutions under appropriate proportions. The ratio of emission intensity at 512 and 572 nm is defined as 1:1.5. For experiments of sensing Zn2+, aqueous solutions of Zn2+ with different concentrations were added into an equal volume of the ratiometric probe solution, and the final concentrations of Zn2+ ions were 0, 0.375, 0.75, 1.5, 3.0, 4.5, 6.0, 9.0, and 12 μM. For accelerating the fluorescent response, catalysts of imidazole and Cd2+ were added with the final concentrations of 60 mM and 5.4 μM, respectively.44 Fluorescence spectra data were collected in 30 min after each addition under ambient condition, and the excitation wavelength was fixed at 460 nm with the slit width of 5 nm. Selectivity and Interference Measurements. For the selectivity experiments, aqueous solutions with various metal ions were added into an equal volume of the ratiometric probe solution or a single-emission probe solution, with a PBS buffer (10 mM) at pH 7.4. For simulating the physiological conditions, the final concentrations were 6.0 μM for Zn2+, 140.0 mM for Na+, 5.0 mM for K+, 2.5 mM for Ca2+, 1.0 mM for Mg2+, 20.0 μM for Fe2+, 25.0 μM for Fe3+, 1.0 μM for Mn2+, Cd2+, Cr3+, and 0.5 μM for Ni2+, Co2+, Pb2+, Cu2+, Ag+, and Hg2+. For the interference experiments, 6.0 μM Zn2+ solutions coexisted with another metal ion with the concentration mentioned above were first prepared and were then mixed with the ratiometric fluorescent probe solution. In all solutions, catalysts of imidazole and Cd2+ were not added. After the incubation time of 12 h, the fluorescent responses of the ratiometric probe and single-emission probes were examined by a fluorescence spectrophotometer with the excitation wavelength of 460 nm. Ratiometric Fluorescent Imaging of Zn2+ in Living Cells. The living HCT116 cells were cultured in the mccoy5A medium to get a suitable density. For Zn2+-spiked cells, the cells were incubated with 6.0 μM ZnCl2 and 12 μM pyrithione for 2 h. Prior to the imaging experiments, the cells were washed with PBS three times. The controlled imaging experiments were carried out with HCT116 cells (with/without Zn2+), incubated with the mixture of 512QD@SiO2 and 572QD@SiO2 for 12 h at 37 °C. The ratiometric fluorescent imaging experiments for intracellular Zn2+ were carried out with HCT116 cells (with/ without Zn2+), incubated with the ratiometric probe for 12 h at 37 °C. The excitation wavelength of the laser was 488 nm and the emissions were centered at 520 ± 10 nm and 575 ± 10 nm of the double channels. The fluorescence for the single cell (totally 10 cells in each image) was analyzed by ImageJ (NIH, http://rsb.info.nih.gov/nih-image/index.html).
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RESULTS AND DISCUSSION The ratiometric fluorescent probe is prepared by first synthesis of amino modified 512QD@SiO2 and 572QD@SiO2 nanoparticles via a reverse microemulsion approach and subsequent deposition of TSPP on the surfaces of mixed nanoparticles by electrostatic adsorption, as shown in Scheme 1. In our sensing system, encapsulation of QDs by silica is designed to play the key roles in preventing the direct contact between QDs and Zn2+ but affording FRET from dual-color QDs to TSPP. The effect of Zn2+ on fluorescence of QD@SiO2 is examined. It is found that the fluorescence signals of 512QD@SiO2 and 572 QD@SiO2 are insensitive to Zn2+ in a wide range of concentration (Figure S1 in the Supporting Information).
Figure 1. (A) Fluorescence spectra of the mixture of 512QD@SiO2 and572QD@SiO2 in the absence (square)/presence (triangle) of 6.0 μM Zn2+. Inset: evolution of emission intensities of 512QD@SiO2 and572QD@SiO2 versus time in a mixture solution with 6.0 μM Zn2+. (B) Evolution of fluorescence spectra of 512QD@SiO2 and 572QD@ SiO2 with the TSPP concentration (0, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 12, and 20 μM).
Figure 1A shows the emission characteristic of a mixture of 512 QD@SiO2 and 572QD@SiO2 with fixed concentrations of nanopaticles under a single wavelength excitation of 460 nm, exhibiting two well-resolved emission peaks at 512 and 572 nm, respectively. It is found that, in the absence/presence of 6.0 μM Zn2+, emission intensities of 512QD@SiO2 and 572QD@SiO2 are kept constant in the mixture solutions. Furthermore, both emission intensities of 512QD@SiO2 and 572QD@SiO2 are invariable as a function of incubation time (the inset of Figure 1A). In contrast, by mixing 512QDs and 572QDs directly, the fluorescence of 512QDs or 572QDs fluctuates with the time in the absence of Zn2+. In the presence of Zn2+, both emission intensities of 512QDs and 572QDs slightly increase in 0.5 h due to the formation of ZnS passivation layer,28,29 but thereafter decrease gradually with the time (Figure S2 in the Supporting 5320
DOI: 10.1021/acs.analchem.5b00514 Anal. Chem. 2015, 87, 5318−5323
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Analytical Chemistry Information). These results indicate that the fluorescence signal of QDs is much improved by the encapsulation of silica shell. Figure 1B shows the evolution of emission intensities of 512 QD@SiO2 and 572QD@SiO2, respectively, upon addition of TSPP. It is found that both emission intensities of 512QD@SiO2 and 572QD@SiO2 decrease steadily with the increased TSPP concentration. At a TSPP concentration of 3.0 μM, the emission intensity decreases to 40% for 512QD@SiO2 and 50% for 572QD@SiO2, respectively. This result indicates that the FRET processes take place from different-colored QDs to TSPP, even with the encapsulation of a silica shell. The architecture of the ratiometric probe is investigated in detail. TEM images show that each QD (512QD or 572QD) is well embedded into a 7.0 nm-thick silica shell, and the ultrathin silica coated nanoparticles are monodisperse (Figure S3 in the Supporting Information). Zeta potential measurements show that Zeta potential of either 512QD@SiO2 or 572QD@SiO2 decreases gradually with the increased TSPP concentration, verifying the electrostatic adsorption of TSPP onto the surface of QD@SiO2 (Figure S4 in the Supporting Information). FTIR spectra show characteristic vibration bands at 1096 and 801 cm−1 for silica, band at 1558 cm−1 for amine groups, and bands at 738 and 636 cm−1 for TSPP on the surface of the silica shell, revealing clearly the architecture of our proposed probe (Figure S5 in the Supporting Information). The effect of pH (from 4.0 to 9.0) on the fluorescence of as prepared ratiometric probe is examined. It is found that the fluorescence signals from both 512QD@SiO2 and 572QD@SiO2 are stable in the range of pH 5.0−8.0 (Figure S6 in the Supporting Information), indicating that this probe is suitable for further usage under physiological conditions. To make this probe feasible and biocompatible for cell imaging studies, pH 7.4 is selected. In addition, the stability of the ratiometric probe against time is investigated. The result indicates that the ratiometric probe is stable at pH 7.4 for 20 h (Figure S7 in the Supporting Information). The response of the ratiometric fluorescent probe to Zn2+ is determined by the chelation of TSPP with Zn2+. The rate of native TSPP-Zn2+ reaction is fairly slow, but it can be accelerated by some catalysts.44 Imidazole and Cd2+ are added into the probe solution as the catalysts (Figure S8 in the Supporting Information). The catalysts do not affect the fluorescence signals of our probe but shorten the response time to be 30 min. As shown in Figure 2A, the probe exhibits wellresolved dual emissions from 512QDs and 572QDs. In the absence of Zn2+, the ratio of emission intensity of 512QDs to that of 572QDs (I512/I572) is 1:1.5. Upon addition of Zn2+, the emission intensity at 512 nm gradually increases, whereas that at 572 nm decreases continuously. At a Zn2+ concentration of 6.0 μM, the value of I512/I572 becomes 1.6:1. As can be seen in Figure 2B, the relative emission intensity ratio is closely related to the amount of Zn2+, ranging from 0.3 to 6.0 μM, which can be used for quantitative determination of Zn2+ with a correlation coefficient of 0.996. The limit of detection (LOD) is defined as 60 nM following the 3σ IUPAC criteria, which is better than those of previously reported QDs-based probes and comparable to those of some ratiometric molecular probes (Table S1 in the Supporting Information). For comparison, the fluorescent responses of single-emission probes (512QD@SiO2/ TSPP or 572QD@SiO2/TSPP) to Zn2+ are examined and the linear relationships are also presented in Figure 2B. Clearly, the ratiometric fluorescent probe possesses higher sensitivity to Zn2+ than the single-emission probes. The LOD for the
Figure 2. (A) Fluorescence spectra of the ratiometric probe treated by various amounts of Zn2+. (B) Evolution of relative emission intensities of I512 (square), I572 (triangle), and their ratio (diamond) with the Zn2+ concentration for the ratiometric probe. For comparison, Zn2+ induced evolutions of relative emission intensity for the 512QD@SiO2/ TSPP probe (circle) and the 572QD@SiO2/TSPP probe (hexagon) are also presented. Inset: photographs of aqueous solutions with (a) the 512 QD@SiO2/TSPP probe and (b) the ratiometric probe upon adding different amounts of Zn2+ under a UV lamp. The Zn2+ concentrations in the solutions from left to right are 0, 1.5, 3.0, 6.0, and 9.0 μM, respectively. 512
QD@SiO2/TSPP probe is 130 nM, and that for the 572QD@ SiO2/TSPP probe is 220 nM. Benefiting from the well-resolved dual emissions and the relatively large range of emission ratios of 512QDs and 572QDs, the ratiometric probe solutions display continuous color changes from yellow to green, which can be clearly distinguished by the naked eye under a UV lamp. Unlike the ratiometric probe, the color changes of single-emission turn-on probe (512QD@SiO2/TSPP) solutions upon addition of Zn2+ are hard to observe (the inset of Figure 2B). The comparison clearly shows that the ratiometric probe has higher reliability than a single-emission probe for visual detection. As aforementioned above, the mechanism for the ratiometric probe is based on dual independent FRET processes. For FRET systems, the efficiency is controlled by spectral overlap between the donor emission and the acceptor absorption. As can be seen in Figure 3, there is a sufficiently large spectral overlap between the 512QDs emission and the absorption band centered at 515 nm of TSPP, affording an efficient FRET-1. The spectral overlap between the 572QDs emission and the absorption bands centered at 555 and 592 nm of TSPP is small, resulting in an inefficient FRET-2. Upon addition of Zn2+, the
Figure 3. Fluorescence spectra of 512QDs and 572QDs and evolution of absorption spectra of TSPP by chelating with different amounts of Zn2+. 5321
DOI: 10.1021/acs.analchem.5b00514 Anal. Chem. 2015, 87, 5318−5323
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Analytical Chemistry
Ag+, or Hg2+ quenches the fluorescence of both single-emission probes due to the strong electronic interactions between such cations and 512QDs (or 572QDs) despite the silica encapsulation.47,48 The selective Zn2+ detection by the ratiometric probe, free of the Cu2+, Ag+, or Hg2+ interference, is thus ascribed to the built-in correction for environment effects. Furthermore, in considering the complexity of the intracellular system, the selectivity for Zn2+ coexisting with various metal ions is examined for this ratiometric probe (Figure 4B). Clearly, these potential metal ion interferences show negligible effects on the ratiometric fluorescent signal for Zn2+ detection, indicating that the ratiometric probe is advantageous in reliability over the single-emission probe for applications in cell imaging studies. The silica-encapsulated QDs can be considered to be low toxicity and biocompatible for living cells.29 Figure S10 in the Supporting Information shows the double-channel fluorescent images for HCT116 cells incubated with the mixture of 512 QD@SiO2 and 572QD@SiO2 for 12 h at 520 ± 10 nm and 575 ± 10 nm. The yellow/green emission ratio for the pristine mixture is 1.29:1 (Figure S11 in the Supporting Information). After the incubation, HCT116 cells show both bright yellow emission and green emission, with the calculated yellow/green emission ratios of 1.24:1 and 1.22:1, in the absence/presence of Zn2+, respectively. These results suggest the ratios of dual-color silica-encapsulated QDs uptaken by the cells are almost equal, and the fluorescence of the mixture maintains its insensitivity toward labile Zn2+ in a cell environment. The application of our probe for ratiometric fluorescent imaging of Zn2+ in living HCT116 cells is thus carried out. Figure 5 shows the double-
chelation of TSPP with Zn2+ induces the specific evolution of absorption in the range of the Q-band.45,46 The absorbance at 515 nm decreases continuously, while those at 555 and 592 nm increase gradually. The spectral overlap between the 512QDs emission and the absorption band centered at 515 nm of ZnTSPP becomes smaller, while that between the 572QDs emission and the absorption bands centered at 555 and 592 nm of ZnTSPP becomes larger. Accordingly, the FRET-1 efficiency is gradually decreased, accompanying with fluorescence enhancement of 512QDs, whereas the FRET-2 efficiency is increased, leading to fluorescence quenching of 572QDs. The independence of the dual FRETs is confirmed by the consistent fluorescent responses to Zn2+ between the single-emission probes and the ratiometric probe by monitoring the emission intensity at 512 and 572 nm, respectively (Figure 2B). The selectivity for Zn2+ of this ratiometric probe as well as single-emission probes (512QD@SiO2/TSPP and 572QD@ SiO2/TSPP) is investigated, and various metal ions such as abundant cellular cations (Na+, K+, Ca2+, Mg2+), trace cations in organisms (Fe2+, Fe3+, Zn2+, Cu2+, Ni2+, Co2+, Cr3+, Mn2+), and toxic heavy metal cations (Cd2+, Pb2+, Ag+, Hg2+) are tested under physiological conditions. As shown in Figure 4A, a
Figure 4. (A) Selectivity of single-emission probes and the ratiometric probe in 10 mM PBS buffer solution at pH 7.4 for various ions. (B) Effect of coexisting ions on the signal (I512/I572) of the ratiometric probe in the presence of Zn2+. Final concentrations of the ions: Zn2+, 6.0 μM; Na+, 140 mM; K+, 5.0 mM; Ca2+, 2.5 mM; Mg2+, 1.0 mM; Fe2+, 20 μM; Fe3+, 25 μM; Cr3+, Mn2+, and Cd2+, 1.0 μM; Ni2+, Co2+, Pb2+, Cu2+, Ag+, and Hg2+, 0.5 μM.
Figure 5. Fluorescent images of live HCT116 cells incubated with the ratiometric probe: top row, treatment without Zn2+; bottom row, treatment with 6 μM ZnCl2 and 12 μM pyrithione. (A, D) Images obtained through the yellow channel. (B, E) Images obtained through the green channel. (C, F) Overlay of fluorescent images from the two channels and the corresponding bright field image.
remarkable enhancement of I512/I572 is induced by Zn2+, and no obvious changes of I512/I572 are observed for the other metal ions, demonstrating the excellent selectivity of this ratiometric probe for Zn2+. In contrast, the single-emission probes suffer from the interference of Fe3+, Cu2+, Ag+, and Hg2+. Fe3+ enhances but Cu2+, Ag+, or Hg2+ quenches the fluorescence of both 512QD@SiO2/TSPP and 572QD@SiO2/TSPP. The Fe3+ induced fluorescence enhancement of single-emission probes is because that Fe(III)TSPP exhibits all decreased absorbance centered at 515, 555, and 592 nm, respectively, compared to TSPP, leading to simultaneously reduced efficiencies of FRET1 (from 512QDs to Fe(III)TSPP) and FRET-2 (from 572QDs to Fe(III)TSPP) (Figure S9 in the Supporting Information). Cu2+,
channel fluorescent images for HCT116 cells incubated with the probe for 12 h. In the absence of Zn2+, HCT116 cells show a strong yellow emission (Figure 5A) and a weak green emission (Figure 5B), with the yellow/green emission ratio of 2.61:1 (Figure S11 in the Supporting Information). After the cells are treated with 6.0 μM ZnCl2 and 12 μM pyrithione, a strong quenching of the yellow signal (Figure 5D) and a remarkable enhancement of the green signal (Figure 5E) can be clearly observed, and the yellow/green emission ratio becomes 0.73:1, which agrees well with the evolution of dual-emissions of this probe in an aqueous solution. These preliminary results demonstrate that this ratiometric fluorescent probe is practi5322
DOI: 10.1021/acs.analchem.5b00514 Anal. Chem. 2015, 87, 5318−5323
Article
Analytical Chemistry cally useful for tracking intracellular labile Zn2+ with satisfying resolution.
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CONCLUSION In summary, by preparing a mixture of dual-emission silicaencapsulated QDs, in conjugation with TSPP, we have developed a selective and sensitive strategy for the ratiometric fluorescent detection of Zn2+. Dual independent FRET processes are involved in the ratiometric detection of Zn2+, which is advantageous in specificity and sensitivity over singleemission probes. A perfect visual identification for Zn2+ by distinct color changes is achieved by this probe in aqueous solutions. This ratiometric probe exhibits well-resolved dual emissions and a large range of emission ratios and thus can be successfully applied for imaging and biosensing of Zn2+ in living cells. This work provides a method for designing QDs-based ratiometric fluorescent assays with high specificity and sensitivity for in vivo imaging and biosensing of metal ions, which can be eventually useful for metalloneurochemistry studies.
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ASSOCIATED CONTENT
S Supporting Information *
Additional fluorescence data and spectra, TEM images, zeta potential data, FT-IR spectra, comparison of principles and analytical parameters of different fluorescent Zn2+ probes, and absorbance spectra. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b00514.
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AUTHOR INFORMATION
Corresponding Author
*Fax: (86) 25-83792349. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is financially supported by NSFC and Jiangsu Province (Grants 21375015, 21005017, and BK20141334).
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DOI: 10.1021/acs.analchem.5b00514 Anal. Chem. 2015, 87, 5318−5323
