Microchim Acta DOI 10.1007/s00604-012-0828-z
ORIGINAL PAPER
Synthesis of ratiometric fluorescent nanoparticles for sensing oxygen Xiao-Hui Wang & Hong-Shang Peng & Zhuo Chang & Ling-Ling Hou & Fang-Tian You & Feng Teng & Hong-Wei Song & Biao Dong
Received: 5 January 2012 / Accepted: 22 April 2012 # Springer-Verlag 2012
Abstract A facile reprecipitation-encapsulation method is used for the preparation of ratiometric fluorescent nanoparticles (NPs) for sensing intracellular oxygen. The surface of the NPs is modified in-situ with poly-L-lysine, which renders good biocompatibility and enables easy internalization into living cells. The sensor NPs contain a red fluorescent probe whose fluorescence is sensitive to oxygen with a quenching response of 77 % on going from nitrogen saturation to oxygen saturation, and a reference dye giving a green signal that acts as an oxygen-independent reference. The ratio of the two emissions serves as the analytical information and is sensitive to dissolved oxygen in the 0–43 ppm concentration range. When incorporated into cells, the ratio of the signals increases by 400 % on going from oxygen-saturated to oxygen-free environment. Keywords Ratiometric fluorescence . Nanoparticles . Oxygen sensing . Intracellular X.-H. Wang : H.-S. Peng (*) : F.-T. You : F. Teng Key Laboratory of Luminescence and Optical Information, Ministry of Education, Institute of Optoelectronic Technology, Beijing Jiaotong University, Beijing 100044, China e-mail:
[email protected] Z. Chang : L.-L. Hou College of Life Sciences & Bioengineering, Beijing Jiaotong University, Beijing 100044, China H.-W. Song : B. Dong State Key Laboratory on Integrated Optoelectronics, College of Electronic Science and Engineering, Jilin University, Changchun 130012, China
Introduction Oxygen is a key component for many physiological processes, and measuring dissolved oxygen in living cells is of great importance to understand the complex physiological processes and the diagnosis of major diseases [1–4]. It has been found that hypoxia is closely related to tumor growth, diabetic retinopathy, rheumatoid arthritis and other diseases in the clinical development process. The interests in the methods for measuring dissolved oxygen concentration have been mainly focused on fluorescence-based approaches, which are non-invasive, highly sensitive and do not consume oxygen [5, 6]. Recently, many efforts have been dedicated to nanosized oxygen sensors. The widely adopted oxygen probes are transition metal complexes, whose fluorescence is severely quenched by oxygen molecules [7–10]. After being embedded into nanoparticles (NPs), these oxygen probes are isolated from the outside environment by the particle matrix. Therefore, photostability and cell toxicity of probe dyes are greatly improved in NPs [11–14]. Although oxygen can be sensed by single-intensity-based methods [15–17], more robust signals are obtained by ratiometric fluorescence methods [18, 19]. The built-in calibrations could circumvent drawbacks caused by the local distribution of probes and by drifts of light sources and detectors. In this work we report ratiometric fluorescent NPs for sensing oxygen prepared by a facile reprecipitationencapsulation method [20–22]. The ratiometric NPs comprise the oxygen probe platinum (II) octaethylporphine (PtOEP) and the reference dye coumarin 6 (C6). Upon single wavelength excitation, PtOEP emits red fluorescence which is very sensitive to oxygen, while C6 results in green fluorescence as
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an oxygen-independent reference. Furthermore, the sensing NPs are in-situ surface modified with poly-L-lysine and hence can be effortlessly introduced into cells. The merits of easy synthesis, nanosized dimension, ratiometric emission as well as good cellular uptake make the NPs very promising nanosensors for intracellular oxygen.
Experimental Reagents Pt(II) Octaethylporphine (PtOEP ), Coumarin 6 (C6 ), polyL-lysine (PLL, Mw 04–15 kDa ), Poly(methyl methacrylate) (PMMA, Mw 0150 kDa) and THF were purchased from Sigma-Aldrich. 2-bis(trimethoxysilyl)decane (BTD) was obtained from Gelest. All chemicals were used without further purification. Doubly distilled water was used in all experiments. Preparation of PLL-modified sensor NPs Firstly, PtOEP, C6, PMMA and BTD were dissolved in THF, in a 0.3:3:46.7:50 weight ratio, and at a total concentration of 1000 ppm. Then, using a microsyringe, a 200 μL volume of the solution was rapidly injected into 8 mL of water containing 0.16 mg PLL (pH 9, adjusted by ammonium hydroxide) under sonication. The resulting suspensions were left for 2 h, and further dialyzed against distilled water for 24 h to remove the organic solvent. The resulting solution (containing around 41 mg of NPs per liter) was used for further experiments including spectral characterizations, TEM, zeta potential and cellular observations. Characterization The specimens for transmission electron microscopy (TEM) were prepared as follows: one drop of the NPs aqueous dispersion was placed on a fresh copper mesh and dried
Fig. 2 The transmission electron microscopy (TEM) image of the sensor NPs
gradually at room temperature. TEM images were recorded on a Tecnai 20 transmission electron microscope (www.fei. com) with an acceleration voltage of 120 KV. Zeta potentials were determined by photon correlation spectroscopy using a Zetasizer Nano instrument (www. malvern.com). The measurements were performed at 25 °C with a detection angle of 90 °. The UV-visible absorption spectrum and the steady-state fluorescence spectrum were recorded with a Shimadzu UV-2101PC spectrophotometer (www.ssi.shimadzu.com) and PerkinElmer LS55 florescence spectrometer (www. perkinelmer.com) respectively. NPs calibration, reversibility and experimental setup The calibration and reversibility were both carried out in a cell filled with 2 mL of NPs solution. The cell was sealed with parafilm (Chicago, IL, USA), through which two 1.5-in. needles were inserted as gas inlet and outlet ports. Different dissolved oxygen (DO) concentrations 5
0.0 300
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Fig. 1 Normalized absorption spectra of PtOEP and C6 in THF solution
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Fig. 3 Zeta potential of sensor NPs before (line A) and after PLL coating (line B)
Synthesis of ratiometric fluorescent nanoparticles N2
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Wavelength/nm Fig. 4 Emission spectra of the sensor NPs suspended in aqueous solution at 381 nm excitation at various oxygen concentrations. The inset shows photographs of the sensor NPs in aqueous solution saturated with oxygen (left) and nitrogen (right) upon 381 nm illumination
were obtained by flowing an O2 (99.6 %) /N2 (99.6 %) gas mixture with various ratios, provided by a WITT gas mixer (type KM60-2, www.wittgas.com, Germany), in the range of 1–25 % with an accuracy of 1 % absolute. All spectra measurements were performed at room temperature. The DO concentrations in NPs solution were deduced according to that in oxygen-saturated solutions (43 ppm) based on the solubility equation of oxygen in water [23]. As for the reversibility experiments, nitrogen and oxygen gases were alternately flowed into the cell over 60 min. Cell culture, NPs incubation and intracellular measurements HepG2 (human hepatocellular liver carcinoma cell line) cells were used for the intracellular studies in the experiment. The cells were cultured in 6 cm culture dishes with 4 mL H-DMEM medium containing 10 % FBS and incubated at 37 °C in a 5 % CO2 environment. During the culturing of cells, fresh medium was replaced every 2 days. The above HepG2 cells were further cultured for 1 day in confocal culture dishes to reach a density of 100,000 cells
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Fig. 6 Photobleaching of PtOEP in THF (square) and NPs (circle). The emission spectra were recorded every 10 min under the 381 nm illumination, and data were obtained from the intensity of 650 nm peak
per dish. Then, 0.8 mL of the sensing NPs suspension and 1.2 mL of the H-DMEM medium were co-added into the culture dishes, followed by 12 h incubation to load these NPs into cells [24]. After loading, the cells were washed three times with PBS before microscopy viewing. Intracellular imaging was conducted on an Olympus FV1000 confocal laser scanning microscope (www.olympus. com). Acquisition of each spectral signal was done in Kalman laser mode. The ratiometric NPs were excited at 405 nm and 543 nm with emission collected at 475–550 nm and at 620– 680 nm, respectively. In all the experiments, fluorescent and differential interference contrast images were collected with a 40 x immersion objective using 0.5 μm steps. The resulting zstacked images were analyzed using an FV1000 Viewer (Olympus).
Results and discussion Sensing mechanism of the ratiometric sensor NPs Figure 1 displays the absorption spectra of PtOEP (probe) and C6 (reference). As shown in the figure, there are a few overlaps between the absorption peak of PtOEP and that of 5
R2=0.9987
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Fig. 5 Stern-Volmer plot of fluorescence intensity ratios. The experiment data were calculated from Fig. 4
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Fig. 7 Reversibility of sensors NPs response to dissolved oxygen. The fluorescence ratios are plotted versus the experimental time
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Fig. 8 Confocal microscope images (a, b) and bright field image (c) of HepG2 cells loaded with the sensor NPs in ambient atmosphere. The green fluorescence of C6 of ratiometric NPs was recorded using a
560 nm emission band-pass filter with a 405 nm excitation (a), and the red fluorescence of PtOEP using a 750 nm emission band-pass filter with a 543 nm excitation
C6. A dual emission is therefore expected to occur from asconstructed fluorescent NPs under 381-nm excitation.
emission is highly sensitive to oxygen, whilst that of 510 nm emission is kept constant. The high oxygen sensitivity is visually detectable with the optical contrast of green fluorescence: the aqueous dispersion switches from yellowgreen to orange when saturated gas changes from oxygen to nitrogen (see inset in Fig. 4). This effect is quite favorable in that the ratio of the two intensities can be measured and related to the oxygen level. By defining R as the ratio of the emission intensity of PtOEP (at 650 nm) to that of C6 (at 510 nm), the sensitivity of the nanosensor can be expressed by the overall quenching response to dissolved oxygen [25],
Synthesis and characterization of sensor NPs The ratiometric fluorescent NPs were obtained by the modified encapsulation-reprecipitation method. In essence, a THF solution of PMMA, BTD and the two dyes was injected into basic water containing PLL, PMMA and BTD were used as matrix and silica-based encapsulation agent, respectively. As a result of the sudden increase in water/THF ratio, these hydrophobic species aggregated to form particles; meanwhile, basecatalyzed hydrolysis and condensation of BTD led to the encapsulation of NPs. With the formation of a silica outer layer, positively charged PLL molecules were electrostatically absorbed onto the surface of negatively charged NPs (with silanol groups), and surface modification was consequently finished. As the ratio of the probe to reference should be of comparable emission intensity at ambient atmosphere, the doping ratio of PtOEP/C6 in NPs was empirically determined to be best at 0.3 % and 3 %. Figure 2 shows TEM images of the NPs, indicating that they are almost spherical with a size distribution of 150– 170 nm. Surface modification is demonstrated by the transition of zeta potentials of NPs before and after PLL coating (Fig. 3), specifically from −37.4 mV to 53.9 mV. Positively charged NPs are advantageous for their internalization into living cells, because of the negative nature of the membrane of cell.
Q ¼ ðRN 2 RO2 Þ=RN 2
ð1Þ
where RN 2 and RO2 represent the emission intensity ratio of the sensor in fully deoxygenated and fully oxygenated solutions, respectively. The as-obtained value of Q is around 77 %, which is comparable with that of silica-based NPs [26], but lower than that of polymer NPs [12]. Unlike hydrophobic surface where oxygen is prone to be partitioned out of solution into the gas phase within the sensor particle, the hydrophilic surface of the silica-encapsulated NPs, though having a hydrophobic core, results in inefficient partitioning and consequent reduced sensitivity [12, 17].
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Oxygen sensitivity and calibration of the sensor NPs Oxygen-sensitivity of the ratiometric fluorescent NPs was studied by purging with a gas mixture with various N2/O2 ratios. Figure 4 shows the response of the emission spectra towards DO under 381 nm excitation. The 510 nm and 650 nm emissions are attributed to C6 and PtOEP respectively. From the top to the bottom lines, the DO concentrations are 0.0 (nitrogen saturated), 0.43, 0.86, 1.72, 3.01, 4.3, 6.45, 8.6, 10.75, 21.5 and 43 ppm (oxygen saturated) in sequence. It can be seen clearly that the intensity of 650 nm
Intensity (a.u.)
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Fig. 9 Fluorescence spectra of HepG2 cells loaded with sensor NPs in N2- and O2-saturated Hanks buffer solution. The cells were digested with trypsin solution and excited with 381 nm light
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In solid state systems, the oxygen-quenching process is usually described by the nonlinear Stern-Volmer equation due to matrix heterogeneity effects [26, 27], R f1 f2 ¼ þ R0 1 þ k ð1Þ ½O2 1 þ k ð2Þ ½O2 SV SV
ð2Þ
where R0 is the emission intensity ratio in the absence of oxygen, R the ratio at a given DO concentration, f1 and f2 the emissive fraction of the probes in different environment, kSV the Stern-Volmer quenching constants of the different components, and [O2] the DO concentration. Figure 5 depicts the Stern-Volmer plot of the fluorescent intensity ratios versus DO concentrations for the ratiometric sensor NPs. The line is the fit of the function described by Eq. 2, and yields k(1)SV and k(2)SV, 0.14 and 0.01, respectively. The data fit well by Eq. 2 with a correlation coefficient of > 0.9987, indicating heterogeneous microenvironments inside the hybrid nanosensor. Stability and reversibility of the ratiometric sensor NPs To test the long-term stability of the nanosensors, aqueous suspension of NPs dialyzed again after storing for 4 weeks indicated negligible leakage of dyes, as determined by UV– vis absorption spectroscopy (data are not shown here). The silica encapsulation efficiently prevents the release of dyes from the particle matrix, which is consistent with previous results [20–22]. It is known that dyes embedded inside NPs usually show improved photostability due to the protection of the matrix. In the case of the ratiometric sensor NPs, the emission intensity of PtOEP is bleached by ~ 30 % under intensive continuous illumination of 381 nm light for 1 h, in comparison to that of 80 % in THF solution over the same period (Fig. 6). Fluorescent spectra of the ratiometric NPs dispersion were recorded after alternately purging with N2 and O2 for 10 min under 381 nm excitation. As depicted in Fig. 7, the nanosensors show complete recovery each time that the sensing environments are changed between N2 and O2. Intracellular study The sensor NPs were introduced into living HepG2 cells grown on culture dishes to demonstrate the feasibility of intracellular sensing of oxygen. Figure 8 displays both the bright field and confocal images of the cells after 12 h incubation with NPs. It can be seen clearly that many particles are incorporated into cells, postulated by a vesicular uptake mechanism, and mainly localized inside the cytoplasm but not the nucleoplasm. The efficient cellular uptake as well as intracellular accumulation lay a strong foundation for future sensing intracellular oxygen.
It should be noted that the confocal images in Fig. 8 are taken under two-wavelength excitation, because the ideal 381 nm laser is unavailable. Therefore, intracellular oxygen is no longer sensed using the ratiometric sensor NPs with the confocal microscope. As an alternative, fluorescence of the HepG2 cells loaded with sensor NPs, in N2- and O2saturated solutions respectively, was measured to verify their responsiveness to intracellular oxygen. It can be seen clearly from Fig. 9 that the peak ratio is highly sensitive to intracellular oxygen with an increase of up to ~ 400 % from oxygen to deoxygenated environment. This result indicates that the ratiometric NPs are very promising as a means for sensing intracellular oxygen. In addition, it is noticed that the intensity ratio of red fluorescence to green fluorescence is much smaller than that in Fig. 4. Considering that the ratiometric fluorescence is derived from NPs confined within digested cells, the lowered peak ratio may be interpreted as follows: PtOEP is not as photostable as C6 under the excitation of 381 nm, while the greatly reduced quantity of NPs after cellular uptake exaggerates the decrease of red fluorescence. Further work on the spectral properties of intracellular NPs is required for the practical sensing of intracellular oxygen, and is also being carried out in our group.
Conclusions In summary, a ratiometric fluorescent nanoparticle for sensing oxygen was prepared. The sensor NPs are composed of the oxygen probe PtOEP and the reference dye C6. The ratiometric fluorescence is very sensitive to oxygen with a quenching response of 77 %, and can be well fitted by a nonlinear SternVolmer equation. Because of the PLL modification, the sensor NPs could be easily introduced inside living cells. Towards intracellular oxygen, the ratiometric NPs are very sensitive, and a threefold increase of the peak ratio is observed from oxygen-saturated to oxygen-free environment. Acknowledgment This work was financially supported by the National Natural Science Foundation of China (grant nos. 61078069 and 10979009), National Science Fund for Distinguished Young Scholars (61125505) and Fundamental Research Funds for the Central Universities (2010JBZ006).
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