Dual-Shell Fluorescent Nanoparticles for Self-Monitoring of pH

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Dual-Shell Fluorescent Nanoparticles for Self-Monitoring of pH-Responsive Molecule-Releasing in a Visualized Way Lingang Yang, Chuanfeng Cui, Lingzhi Wang, Juying Lei, and Jinlong Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b05872 • Publication Date (Web): 05 Jul 2016 Downloaded from http://pubs.acs.org on July 7, 2016

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ACS Applied Materials & Interfaces

Dual-Shell Fluorescent Nanoparticles for SelfMonitoring of pH-Responsive Molecule-Releasing in a Visualized Way Lingang Yang, Chuanfeng Cui, Lingzhi Wang*, Juying Lei, and Jinlong Zhang* Key Laboratory for Advanced Materials and Institute of Fine Chemicals, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237 (P. R. China)

KEYWORDS：Carbon Dot, PMAA, Fluorescence Resonance Energy Transfer, pH-Sensitive, Visible Release

ABSTRACT: The rational design and controlled synthesis of smart device with flexibly tailored response ability is all along desirable for the bio-application but long remains a considerable challenge. Here, a pH-stimulated valve system with a visualized “on-off” mode is constructed through a dual-shell fluorescence resonance energy transfer (FRET) strategy. The dual shells refer to carbon dots and fluorescent molecules embedded polymethacrylic acid (F-PMAA) layers successively coating around a SiO2 core (ca. 120 nm), which play the roles as energy donor and acceptor, respectively. The total thickness of the dual-shell in the solid composite is ca. 10 nm. The priorities of this dual-shell FRET nanovalve stem from three facts: (1) the thin shell allows the formation of efficient FRET system without chemical bonding between energy donor and

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acceptor; (2) the maximum emission wavelength of CD layer is tunable in the range of 400-600 nm, thus providing a flexible energy donor for a wide variety of energy acceptors; (3) the outer F-PMAA shell with a pH-sensitive swelling-shrinking (on-off) behavior functions as a valve for regulating the FRET process. As such, a sensitive and stable pH ratiometric sensor with a working pH range of 3-6 has been built by simply encapsulating pH-responsive fluorescein isothiocyanate (FITC) into PMAA; a pH-dependent swelling-shrinking shuttle carrier with a finely controllable molecule-release behavior has been further fabricated using rhodamine B isothiocyanate (RBITC) as the energy donor and model guest molecule. Significantly, the controlled releasing process is visually self-monitorable.

Introduction Fluorescence resonance energy transfer (FRET), the non-radiative and distance-dependent energy transfer between two chromophores, is known as the spectroscopic ruler for measuring the tiny distance variation between fluorescent molecules.1-4 The distance between the donor and the acceptor should be less than 10 nm, thus allowing the excited-state energy to be transferred from the donor to the acceptor via a dipole-dipole coupling.1-4 Benefitting from the extreme sensitivity to distance, FRET based sensors have been intensively studied and widely applied to the analysis of ions, organics and bio-events.5-14 Besides the distance, the fluorescence emission of energy donor and the absorbance of energy acceptor should be overlapped,1-4 which however has significantly restricted the application of FRET system due to the shortage of available chromophores. Quantum dots (QDs) including CdS, CdSe and CdTe are desirable energy donor concerned with their tunable fluorescence


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emission.15-19 Unfortunately, the cytotoxicity has long obstructed their applicability.20,21 Recently, non-metal carbon dots (CD) have been intensively reported and applied to fabricate versatile sensors due to the easy preparation and abundant resources.22-25 Most importantly, it has flexible emission dependent on the excitation wavelength and existing form (colloidal suspension, film, porous nanoparticle), making it a good alternative energy donor for the construction of versatile FRET system.26,27 Meanwhile, polymethacrylic acid (PMAA) composed of polymeric backbones with ionic carboxyl pendant groups is known as one of the most commonly studied pH-responsive hydrogels. In an appropriate pH range, the pendant groups ionize and form fixed charges on the polymer network, resulting in the swelling of the hydrogel due to the electrostatic repulsive force.28 Little pH-variation can cause a significant swelling-shrinking behavior, making PMAA active for controlling the in-and-out of guest molecules. As such, a controlled release of preembedded guest molecules could be achieved on PMAA. However, there is a lack of efficient monitoring means to in situ follow the stimuli-responsive variation of polymeric network, making it hard to real-time understand and regulate the molecule-releasing process. Enlightened by the high distance-sensitivity of FRET, the effective monitoring and controlling of the stimuliresponsive behavior of PMAA is expected to be achieved through an appropriate combination of FRET and PMAA. Here, a novel FRET system is designed and fabricated by successively coating CD-doped SiO2 and fluorophores-embedded PMAA layers with a total thickness of ca. 10 nm around a SiO2 core, where CD and encapsulated fluorescent molecules play the roles of energy donor and acceptor, respectively. The emission of CD can be flexibly tuned in the range of 400-600 nm by varying its doping concentration in the inner layer, which allows the construction of versatile FRET systems


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by freely altering the guest molecules in PMAA as demonstrated by FITC and RBITC with the maximum absorption wavelengths of 490 nm and 554 nm. A high FRET efficiency (> 70%) and a short distance (< 4 nm) between energy donor and acceptor have been achieved from this dualshell FRET strategy. Finally, a pH sensor with a working pH range of 3-6 was fabricated from FITC-containing FRET system; a visualized molecule-release was achieved from RBITCembedded particle, where the PMAA shell with finely controlled pH-sensitive swellingshrinking ability is served as a shuttle valve to regulate the FRET efficiency. Experimental section Materials. All chemicals, including anhydrous citric acid (AR), hexane (AR), tetraethyl orthosilicate (TEOS) (AR), ammonium hydroxide (AR), ethyl alcohol (AR), acetonitrile (AR), N, N'-methylene diacrylamide (MBA) (AR) and methacrylic acid (MAA) (AR) were used as received without any further purification. N-(2-aminoethyl)-3-aminopropyl methyl dimethoxy silane (AEAPMS) (96%), fluorescein isothiocyanate (FITC) (HPLC), rhodamin B isothiocyanate (RBITC) (HPLC) and vinyltriethoxysilane (VTES) (97%) were purchased from Sigma-Aldrich, and deionized distilled water was used for all experiments. Synthesis of Si-CDs. Methylmethoxysilane-modified carbon dots (Si-CDs) were prepared according to the method addressed by Liu’s group.1 Generally, 10 mL AEAPMS was added into a 100 mL three-necked flask and was degassed with flowing nitrogen for 30 min. As soon as the temperature was raised to 242 °C, 0.5 g anhydrous citric acid was added rapidly into the solution and vigorous stirring was indispensable. After being maintained at 242 °C for 2 min, the mixture was naturally cooled to the room temperature and then, washed with hexane for several times to obtained the final product as an orange oil.


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Synthesis of SiO2 nanoparicles (NPs). SiO2 NPs were synthesized through a Stöber route at room temperature. Typically, 3.5 mL ammonium hydroxide and 20 mL ultrapure water were dispersed into 76.5 mL ethyl alcohol, stirred and named solution A. Besides, 5 mL TEOS was dispersed into 95 mL absolute ethyl alcohol, stirred and named solution B. After 30 min, solution A and solution B were mixed together and keep stirred for further 6 h and an oyster white emulsion was obtained. The SiO2 NPs were collected by centrifugation and washed with ethanol and water for three times, respectively. The SiO2 NPs were re-dispersed in ethyl alcohol (20 mL) for further use. Coating with Si-CDs and decorated with vinyl group (SiO2@CD-X, X = VVTES:VCD). The synthetic procedure is modeled on SiO2@CD-2 where the volume ratio between VTES and CDs is 2. First, 5 mL SiO2 ethyl alcohol dispersion prepared above, mixed with 135 mL ultrapure water, 1.98 mL ammonium hydroxide and 40 mL ethyl alcohol, and keep stirring for 30 min. After that, 0.4 mL VTES and 0.2 mL Si-CDs in ethanol (0.4 mL) were added. It should be note that for the preparation of SiO2@CD-X with different doping amounts of CDs, the volume of VTES was fixed while the amount of CDs were varied. After reaction for 12 h at room temperature, the SiO2@CD-X was collected by centrifugation and dried in 60 °C vacuum drying oven after being washed with ethanol and water for three times, respectively. The modified SiO2@CD-X NPs were grinded for further use. Preparation of FRET-based nanoparticles. The FRET-based dual-shell NPs were synthesized through a distillation-precipitation polymerization process. First, 200 mg modified SiO2 NPs prepared above were dispersed in 80 mL acetonitrile by ultrasonication to form a homogeneous emulsion. Then, added 16 mg FITC (or RBITC) as FRET energy acceptor, 0.088 g MBA as cross-linking agent, 0.82 mL MAA as monomer and 0.02 g AIBA as initiator. The


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reaction system was transferred to a distilling apparatus with 90 °C oil bath before all the drugs were dissolved well. The reaction was finished 6 h later and nearly half of the acetonitrile was distilled. When the reacting solution was cooled to room temperature, the final product was collected by centrifugation and washed with diluted hydrochloric acid (pH = 2) to remove unreacted material and unencapsulated dye molecules. The FRET-based hybrid fluorescent nanoparticles were dried in 60 °C vacuum drying oven and grinded for further test. pH Stimuli-Responsive Molecule-Releasing. The FRET-based hybrid dual-shell fluorescent NPs were characterized by spectrofluorometry to assess the pH-sensitivity in phosphate-buffered saline (PBS, 0.1 M). Typically, 10 mg of hybrid NPs were dispersed in 10 mL of PBS and the pH of the suspension was adjusted to 3, 4, 5, 6, 7, 8, 9, 10, 11, respectively. Then, the suspensions with different pH value were transferred into a gas bath thermostatic oscillator to allow the molecule releasing via a diffusion-controlled mechanism. It's worth noting that the fluorescence emission spectra of all the suspensions are recorded at fixed time-intervals. In the pH-changing circulation experiment, SiO2@CD@ RBITC-PMAA dual-shell NPs which had been pre-treated in basic PBS (pH = 11) for 48 h were dispersed in near neutral PBS with pH = 6.5. After 1 h, the solution was instead of basic PBS (pH = 10) and keeping for 0.5 h, then a near neutral PBS (pH = 6.5) is provided again. Thus circulation experiment was repeated for 5 times. It's worth noting that the volume of the solution must be kept the same and the emission spectrum was recorded before the changing of pH every time. Characterization. The transmission electron microscopy (TEM) was conducted on a JEOL transmission electron microscope (JEM-2100EX, 200 KV). Scanning electron microscope (JSM6360LV) was employed to characterize the morphologies of the samples. Diffuse reflectance spectra of UV-Vis were acquired at room temperature by means of a Shimadzu UV-Vis 2450


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scan spectrophotometer equipped with an integrating sphere assembly and BaSO4 was used as the reference. The one-photon fluorescent emission spectra were obtained using a RF-5301PC luminescence spectrometry at room temperature. The CIE 1931 color coordinates as described with chromaticity (x, y) on the CIE diagram were calculated by using the CIE1931xy.V.1.6.0.2a software. Confocal fluorescence images were obtained using an OLYMPUS Fluo View FV1000 confocal fluorescence microscope laser with a 60× oil-immersion objective lens.

Results and Discussion Preparation and characterization of SiO2@CD@PMAA dual-shell nanoparticles. Typically, for the fabrication of dual-shell nanoparticles (NPs), athoxysilyl-modified carbon dot (Si–CD) prepared through the pyrolyzation of citric acid with N-(2-aminoethyl)-3-aminopropyl methyldimethoxy silane (AEAPMS) as coupling agent27 was coated around SiO2 NPs together with vinyltriethoxysilane (VTES) by a modified Stöber method (SiO2@CD-X, X = VVTES:VCD), and then followed with a further coating of PMAA shell from the polymerization of MAA,29 as illustrated in Scheme 1. Scanning electronic microscopy (SEM) image (Figure 1A) shows that the naked SiO2 NPs are monodisperse and have a uniform particle size of ca. 120 nm. Using sample SiO2@CD-4 as an example, the particle shows no obvious change after coating with CD (Figure 1B) and has a size-increase of ca. 20 nm after the second coating of PMAA (Figure 1C). The transmission electronic microscope (TEM) images indicate that the particles before and after dual-time coating both have a good monodispersity and size-uniformity (Figure 1D-F). Besides, a core-shell structure is clearly observed after the second coating, accompanied by a surfacecoarsening. The dual-layer shell is ca. 10 nm thick, which is accordant with the results from the SEM image. A DLS diagram (Figure S1) is further given to show the size distribution of SiO2


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NPs (black), SiO2@CD-4 NPs (red) and SiO2@CD@PMAA dual-shell NPs (blue), respectively. The FT-IR spectra (Figure 1G) of all samples show bands at 1104, 962, 798 and 472 cm-1, characteristics of SiO2 core. The FTIR spectrum of SiO2@CD-4 shows an additional band at 1635 cm-1 ascribed to the vibration of CONH2, demonstrating the successful coating of CD. In the FTIR spectrum of SiO2@CD@PMAA, the bands at 1709, 1540 and 1390 cm-1 can be assigned to the carboxyl vibrations, the bands around 2930 and 1476 cm-1 belong to the methyl vibrations, and the peak at 3420 cm-1 stems from the hydroxyl vibration, which confirm the presence of PMAA after polymerization. The UV-Vis diffuse reflectance spectra (Figure 2A) of all SiO2@CD-X NPs show a wide absorption band at around 360 nm, which is well in accordance with previous reports,24,27 and further demonstrates the successful coating of CD around SiO2 core. The SiO2@CD-X NPs excited at 360 nm presents emission bands in the range of 400-600 nm, which shows a bathochromic shift with the increasing doping amount of CD (Figure 2A).24 Specifically, SiO2@CD-4 NPs prepared at VVTES/VCD = 4 show a single emission peak centered around 460 nm, while a new band arises around 520 nm when the VVTES/VCD ratio is decreased to 2, whose intensity further increases for sample SiO2@CD-1. The elaborate tailoring of emission wavelength thus provides a flexibly alternative energy donor for the further construction of FRET system. Fluorescence of SiO2@CD@FITC-PMAA dual-shell NPs. Here, the 10 nm-thick dual-shell is elaborately designed and fabricated to satisfy the requirement for the formation of FRET. When FITC, a commonly used pH-sensitive dye with an absorption band centered around 490 nm (Figure 3A, red), is encapsulated into PMAA shell, SiO2-CD-4 with the emission band centered at 460 nm is chosen as the energy donor according to the energy overlapping principle


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(Figure S 2A).1-4 The synthesis is simply carried out by extra introducing FITC molecules into the second coating system to form SiO2@CD@FITC-PMAA dual-shell NPs. When excited at 360 nm, the composite shows bimodal emission centered at ca. 460 and 520 nm (Figure 3A, black), which originate from CD and FITC, respectively, suggesting the successful FRET from CD to FITC. E = 1 − ⁄

1

= 8.785 × 10 η κ λ

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λ = ∑ λ " λ λ ∆λ / ∑ λ ∆λ &' = (' + * ; ' = 1⁄&' − 1

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To more clearly understand the FRET process in the above dual-shell NPs, calculation of the energy transfer efficiency was carried out, as well as the average distance between energy donor and acceptor. The energy transfer efficiency, denoted as E, is calculated by comparing the fluorescence intensity of the donor alone (FD) and in the presence of the acceptor (FDA) according to Equation 1.2-4 A quite high energy transfer efficiency (E) of 74.2 % is thus calculated (see SI in detail). In a Förster model, Förster radius Ro is the distance between the donor and acceptor where the FRET efficiency is 50%. Here, it is calculated as 3.14 nm according to equations 2 and 3.2-4 The average donor-acceptor distance in the as-synthesized dual-shell NP is then further calculated out according to equation 4 (2.63 nm).2-4 Fluorescence of SiO2@CD@RBITC-PMAA dual-shell NPs. The flexibly tunable emission wavelength of CD in a wide range allows the substitution of energy acceptor with different absorption. Here, pH-stable fluorescent RBITC with the maximum absorption wavelength at 554 nm was further chosen as an energy acceptor (Figure 3B, red). SiO2@CD-1 with a maximum emission wavelength at 520 nm is adopted as the energy donor. The synthesis is the same as that of SiO2@CD@FITC-PMAA dual-shell NPs. As shown in Figure 3B (black), the simultaneous


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emission of CD and RBITC indicates the successful encapsulation of RBITC molecules and the effective energy transfer from CD to RBITC. Benefitting from the well overlapping between the emission band of SiO2@CD-1 and the absorbance band of RBITC (Figure S 2B), excellent energy transfer efficiency of ca. 89.4 % is achieved, and the average donor-acceptor distance of the as-synthesized dual-shell NPs is further calculated as 3.17 nm, with a Förster radius Ro of 4.96 nm. pH-sensitivity of SiO2@CD@FITC-PMAA dual-shell NPs. The influence of pH to the FRET process was further investigated using SiO2@CD@FITC-PMAA as a demonstration. With the fluorescence spectrum at pH =7 as the reference, the peak intensity of CD increases with the decreasing pH, accompanied by the peak decreasing of FITC (Figure 4A), which should be caused by the formation of lactonized FITC with poor fluorescence in acid environment. In contrast, the fluorescence intensity of CD increases and the emission of FITC decreases with the increasing pH in basic environment, with a less significant variation magnitude. After 24 h, the peak patterns in acidic system almost keep fixing, while the increasing of CD peak and decreasing of FITC peak become more obvious in basic conditions (Figure 4B). To clearly understand the variation of FRET process with pH and time, the emission intensity ratios of CD to FITC (ICD/IFITC) were further plotted against the pH value (Figure 4C). It is obvious that ICD/IFITC linearly decreases with the increasing pH from 3 to 6. The ratio at pH = 7 is almost the same as that at pH = 6, which then slowly increases with the increasing pH from 7 to 11 with a smaller slope. The curve in the acid environment keeps the same after 24 h, further verifying the linear decreasing of ICD/IFITC along with the increasing pH value from 3 to 6 is attributed to the pH-sensitive fluorescence of FITC,29 and there is no leakage of fluorescent molecules from the PMAA shell in the acidic system. In contrast, the slope in basic conditions is significantly


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improved after 24 h, suggesting a decreased FRET efficiency. Since FITC is strongly fluorescent in basic conditions, the decreased efficiency should be attributed to the slow diffusion of FITC into the solvent from a swollen PMAA.30 Consequently, the average distance between CD and FITC is lengthened and the FRET efficiency is declined. Moreover, the inflection point at pH = 6 shown in Figure 4C regardless of the time is well accordant with the critical point for the swelling of PMAA, demonstrating a high sensitivity of our dual-shell FRET system to the swelling-shrinking process. Self-monitored PMAA-Swelling and Molecule Release. The polymeric backbone of PMAA is swollen in basic environment, which may cause the possible release of encapsulated guest molecules. The distance-sensitivity of FRET system provides the chance for self-monitoring of the PMAA-swelling and the molecule-release through the spectrofluorometer. Here, the relation between the swelling degree of PMAA and the efficiency of FRET in different pH environment was studied using SiO2@CD@RBITC-PMAA NPs encapsulated with pH-stable RBITC as a demonstration, with the pH value varying from 6 to 11 and a time-interval of 12 h. In the SiO2@CD@RBITC-PMAA dual-shell system, RBITC plays roles of the FRET energy acceptor and the released object simultaneously. It is obvious that the emission bands of the composite at different pH are almost the same in the beginning (Figure 5A, 0 h). After 12 h, the fluorescence spectra of composites vary depending on the pH value, where the CD peak increases and RBITC peak decreases with the increasing pH value (Figure 5B), indicative of a decreased FRET efficiency in the basic conditions. The variation tendency in the solution with pH > 8 becomes more distinct after 24 and 40 h (Figure 5C, D), while those in nearly neutral conditions (pH = 6-8) almost keep fixing regardless of the time. The decreased FRET efficiency with the increasing pH suggests an enlarged distance between CD and RBITC. The emission intensity ratios of CD to


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RBITC (ICD/IRBITC) at different pH were further plotted against time (Figure 5E), which all keeps increasing within a long period of ca. 320 h except the constant value at pH = 6 (not shown). The ICD/IRBITC ratios at pH = 7 and 8 keep linearly increasing with the prolonging time, while those at higher pH values all show a jump within the first 48 h, then followed by a much slow linear increase with a slope similar to those in the neutral environment. Therefore, the variation in FRET efficiency in the basic system with pH > 8 should be a combinational result of initially exquisite PMAA-swelling and a later slow diffusion of RBTIC from PMAA to the solution. Moreover, all the solutions with different pH values after 12 h show typical pink color of RBITC according to its absorption band centered at ca. 530 nm (Figure 3B), which appear orange to pale blue under the UV-light irradiation as a result of FRET from CD to RBITC (Figure 5F). Therefore, our dual-shell FRET system could be used for the real-time monitoring of pH-stimuli molecule-release simply through visualized fluorescence, which has never been achieved previously. To more clearly demonstrate the performance of PMAA as a pH-responsive valve and the in situ monitoring efficiency of the dual-shell FRET system, the emission spectra of SiO2@CD@ RBITC-PMAA in basic (pH = 10) and acid (pH = 6.5) solutions buffered with PBS are recorded and the corresponding ICD/IRBITC ratios are calculated with fixed time-intervals after the pHadjusting, where the composite was pre-treated in basic PBS (pH = 11) for 48 h. The ICD/IRBITC ratio decreases from 1.95 to 1.65 with the pH value decreasing from 10 to 6.5, which reverses to 1.95 when the pH value re-adjusts to 10. A total of 5 cycling was carried out, where the ICD/IRBITC ratio finely oscillate up and down with the varied pH value. Besides, the ICD/IRBITC ratio gradually increases with the incremental cycling times, regardless of basic or acid environment, which should be attributed to the slow release of embedded RBITC with the


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prolonging time. In contrast, the oscillation amplitude does not vary much, suggesting a finely controlled reversible swelling-shrinking behavior of PMAA and an excellent in situ monitoring efficiency of our dual-shell FRET system (Scheme 2). The confocal fluorescent microscopic imaging was further applied to demonstrate the coding and labelling performances of the dual-shell NPs (Figure 7). Here, two kinds of SiO2@CD@PMAA dual shell NPs doped with FITC and RBITC respectively are mixed and excited upon a 404 nm argon-ion laser, where two distinguishable bright colors are simultaneously and clearly observed (Figure 7A). Besides, these hybrid colloids also show bright colors under 800 nm argon-ion laser, which should be attributed to the two-photon fluorescence of CD (Figure 7B). In consideration of the advantages of two-photon fluorescence including deeper penetration depth (>500 µm), lower tissue auto-fluorescence, as well as reduced photodamage and photo-bleaching, these dual-shell multifluorescent colloids are expected to find a variety of biological applications. Furthermore, the variation of FRET can be indicated by confocal fluorescent microscopic imaging with Ratio channel in different colors. As shown in Figure 7C, the Ratio channel (IRBITC/ICD) of SiO2@CD@RBITC-PMAA dual-shell NPs before releasing exhibited yellow dots, indicating a higher Ratio value and verifying high energy transfer from CD to RBITC. In contrast, the blue white dots in Figure 7D proved the poor FRET with a lower Ratio value in SiO2@CD@RBITC-PMAA dual-shell NPs after releasing. Mechanism of FRET. The possible mechanism for the formation of FRET in the dual-shell NP was further studied through the transient-state photoluminescence (PL) spectrum. The PL lifetime of CD in the SiO2@CD@RBITC-PMAA composite with the emission at 520 nm were measured at pH = 8, which prolongs with the swelling time from 0 (2.47 ns, Figure 8A), 2 (6.22


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ns, Figure 8B) to 5 days (7.42 ns, Figure 8C), ascribing to the slow diffusion of RBITC from PMAA into solution as discussed above. The PL decay can be well fitted by a double-exponential model shown in equation 5, where I/(t) is the intensity of PL decay signal, τ1 and τ2 are the decay time, and A1 and A2 are the corresponding magnitude.31, 32 , - = . exp − -⁄2 + . exp − -⁄2

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The double-exponential model suggests that two emissive states are involved in the PL decay. Here, the faster decay component (τ1) should be attributed to initially populated core-state recombination, and the much slower component (τ2) is originated from surface-related indirect recombination with a relative low surface traps energy level compared to the core-state.31, 32 The variation of τ1 and τ2 along with the swelling time are further illustrated with pie charts (Fig. 8D8F), where the proportion of surface-related τ2 gradually increases from 36.63% to 61.37%, accompanied by a significantly declined lifetime from 5.31 to 11.52 ns. In contrast, the difference of τ1 is negligible compared with the distinct falling proportion. The above results clearly demonstrate that the energy transferred from CD to RBITC is derived from the surfacerelated emissive state rather than the initially core-state (Figure S3). Conclusions In summary, a dual-sell strategy is developed to construct a pH-sensitive FRET system, where a 10 nm-thick dual-layer composed of CD and fluorescent molecules-embedded PMAA is coated around SiO2 colloid through a layer-by-layer process. The pH-triggered swelling-shrinking of PMAA is utilized to regulate the distance between the energy donor and acceptor. The easily tailored emission of CD in the range of 400-600 nm provides a flexibly optional energy donor for versatile energy acceptors. The encapsulation of pH-responsive FITC results in a stable


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ratiometric sensor operating in the acid conditions, where a stable detection performance is achieved due to the shrinkage of PMAA. The adoption of RBITC produces a fluorescently visible molecule releasing system in the basic range. Besides, the hybrid possesses two-photon imaging ability. This two-shell strategy describes the blueprint of rational engineering and controlled synthesis of stimuli-responsive FRET system to achieve more smart function and extended application.

Figure 1. SEM images (A, B, C), TEM images (D, E, F) and FT-IR spectra (G) of SiO2 NPs, SiO2@CD-4 NPs and SiO2@CD@PMAA dual-shell NPs, respectively.


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Figure 2. Absorbance spectra and normalized fluorescent emission spectra (Ex: 360 nm) (A) and CIE chromaticity diagram (B) of SiO2@CD-X NPs with different ratio of CD.

Figure 3. (A) Normalized absorbance spectrum of FITC and emission spectra of SiO2@CD4@FITC-PMAA in PBS with pH=7. (B) The normalized absorbance spectrum of RBITC and emission spectra of SiO2@CD-1@RBITC-PMAA in PBS with pH=7.

Figure 4. Emission spectra (A: 0 h, B: 24 h) and plot (C) of emission intensity ratio between CD and FITC of SiO2@CD@ FITC-PMAA NPs in PBS (0.1 M sodium phosphate) with different pH values.


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Figure 5. Emission spectra of SiO2@CD@ RBITC-PMAA after dispersed in PBS for 0 h (A), 12 h (B), 24 h (C) and 40 h (D). (E) plot of emission intensity ratio between CD and RBITC of SiO2@CD@ RBITC-PMAA dual-shell NP after shaking for certain time in PBS at different pH condition and (F) the optical pictures of SiO2@CD@RBITC-PMAA in PBS solutions with different pH value (from left to right: 3-11) after 120 h. (Up: under sunlight; down: under UVlight (360 nm)).


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Figure 6. (A) Emission spectra of SiO2@CD@ RBITC-PMAA dual-shell NPs and (B) plot of emission intensity ratio between CD and RBITC after being dispersed in pH-changed PBS (reciprocated switching between 6.5 and 10; the dispersing time in pH=6.5 is 1 h and 0.5 h for pH=10). The composite was pre-treated in basic PBS (pH = 11) for 48 h.


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Figure 7. Confocal fluorescence images of a mixture of two types (FITC: green; RBITC: red) of SiO2@CD@PMAA dual-shell NPs. (A) One-photon under 404 nm argon-ion laser excitation, (B) Two-photon under 800 nm argon-ion laser excitation. The Ratio channel (IRBITC/ICD) of SiO2@CD@RBITC-PMAA dual-shell NPs with rich FRET (C: before releasing) and poor FRET (D: after releasing). The inserted color strip represents different Ratio value. Purple indicates lower Ratio value, red indicates higher Ratio value.

Figure 8. PL decay curves of CD after the SiO2@CD@ RBITC-PMAA dual-shell NP in PBS (pH=8) being shaken for 0 d (A), 2 d (B) and 5 d (C). Figure D, E and F are the corresponding pie charts of decay times (τ1, τ2) after being shaken for 0 d, 2 d and 5 d, respectively.

Scheme 1. Schematic diagram for preparing the pH sensitive FRET system.


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Scheme 2 Illustration for the pH sensitive FRET process.

ASSOCIATED CONTENT Supporting Information. Calculation of the energy transfer efficiency and the average donoracceptor distance between two FRET dye molecules; overlapped spectra between fluorescent emission of energy donor and the absorbance of acceptor; proposed mechanism of the multiroutes of energy decay and energy transfer. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author Jinlong Zhang* E-mail: [email protected]; Tel.:86-021-64252062 Lingzhi Wang* E-mail: [email protected]; Tel.:86-021-64252062 Author Contributions


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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT This work was supported by the National Nature Science Foundation of China (U1407102, 21173077 and 21377038), the National Basic Research Program of China (973 Program, 2013CB632403), the Science and Technology Commission of Shanghai Municipality (14ZR1410700 and 14230710500) and the Fundamental Research Funds for the Central Universities. REFERENCES (1) Stryer, L.; Haugland, R. P. Energy Transfer: A Spectroscopic Ruler. Proc. Natl. Acad. Sci. U. S. A. 1967, 58, 719-726. (2) Stryer, L. Fluorescence Energy Transfer as A Spectroscopic Ruler. Annu. Rev. Biochem. 1978, 47, 819-846. (3) Ray, P. C.; Fan, Z.; Crouch, R. A.; Sinha, S. S.; Pramanik, A. Nanoscopic Optical Rulers beyond the FRET Distance Limit: Fundamentals and Applications. Chem. Soc. Rev. 2014, 43, 6370-6404. (4) Yuan, L.; Lin, W.; Zheng K.; Zhu, S. FRET-Based Small-Molecule Fluorescent Probes: Rational Design and Bioimaging Applications. Acc. Chem. Res. 2013, 46, 1462-1473. (5) Jares-Erijman, E. A.; Jovin, T. M. FRET Imaging. Nat. Biotechnol. 2003, 21, 1387-1395. (6) Lu, D., Chen, H., Yan, X., Wang, L., Zhang, J. Ratiometric Hg2+ Sensor Based on Periodic Mesoporous Organosilica Nanoparticles and Förster Resonance Energy Transfer. J. Photochem. Photobiol. A 2015, 299, 125-130.


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Dual-Shell Fluorescent Nanoparticles for Self-Monitoring of pH

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