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Amplified Fluorescence Quenching of Lucigenin Self-Assembled inside Silica/Chitosan Nanoparticles by Cl− Rui Tian, Yingjuan Qu, and Xingwang Zheng* Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi’an, 710062 Shaanxi, People’s Republic of China S Supporting Information *

ABSTRACT: Fluorescence sensing of an analyte based on the fluorophore collective effect is a reliable, sensitive sensing approach. Many ultralow targets can be detected on the basis of the high sensitivity and signal amplification of the fluorescence sensing system. However, the complicated synthesis procedures, harsh conditions required to design and control the fluorescence molecular probes and conjugated chain length, and the higher cost of synthesis are still challenges. To address these issues, we developed a simple, rapid, and sensitive collective effect based fluorescence sensing platform. In this sensing platform, the fluorophore unit was self-assembled on the wall of the nanopores of the porous structural silica/ chitosan nanoparticles (SCNPs) on the basis of the electrostatic interaction and supermolecular interaction between the fluorophores and SiO− groups and chitosan. Since these selfassembled fluorophores are close enough to communicate with each other on the basis of the space confinement effect of the pore size, many fluorophore units could interact with a single analyte and produce an amplified fluorescence sensing ability. Chloride ion, an important anion in biological fluids, and lucigenin, a typical fluorescent dye, were used as a model to confirm the proof-of-concept strategy. Our results showed that, compared to free-state lucigenin in solution, the assembled-state lucigenin in SCNPs presented an about 10-fold increase in its Stern−Volmer constant when the concentration of Cl− was lower than 10 mM, and this fluorescence nanosensor was also successfully used to sense the chloride ion in living cells. hloride ion (Cl−) is one of the major anions in biological fluids and plays numerous roles in biological systems. The determination of Cl− in biological and environmental samples is of great need. Various methods such as ion-selective electrodes1−3 and fluorescence methods4−6 have been utilized for Cl− measurements. As for the fluorescence method, various molecular fluorescence probe immobilized particles were used as Cl− sensors to improve the analysis feature. These particles include probes encapsulated by liposomes, lipobeads, etc.7,8 However, the small Stern−Volmer constant6,9 will negatively affect their analytical sensing properties; thus, there is a need for more sensitive and stable methods for Cl− detection. In the field of fluorescence sensing, the signal amplification strategy is one of the most promising directions to achieve the detection of the ultralow target species.10−12 For this purpose, much work has been done, such as the design and synthesis of excellent fluorophore molecular probes,13 sensing analytes by preorganized sensing elements,14,15 incorporation of nanomaterials to increase loading of tags,16,17 etc.18 Among these strategies, sensing analytes by the collective effect of the preorganized sensing elements has attracted special interest for its signal amplification ability. In this regard, the fluorescent conjugated polymers are the most popular;19−21 however, the complicated synthesis procedures and the nonspecific interaction between the conjugated polymer and targets make this

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© XXXX American Chemical Society

strategy a challenge in practical application. Similarly, another fluorescence signal amplification scheme widely applied was the use of dye-organized silica nanoparticles;9,22−26 Compared to that based on conjugated polymers, a signal amplification platform based on silica nanoparticles has the advantage of easy synthesis and versatility. Therefore, it was imagined that if there are many nanopores within a single dye-doped silica nanoparticle, the dye molecules could easily communicate each other due to their close proximity to each other; thus, one analyte may interact with many sensing units and lead to signal amplification. However, to our knowledge, no research on this aspect has been reported. In this work, we develop a new and sensitive fluorescence sensing strategy. In this sensing platform, the fluorophore unit was self-assembled on the wall of the nanopores of the porous structural silica/chitosan nanoparticles (SCNPs) on the basis of the electrostatic interaction and supermolecular interaction between the fluorophores and SiO− groups and chitosan. Since these self-assembled fluorophores are close enough to communicate with each other on the basis of the space Received: May 18, 2014 Accepted: August 19, 2014

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confinement effect of the pore size, many fluorophore units could undergo a collective process to interact with a single analyte and produce powerful signal amplification. Here, the Cl− quenching of lucigenin’s fluorescence was used as a model to confirm the proof-of-concept strategy. Our results showed that, compared with the previous reports,27−31 our fluorophore sensing system based on the space confinement effect of the SCNPs is easily prepared and biocompatible and produces an about 10-fold enhancement of the Stern−Volmer constant value compared to that of the free-state lucigenin in solution when the concentration of Cl− is lower than 10 mM. Finally, this fluorescence sensing platform was successfully used to sense Cl− in living cells.

Afterward, the mixture was centrifuged and washed with water many times to remove the free lucigenin molecules, leaving the yellow-green lucigenin-assembled SCNPs (LSCNPs). Cell Culture and Fluorescence Imaging Microscopy. Mouse leukemia cells L1210 were cultivated in 25 mL culture bottles and loaded with the LSCNPs by endocytosis. In brief, the mouse leukemia cells, which were in the logarithmic growth phase, were diluted into a suitable concentration cell suspension using RPM1640 culture medium. Then 4 mL of the above cell suspension and some LSCNPs were mixed and incubated in a 25 mL culture bottle in a 5% CO2 atmosphere at 37 °C. After an incubation period of 4 h, the contents were centrifuged and washed with PBS (10 mM, pH 7.4) three times to remove the LSCNPs that were not phagocytosed by the cells. Then 1 mL of PBS (10 mM, pH 7.4) was added, culturing was continued for another 5 min, and the cells were imaged under the fluorescence microscope. To assess the response of the LSCNPs in living cells, 40 μL of 0.01 M KCl was added to the cell suspension which had already phagocytosed the LSCNPs to change the intracellular Cl− concentration followed by further culturing for 30 min, and the cells were observed under the fluorescence microscope.

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EXPERIMENTAL SECTION Chemicals. All reagents and solvents were purchased in their highest available purity and used without further purification. Lucigenin (C28H22N4O6, N,N-dimethyl-9,9-biacridinium dinitrate), tetraethoxysilane (TEOS), chitosan, and Triton X-100 were purchased from Sigma-Aldrich (United States). Ethanol (95%), n-hexane, cyclohexane, acetone, aqueous ammonia solution (25 wt %), and other reagents were of analytical grade and were purchased from the Xi’an Chemical Reagent Factory. Sodium phosphate buffer (PBS) stock solution (pH 7.4) was prepared by dissolving a suitable amount of analytical grade Na2HPO4−NaH2PO4 in ultrapure water. Millipore Milli-Q (≥18MΩ·cm) water was used in all experiments. Instrumentation. A multiposition magnetic stirrer (IKA, Germany) and high-speed centrifuge (5804R, Eppendorf, Germany) were used for the synthesis of silica nanoparticles. Fluorescence spectra were recorded on a Hitachi F-7000 fluorescence spectrophotometer. UV−vis adsorption spectra were recorded on a UV−vis spectrophotometer (TU1901, China). The transmission electron microscopy (TEM) image of the nanoparticles was obtained by a JEM-2100 transmission electron microscope (Hitachi, Japan). The fluorescence lifetime was determined using an Edinburgh FLS 920 fluorescence spectrometer. Mice leukemia cells L1210 (supplied by the College of Life Science, Shaanxi Normal University) were cultivated in an incubator (PYX-DHS, Shanghai Yuejin Medical Instruments Factory, China). Cell imaging studies used a DMLB2 fluorescence microscope (Leica, Germany). Synthesis of SCNPs and Assembly of Lucigenin. The nanoparticles were prepared using the microemulsion method according to the literature.27 Chitosan was dissolved in 0.10 M acetic acid and stored for future use. A 7.5 mL volume of cyclohexane, 1.8 mL of 1-hexanol, and 1.8 mL of Triton X-100 were mixed, and 200 μL of water was added to form a transparent microemulsion. Then 100 μL of 0.5% chitosan in 0.10 M acetic acid was added to the microemulsion, and the pH of the system was adjusted to neutral with NH4OH. After this, the mixture was stirred for 1 h, and 100 μL of TEOS was then added as a precursor for silica formation, followed by the addition of 80 μL of NH4OH to initiate the polymerization process. The reaction was allowed to continue for 24 h at room temperature to form the SCNPs. Then the SCNPs were isolated from the microemulsion using acetone, centrifuged, and washed with ethanol and water several times to discard unreacted materials. Next the SCNPs were suspended in 1 mL of ultrapure water and mixed with the same volume of lucigenin solution inside a centrifuge tube for dye assembly. The mixture was shaken for 40 min to achieve an ideal dye assembly.

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RESULTS AND DISCUSSION Characterization of the Synthesized SCNPs. The morphology of the SCNPs was characterized by TEM. TEM

Figure 1. TEM image of SCNPs (a) and high-magnification TEM images of an SCNP (b) and a silica nanoparticle (c).

images revealed that the synthesized SCNPs were uniform in size and had a diameter of about 60 ± 5 nm as shown in Figure 1. Compared to the pure silica nanoparticles (SNPs) (as shown in Figure 1c), the SCNPs had a porous structure (as shown in Figure 1b). This result indicated that chitosan can make the nanoparticles porous, which may be attributed to the following aspects: when the pH of the reverse microemulsion system was changed to basic (pH higher than the pKa (6.5) of chitosan), B

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Figure 5. Fluorescence lifetime curve of free (black curve) and assembled (red curve) lucigenin.

on the template of porous structure chitosan hydrogels result in the formation of porous SCNPs. Moreover, the pore diameter of the SCNPs was determined by nitrogen adsorption−desorption isotherm measurements at 77 K on a Micromeritic ASAP 2020M system. Figure 2 presents the nitrogen adsorption−desorption isotherm, and the inset of Figure 2 indicates the Barrett−Joyner−Halenda (BJH) pore size distribution of SCNPs. The results showed that the SCNPs were porous, and there are more micropores than mesopores in the SCNPs. To further investigate whether the chitosan was integrated successfully into the nanoparticles, the surface properties of the as-prepared nanoparticles were measured by ζ potential measurement. The results showed that, in pH 6.0 PBS buffer, the ζ potential of the SCNPs was 49.5 mV, but for SNPs the ζ potential was −22.6 mV, which also demonstrates that chitosan was doped into the silica nanoparticles. Self-Assembly of Lucigenin on the SCNPs. When a suitable amount of chitosan was doped into the silica nanoparticles, the mixture network of chitosan and silica made the nanoparticles porous with more SiO− groups exposed on the surface of the SCNPs. Thus, in neutral medium, the strong electrostatic interactions between the SiO− groups in the SCNPs and the amino groups on the lucigenin molecules induce the assembly of lucigenin molecules on SCNPs on the basis of their stronger electrostatic interactions with each other. According to this consideration, the interaction of the SCNPs with lucigenin was studied. Our result showed that, after addition of lucigenin into the aqueous solution of SCNPs for a given time and further separation of these SCNPs from the

Figure 2. Brunauer−Emmett−Teller (BET) nitrogen adsorption− desorption isotherms of the nanoparticles. Inset: BJH pore size distribution of the SCNPs.

Figure 3. Normalized fluorescence efficiency at different self-assembly times in the absence (gray) and presence (pink) of 1.0 × 10−3 M Cl−.

the chitosan could form hydrogels31 and act as a template for the formation of a porous structure. Then when NH4OH was added to the reverse microemulsion system, its catalysis caused the TEOS to hydrolyze and form Si(OH)4. The loosened porous structure of the chitosan hydrogel allows the Si(OH)4 to assemble on the chitosan template on the basis of the hydrogen bond effect between Si(OH)4 and the amino or hydroxyl groups on chitosan; thus, in the subsequent gel procedure, the localized polymerization reactions of Si(OH)4

Figure 4. Absorption spectra (A) and fluorescence emission spectra (B) of the free (black curve) and assembled (red curve) lucigenin. C

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Figure 6. Fluorescence spectra of the LSCNP (A) and LSNP (B) response to Cl−. For curves 1 and 2 the concentration of Cl− was 0 and 4.0 × 10−3 M, respectively.

Figure 7. Stern−Volmer plot describing the response of F0/F to the Cl− concentration (in 10 mM pH 7.4 PBS buffer). F0 is the fluorescence intensity of the LSCNPs in a Cl−-free PBS buffer solution at pH 7.4, and F is the fluorescence intensity of the LSCNPs at a given Cl− concentration. The concentration of Cl− was 2.0 × 10−5, 4.0 × 10−5, 6.0 × 10−5, 8.0 × 10−5, 1.0 × 10−4, 2.0 × 10−4, 4.0 × 10−4, 6.0 × 10−4, 8.0 × 10−4, and 1.0 × 10−3 M.

Figure 8. Fluorescence spectra of the LSCNP response to Cl−. The concentration of Cl− from curve a to curve n was 0, 1.0 × 10−5, 2.0 × 10−5, 4.0 × 10−5, 6.0 × 10−5, 8.0 × 10−5, 1.0 × 10−4, 2.0 × 10−4, 4.0 × 10−4, 6.0 × 10−4, 8.0 × 10−4, 1.0 × 10−3, 2.0 × 10−3, and 4.0 × 10−3 M, respectively.

result indicated that the assembly of lucigenin on SCNPs is a slow procedure; thus, it can provide a chance to adjust the concentration of lucigenin on the SCNPs by changing the assembly time. Moreover, the stability of the LSCNPs in aqueous solution was studied by monitoring the fluorescence intensity of the LSCNPs. Our results showed that the fluorescence intensity of the LSCNP solution was about 90% of the original value when the LSCNPs were stored in the freezer for 1 month. This result demonstrates that the LSCNPs were stable in aqueous solution and the leakage of lucigenin molecules from the LSCNPs was nearly negligible. This may be attributed to the stronger interaction of the lucigenin with the silica matrix. For obtaining better fluorescence quenching of the LSCNPs by Cl−, the effect of the assembly time of lucigenin on the SCNPs on the fluorescence quenching efficiency of LSCNPs by Cl− was investigated. As shown in Figure 3, while the assembly time of lucigenin increased from 10 to 40 min, the fluorescence quenching efficiency of the LSCNPs by 1.0 × 10−3 M Cl− remained nearly the same and was about 50%. At 40 min, the fluorescence quenching efficiency of the LSCNPs by Cl− reached its highest value. Above 40 min, the fluorescence quenching slowly decreased with an increase of the assembly time. Notably, when the assembly time was longer than 24 h, the fluorescence quenching degree of LSCNPs by Cl− reached its minimum value. Therefore, a 40 min assembly time was

Table 1. Linear Equations of the LSCNP Response to Cl− in PBS Buffer at pH 7.4 concn range of Cl−/M −5

−4

2.0 × 10 to 1.0 × 10 1.0 × 10−4 to 8.0 × 10−4

linear equation

R

y = 3479.1x + 0.9818 y = 549x + 1.297

0.9997 0.9855

solution by centrifuge, yellow-green LSCNPs were obtained; meanwhile, ζ potential analysis showed that the ζ potential of the LSCNPs was about 3 mV higher than that of the SCNPs in the same conditions. This indicated that lucigenin molecules were indeed assembled on the SCNPs. It was also found that, while the SNPs and pure chitosan nanoparticles (CNPs) were treated by a procedure similar to that of the SCNPs mentioned above, only the white SNPs became slightly yellow-green (Figure S1 in the Supporting Information). This result demonstrated that the electrostatic interaction between positively charged lucigenin and negatively charged SiO− groups in the silica matrix was the key force to assemble the lucigenin molecules on the SCNPs. We next studied the effect of the assembly time on the LSCNPs. The result showed (Figure S2 in the Supporting Information) that the fluorescence intensity of the LSCNPs rapidly increased when the assembly time increased from 10 to 60 min. Above 60 min, the increase became very slow. This D

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Scheme 1. Self-Assembly of Lucigenin in SCNPs and Its Cl− Sensing Scheme

matrix, and the hydrophobic interaction between lucigenin and the carbon chain of chitosan. To further confirm this effect, the fluorescence lifetimes of free lucigenin and LSCNPs were measured since the lifetime of the assembled-state lucigenin may provide more information about lucigenin molecules in SCNPs. As shown in Figure 5, compared to the 20.52 ns lifetime of free lucigenin in solution, the lifetimes of immobilized-state lucigenin in SCNPs were 9.71 and 17.71 ns with relative amplitudes of 27.95% and 72.05%, respectively. This result suggested that the lucigenin molecules may have two states in the LSCNPs due to the nonhomogeneous distribution of the lucigenin: one state may localize in the inner part of the silica matrix on the wall of the SCNPs, and the other may exist on the surface part of the silica/chitosan hybrid matrix on the wall of the SCNPs (as shown in Scheme 1). Due to the difference in their locations and microenvironments, the lifetimes of the lucigenin molecules were also different. Lucigenin molecules which were located in the inner part of the nanoparticles were relatively far away from the solvated region; as a result, the lifetime of the lucigenin molecules in this domain may be much shorter. However, lucigenin molecules which existed on the surface, due to the surface effect of the silica/chitosan hybrid matrix, were largely exposed to aqueous solution and were more solvent-accessible; this kind of lucigenin molecule may present a relatively longer lifetime. This was consistent with the results of Santra’s group and McDonagh’s group for the FITCand NIR664-doped fluorescent silica nanoparticles.28,30 Meanwhile, since the quantum yield of the dye will change when it is doped into the silica nanoparticles,32 the quantum yields of the LSCNPs were measured by a comparative method;33 the results showed (Table S1 in the Supporting Information) that the quantum yields of the LSCNPs increased when the lucigenin was assembled for about 40 min, which may be caused by a mechanism similar to that for other dye-doped silica nanoparticles.28 Conversely, the quantum yields of the LSCNPs decreased when the lucigenin was assembled overnight. This may be because, with the extension of the

Figure 9. Fluorescence microscopic image of L1210 cells incubated with LSCNPs.

selected for the subsequent work, and the possible reason for these phenomena will be explained in the section “Fluorescence Sensing Behavior of the LSCNP Response to Cl−”. Optical Behavior of Lucigenin on a Silica/Chitosan Composite Nanomatrix. To characterize the optical behavior of the LSCNPs, the absorption and emission spectra of the LSCNPs were measured and further compared with those of free lucigenin in solution. As shown in Figure 4, although the absorption spectrum (Figure 4A) of LSCNPs has nearly the same outline and the same λmax as that of free lucigenin, the outline of the fluorescence emission spectrum (Figure 4B) of the assembled-state lucigenin on LSCNPs shows an obvious difference from that of free lucigenin. As can be seen in Figure 4B, for the LSCNPs, a new and small emission peak occurred at 485 nm, and the original emission peak at 500 nm was slightly decreased. These phenomena indicated that the microenvironment of lucigenin molecules in SCNPs was changed, which may be related to the rigid structure of SiO2, the electrostatic interaction between lucigenin and the SiO− groups of the silica E

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Figure 10. Fluorescence microscopy images of LSCNP-loaded L1210 cells before (A, B) and after (C, D) addition of the Cl− calibration solution.

was nearly stable. This may be because too much excess chitosan cannot form a hybrid with the SCNPs, so 0.5% chitosan was used in the preparation of the SCNPs in the subsequent studies. In addition, it was reported in the previous work that the lucigenin quenching mechanism of Cl− was the collisional quenching process,34 and the relationship between the fluorescence intensity of the lucigenin and Cl− concentration can be described by the Stern−Volmer equation:

assembly time, many more lucigenin molecules were assembled on the SCNPs, causing self-quenching of the lucigenin. Lastly, the photostabilities of free lucigenin and assembledstate lucigenin in the SCNPs were compared. The results showed that, after 1000 s of continuous irradiation, the fluorescence intensity of free lucigenin decreased by 10%, but the fluorescence intensity of LSCNPs decreased by only about 4% (as shown in Figure S3 in the Supporting Information), which demonstrated that the photostability of the assembledstate lucigenin was improved. Fluorescence Sensing Behavior of the LSCNP Response to Cl−. As shown in Figure 6, the fluorescence of LSCNPs was greatly quenched by 4.0 × 10−3 M Cl−, and the quenching efficiency was about 68% (as shown in Figure 6A). In contrast, for the LSNPs, the fluorescence was nearly not quenched by 4.0 × 10−3 M Cl− (Figure 6B). This may be explained by the electrostatic repulsion effect between the SiO− groups in the silica matrix and Cl−. In SNPs, since there are a lot of SiO− groups, Cl− was electrostatically repelled by the negatively charged SiO− groups, which made it difficult for Cl− to approach the lucigenin in the SNPs, resulting in a weak fluorescence quenching efficiency. As for SCNPs, chitosan creates many nanochannels in the SCNPs, and these nanochannels allow the Cl− to easily diffuse into the pores of the SCNPs, approach the lucigenin located on the surface of the nanopores of the SCNPs, and effectively quench its fluorescence. The results demonstrate that chitosan plays a key role in fluorescence sensing. For better fluorescence quenching sensing behavior, the effect of the chitosan concentration on the sensing of Cl− by LSCNPs was studied. The result showed that (as shown in Figures S4 and S5 in the Supporting Information) the ability of Cl− to quench LSCNPs increased when chitosan’s concentration increased from 0.1% to 0.5%. When the concentration of chitosan was above 0.5%, the fluorescence quenching ability

F0 τ = 0 = 1 + KSV[Cl−] F τ

(1)

where F0, τ0 and F, τ are the fluorescence intensities and lifetimes of the fluorophore in the absence and presence of Cl−, respectively, and KSV is the Stern−Volmer quenching constant. On the basis of this consideration, the Stern−Volmer quenching constant of the LSCNP sensing system was measured. Our result showed that, while the Cl− concentration changed from 2.0 × 10−5 to 1.0 × 10−4 M, the measured KSV for Cl− quenching of LSCNPs was 3479 L·mol−1, which was about 1 order of magnitude larger than that of free-state lucigenin in solution. In addition, while the Cl− concentration changed from 1.0 × 10−4 to 1.0 × 10−3 M, the measured KSV was 549 L·mol−1, which was about 2 times that of lucigenin in solution.34 These results showed that although the assembled-state lucigenin molecules presented a large Stern−Volmer constant for Cl−, their fluorescence quenching plot (shown in Figure 7) became curved at higher concentration of Cl− and more linear at lower Cl− concentration (Table 1). This type of curved Stern− Volmer plot has been observed previously and has been attributed to site heterogeneity for the fluorescence dyes.35 Thus, our results, including the curved Stern−Volmer plot and the lifetime results, suggested that there are two states of lucigenin molecules in the LSCNPs. F

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Lastly, to quantitatively evaluate these large KSV values in the LSCNP response to Cl−, the following experiment was designed and performed. First, dilute solutions of free lucigenin and LSCNPs with the same lucigenin molecule number were prepared, and their original fluorescence intensity was measured. Second, the same amounts of Cl− were added to the above solutions, and the fluorescence intensity of the resulting solutions was measured. Our results showed that, compared to the 19% free lucigenin fluorescence quenching efficiency of 1.0 × 10−3 M Cl−, a 55% LSCNP fluorescence quenching efficiency of Cl− was obtained, which was about 3 times that of the free lucigenin. This result indicated that one Cl− ion could quench about three lucigenin molecules in the LSCNPs. Under the optimum experimental conditions, the fluorescence sensing of Cl− by LSCNPs was studied. The results showed (Figure 8) that when the Cl− concentration increased from 1.0 × 10−5 to 4.0 × 10−3 M, the fluorescence intensities of LSCNPs continuously decreased. In the field of fluorescence sensing, the ratiometric sensing scheme was paid much attention due to its better analytical performance.36,37 For establishing a ratiometric sensor, the introduction of a reference dye into the resulting sensor is the key. In principle, with regard to our designed sensing system described herein, there may be two ways to introduce the inert fluorescent dye into the nanoparticles on the basis of the character of silica and chitosan, respectively: one way is doping the inert fluorescent dye into the silica matrix as for other dyedoped silica nanoparticles,28 and the other way is bonding the inert reference dye with chitosan through the well-established covalent bonding route. Here, as a model, we paid our attention to doping the reference dye into the silica matrix to design the ratiometric sensor due to its simple and well-established route. Tris(2,2′-bipyridyl)dichlororuthenium(II) (Rubpy) was selected as the reference dye and was first doped into the silica to form a small silica core, which was capped with the silica/ chitosan composite as a shell. Lastly, the sensing dye, lucigenin, was assembled onto the composite nanoparticle to form the ratiometric sensor (L/Ru/SCNPs). The sensing performance of the designed ratiometric sensor is shown in Figure S7 in the Supporting Information. The results showed that the dyedoped silica core/chitosan hybrid matrix shell nanoparticles have good Cl− sensing ability too, and that, similarly to LSCNPs, when the Cl− concentration increased from 1.0 × 10−5 to 4.0 × 10−3 M, the fluorescence intensities of L/Ru/ SCNPs continuously decreased. This result demonstrates that a ratiometric sensor based on composite silica/chitosan nanoparticles can be established. More importantly, this result demonstrates that the SCNPs may have other potential applications in ratiometric sensing, etc. Possible Assembly Mechanism of Lucigenin on the SCNPs and Its Signal Amplification Mechanism. On the basis of all of the results mentioned above, the possible Cl− fluorescence quenching signal amplification mechanism of the LSCNPs may be explained by Scheme 1. First, concerning the self-assembly procedure, the lucigenin molecules could move into the inner part of the pore in the SCNPs to interact with the SiO− groups due to the porous structure of the SCNPs and the positively charged feature of the lucigenin molecule. Meanwhile, because of the relatively compact structure of the pure silica domain, only a small part of the lucigenin molecules can be assembled into the pure silica domain in the SCNPs. In contrast, for the hybrid part of silica/

chitosan in the SCNPs, which contained both chitosan and silica, due to their porous structure and large surface area, a lot of lucigenin molecules could self-assemble on the surface on the basis of the electrostatic interaction between the SiO− groups and lucigenin molecules. More importantly, because of this pore structure and the space confinement effect of the pore size, many lucigenin molecules exist in one pore and are close to each other. The self-assembly feature is shown in cartoon A of Scheme 1. Second, as mentioned above, two kinds of lucigenin molecules exist in the LSCNPs, one localized in the inner part of the silica matrix and the other one existing on the surface of the nanopores in the SCNPs. For the former one, due to the stronger electrostatic interaction with the SiO− groups within the silica matrix as well as the compact structure of the pure silica matrix, the lucigenin molecules present a shorter fluorescence lifetime and a relatively smaller KSV response to Cl−. For the latter one, due to the channel confined space effect of the nanopore of the SCNPs, one Cl− could quench multiple lucigenin molecules on the basis of the collective effect of the self-assembled-state lucigenin molecules. Thus, a large KSV was obtained similarly that of the previous signal amplification mechanism.31 Cartoon B in Scheme 1 is used to describe this signal amplification mechanism. Imaging of the LSCNP Response to Cl− in Living Cells. To test the LSCNP sensing ability in living cells, we incorporated them into mouse leukemia cells L1210 by endocytosis.38 By adding 100 μL of a 0.1 mg/mL LSCNP solution to a 4 mL cell suspension and culturing for 4 h in a 5% CO2 atmosphere at 37 °C, the cells could efficiently load the LSCNPs. From the fluorescence microscopic image (Figure 9), we can see that the cells emit clear fluorescence, which shows that the LSCNPs can be loaded into the cells with no damage to the cells. To assess the response of the LSCNPs to Cl− in living cells, we changed the intracellular Cl− concentration by adding external Cl−. A 40 μL volume of 0.01 M KCl was added to 1 mL of the cell suspension which had already phagocytosed LSCNPs followed by further culturing for 30 min, and then the cells were observed with fluorescence microscopy.39 Figure 10 shows the fluorescence microscopy images of the same LSCNP-loaded L1210 cells before (A, B) and after (C, D) addition of the Cl− calibration solution. We can see that images A and B are much brighter than images C and D, which means that the fluorescence of the LSCNPs in the cells was quenched, and this quench was attributable to Cl− entry into the cells. This result showed that the LSCNPs respond to a change in the Cl− concentration in living cells, which can be applied for in vivo, in situ, and real-time monitoring of changes in the intracellular Cl− concentration.

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CONCLUSION In summary, by using the self-assembly technique and the hydrogel porous template technique, porous dye self-assembled silica/chitosan hybrid nanoparticles were prepared and further used to develop a fluorophore collective fluorescence sensing strategy. The key feature of the proposed strategy was the combination of the effective channel mass transfer with the space confinement effect of the self-assembled-state dye in a smaller pore, which forms the basis of the proposed fluorescence signal amplification. Accordingly, because of their high analytical performance, together with their high biocompatibility, SCNPs have been successfully applied for G

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monitoring the changes in Cl− concentration in living cells. In addition, the preliminary ratiometric sensing result showed that the silica/chitosan hybrid nanoparticles have potential application in ratiometric sensing. In addition, the loading ability of the pore and covalent bonding ability of the amino and hydroxyl groups of chitosan in the SCNPs may allow loading of different sensing and functional groups, giving them many other potential applications, such as selective sensing, multicomponent sensing, targeted sensing, etc. Thus, we believe that this work has not only established a reliable approach to determine the Cl− concentration, but also provided a methodology for designing a fluorescence sensing strategy for detecting other species which may play critical roles in biological systems.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-29-81530791. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Grant 21375085), the Cultural Heritage Conversation Science and Technology Research Foundation (Grant 20090106), and the Fundamental Research Funds for the Central Universities (Grants GK201001007 and GK201302018).

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dx.doi.org/10.1021/ac5018502 | Anal. Chem. XXXX, XXX, XXX−XXX