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Orderly Arranged Fluorescence Dyes as a Highly Efficient Chemiluminescence Resonance Energy Transfer Probe for Peroxynitrite Zhihua Wang, Xu Teng, and Chao Lu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b00472 • Publication Date (Web): 19 Feb 2015 Downloaded from http://pubs.acs.org on February 22, 2015

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Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Analytical Chemistry

Orderly

Arranged

Fluorescence

Dyes

as

a

Highly

Efficient

Chemiluminescence Resonance Energy Transfer Probe for Peroxynitrite

Zhihua Wang, Xu Teng and Chao Lu*

State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, China.

Fax/Tel.: +86 10 64411957. E−mail: [email protected].

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ABSTRACT: Chemiluminescence (CL) probes for reactive oxygen species (ROS) are commonly based on a redox reaction between a CL reagent and ROS, leading to poor selectivity towards a specific ROS. The energy−matching rules in chemiluminescence resonance energy transfer (CRET) process between a specific ROS donor and a suitable fluorescence dye acceptor is a promising method for the selective detection of ROS. Nevertheless, higher concentrations of fluorescence dyes can lead to the intractable aggregation−caused quenching effect, decreasing the CRET efficiency. In this report, we fabricated an orderly arranged structure of calcein−sodium dodecyl sulfate (SDS) molecules to improve the CRET efficiency between ONOOH* donor and calcein acceptor. Such the orderly arranged calcein−SDS composites can distinguish peroxynitrite (ONOO−) from a variety of other ROS owing to the energy−matching in CRET process between ONOOH* donor and calcein acceptor. Under the optimal experimental conditions, ONOO− could be assayed in the range of 1.0−20.0 µM, and the detection limit for ONOO− (S/N = 3) was 0.3 µM. The proposed strategy has been successfully applied in both detecting ONOO− in cancer mouse plasma samples and monitoring the generation of ONOO− from SIN−1. Recoveries from cancer mouse plasma samples were in the range of 96–105%. The success of this work provides a unique opportunity to develop a CL tool to monitor ONOO− with high selectivity in a specific manner. Improvement of selectivity and sensitivity of CL probes holds great promise as a strategy for developing a wide range of probes for various ROS by tuning the types of fluorescence dyes.

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INTRODUCTION Chemiluminescence (CL) probes for reactive oxygen species (ROS), such as superoxide anion (•O2−), singlet oxygen (1O2), hydroxyl radical (•OH), hypochlorite (ClO−), and peroxynitrous acid/peroxynitrite (ONOOH/ONOO−) have been widely used for the detection of ROS.1 However, the reported CL probes for ROS are usually based on a redox reaction between a CL reagent (e.g., luminol and lucigenin) and ROS.2-5 Generally, these CL reagents can respond to different ROS, and thus it remains a great challenge to selectively detect a specific ROS. Among a large number of ROS, 1O2 can produce a weak CL at 634 nm, 703 nm and 1270 nm;6 while the emission wavelength of an excited state of ONOOH (i.e., ONOOH*) is 350−450 nm.7 Obviously, chemiluminescence resonance energy transfer (CRET) can take place between 1O2/ONOOH* and a suitable acceptor. Currently,

fluorescence

dyes

are

often

used

as

CL

energy

acceptors

for

the

luminol−H2O2−horseradish peroxidase−CL system, where xanthene fluorescence dyes could absorb the excited−state luminol energy at 425 nm and re−emit stronger light at longer wavelengths (usually at 510−520 nm).8,9 However, the concentrations of fluorescence dyes need be cautiously controlled in the low concentration range because higher concentrations of fluorescence dyes can lead to the intractable aggregation−caused quenching effect.10-12 Therefore, it represents a significant challenge to explore an efficient strategy to improve the CRET efficiency. Layered double hydroxides (LDHs) are an interesting class of inorganic layered solid host matrices with structurally positively charged layers and interlayer balancing anionic species and water molecules.13-15 LDH materials feature a tunable layer charge density, variable elemental composition, and high chemical stability.16,17 Fluorescence dyes in the LDH galleries can improve the fluorescence efficiency and reduce aggregation−caused quenching effect in comparison to the aqueous fluorescence molecules alone owing to highly ordered and preferentially oriented fluorescence molecules between LDH sheets.18-21 On the other hand, fluorescence dye molecules on the surface of LDHs could be arranged in an oriented and planarity manner, where the intermolecular π–π stacking interactions among aromatic rings were well suppressed.22 Nowadays, 3



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making use of the unique advantages of LDH−enhanced fluorescence emissions, some groups have reported their potential applications in chemical sensing.23-25 For example, Wei and her co−workers developed a type of optical pH sensor from pH 5.02 to 8.54 based on a highly oriented photoluminescent film, which was fabricated by co−intercalating fluorescein and 1−heptanesulfonic acid sodium into a LDH matrix.24 Lu and his co−workers fabricated an ordered ultrathin film with blue luminescence composed of a styrylbiphenyl derivative and LDH nanosheets, and the fluorescence intensity of the as−prepared film decreased linearly upon increasing the concentrations of Hg2+.25 However, these methods usually need basic media because LDH layers can be dissolved in acidic solutions, limiting their applicability. These disadvantageous properties of LDH−improved fluorescence could provoke our interests in exploring the properties of fluorescence dye molecules assembled on the surface of the LDHs when the structure of the LDHs was collapsed in acidic solutions. In this work, we fabricated a fluorescence dye−LDH nanocomposite by embedding trace calcein molecules into the sodium dodecyl sulfate (SDS) bilayer bunches on the LDH exterior surfaces. When the as−prepared nanocomposite colloidal solution was dissolved in acidic solution, it was transformed from turbid to transparent. Interestingly, fluorescence spectrum, fluorescence lifetime and steady−state fluorescence polarization measurements demonstrated that the conformational structure of calcein−SDS molecules was orderly after the structure of LDHs was collapsed in acidic solution. In addition, the obtained orderly arranged structure of calcein and SDS (calcein@SDS) can accept the energy from ONOOH* donor to produce a strong CL emission owing to the energy−matching in CRET process between ONOOH* donor and calcein acceptor (Figure 1). Therefore, the fabricated calcein@SDS composite can distinguish ONOO− from a variety of other ROS. Validation of the proposed approach was checked by determining ONOO− in cancer mouse plasma samples and monitoring the generation of ONOO− from SIN−1. Our work extends the scope of LDH−improved fluorescence intensities and opens up new possibilities for highly selective and sensitive detection of ONOO− using an efficient CRET technique. 4


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Figure 1. CL features of the calcein@SDS solution in the presence of different ROS.

EXPERIMENTAL SECTION Chemicals and Solutions. All reagents were of analytical grade and used without further puriﬁcation. Analytical grade chemicals including Al(NO3)3·9H2O, Mg(NO3)2·6H2O, NaOH, NaClO, FeSO4, KO2, 1,4−diazabicyclo[2.2.2]octane (DABCO), sodium azide (NaN3), ascorbic acid, and glucose were purchased from Beijing Chemical Reagent Company. Sodium dodecyl sulfonate (SDS) was purchased from Guangdong Chemical Reagent Company. Thiourea, calcein, and uric acid were obtained from Tianjin Chemical Reagent Company. Nitro blue tetrazolium chloride (NBT) was purchased from Nacalai Tesque Inc. (Tokyo, Japan). Glutamine, homocysteine, cysteine and bilirubin were purchased from Sigma–Aldrich (St. Louis, USA). A mixed working solution of 0.05 M H2O2 and 0.03 M HCl was freshly prepared by volumetric dilution of commercial 36% (v/v) HCl (Beijing Chemical Reagent Company) and 30% (v/v) H2O2 (Beijing Chemical Reagent Company) with deionized water. A 0.1 M NaNO2 stock solution was prepared by dissolving 0.69 g NaNO2 (Tianjin Chemical Reagent Company) in 100 mL deionized water. Working solutions of NaNO2 were freshly prepared by diluting the NaNO2 stock solution with deionized water. ClO− was 5



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prepared by diluting commercial NaClO solution in deionized water and assayed by using a spectrophotometer with ε

293 nm

= 350 cm-1M-1. A 5 mM 3−morpholinosydnonimine (SIN−1,

Toronto Resseach Chemicals Inc.) solution was prepared by dissolving 41 mg SIN−1 into 40 mL NaOH solution (0.05 M). Synthesis of Mg−Al−NO3 LDHs. Mg−Al−NO3 LDHs were synthesized by the coprecipitation method at pH 10 under low supersaturation conditions. The preparation was performed under a N2 atmosphere to exclude the aqueous CO2. CO2−free deionized water was prepared by boiling deionized water under a N2 atmosphere. Solution A: Mg(NO3)2·6H2O and Al(NO3)3·9H2O with a Mg/Al molar ratio 3 (0.045 mol Mg(NO3)2·6H2O and 0.015 mol Al(NO3)3·9H2O) were dissolved in 60 mL of CO2−free deionized water. Solution B: 0.12 mol NaOH was dissolved in 60 mL of CO2−free deionized water. The two solutions were simultaneously added slowly to a 250 mL flask under vigorous stirring maintaining pH 10 at room temperature. The resulting white slurry was aged for 24 h at 65˚C under N2 atmosphere. Afterward, the precipitate was collected by centrifugation, washed thoroughly with de−CO2 deionized water for three times. The resulting colloidal solution of Mg−Al−NO3 LDHs was diluted to original concentration. Synthesis of SDS−LDHs. SDS−LDHs were prepared following the anion−exchange methods. 3.6048 g SDS was dissolved in 100 mL de−CO2 deionized water, and then added 30 ml Mg−Al−NO3 LDH colloidal solution. The anion exchange was carried out under stirring at 60˚C under N2 atmosphere for 24 h. The resulting product SDS−LDHs suspension was stored at 4˚C until further use. Immobilization of Calcein on the Surface of SDS−LDHs. For calcein was immobilized on the surface of SDS−LDHs, 0.0060 g calcein was added into 10 mL SDS−LDHs with a gentle stirring for 20 min at room temperature. In comparison, 0.0078 g calcein and 0.3605 g SDS were simultaneously dissolved in 10 mL de−CO2 deionized water. The prepared calcein−SDS solution was mixed with 3 ml Mg−Al−NO3 LDH colloidal solution. Mouse Plasma Sample Pretreatment. Normal female BALB/c mice were purchased from the 6


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Academy of Military Medical Sciences (Beijing, China). Next, some of the obtained mice were treated with 4T1 breast cancer by the Life Science and Technology College, Beijing University of Chemical Technology. The mouse blood was collected in ethylene diamine tetraacetic acid (EDTA) blood sampling tube to prevent coagulation. The collected blood samples were pretreated following the literature procedure.26 Briefly, the samples were centrifuged at 4000 rpm for 15 min at 4 ˚C. The supernatant solution was decanted and stored at −80 ˚C in refrigerator. Before the analysis, the as−prepared solution was centrifuged twice at 10000 rpm for 15 min each time at 4 ˚C. Finally, the supernatant solution was diluted to an appropriate concentration with deionized water and analyzed immediately by the proposed method. The standard addition method was applied in the detection of ONOO− in mouse plasma samples. Apparatus. Transmission electron microscope (TEM) images were obtained using a Tecnai G220 transmission electron microscope (FEI Co., Netherlands). The powder X−ray diffraction (XRD) measurements were performed on a Bruker (Germany) D8 ADVANCE X−ray diffractometer equipped with graphite−monochromatized Cu/Kα radiation (λ=1.5406 Ǻ). And a 2θ angle of the diffractometer was stepped from 2° to 70° at a scan rate of 0.02 °/s. The CL detection was conducted on a BPCL luminescence analyzer (Institute of Biophysics, Chinese Academy of Science, Beijing, China). The CL spectrum of this system was measured with high−energy cutoff filters from 460 to 580 nm between the flow CL cell and the photomultiplier tube (PMT). Zeta potential measurements were determined using a Malvern Zetasizer 3000HS nanogranularity analyzer. Steady−state polarized photoluminescence measurements were recorded in a Renishaw Micro−Raman Spectroscopy InVia Raman system (England), which is equipped with a charge−coupled device (CCD) detector and a Renishaw laser excited at 514 nm. The fluorescence lifetime (Edinburgh Instruments’ LifeSpec ps spectrometer) was measured by exciting at 490 nm with a nanosecond flashlamp. The percentage contribution of each lifetime component to the total decay was calculated with the F900 Edinburgh instruments software. Fluorescence spectra were performed using a Hitachi F−7000 fluorescence spectrophotometer (Tokyo, Japan). UV−visible 7



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spectra were recorded on a USB 4000 miniature fiber optic spectrometer in absorbance mode with a DH−2000 deuterium and tungsten halogen light source. CL Measurements. The schematic diagram of the CL system was shown in Figure S1. To investigate the effect of calcein under different conditions on ONOO− CL system, calcein or calcein@SDS solution was injected into the ONOO− solution through a valve injector with a 110 µL injection loop. The standard ONOOH solution was produced on−line by the reaction between acidified H2O2 (0.02 M) and NaNO2 (0.01 mM), and then it mixed with NaOH (0.2 M) through a three−way connector to produce ONOO−. NaOH solution was pumped by the peristaltic pump at a rate of 0.8 mL/min. The flow rates for the NaNO2 and acidified H2O2 were 1.6 mL/min. The CL signals were monitored by PMT (−950 V) adjacent to the flow CL cell. The PMT signals were imported to the computer for data acquisition. For the determination of ONOO− in real samples, the sample or spiked sample was pumped into the flow cell at a rate of 0.8 mL/min. The calcein@SDS solution was injected into the sample or spiked sample solution through a valve injector with a 110 µL injection loop (Figure S2).

RESULTS AND DISCUSSION Interlayer Configurations and Surface Morphology. The XRD patterns of NO3−LDHs and SDS−LDHs were shown in Figure S3. All the patterns of these samples can be indexed to a hexagonal lattice.27 The interlayer spacing can be calculated by averaging the positions of the three harmonics: d = (1/3) (d003 + 2d006 + 3d009).28 For the pristine NO3−LDHs, the interlayer spacing was 0.85 nm, corresponding to the basal spacing of NO3− incorporated in hydroxide layers.29 Upon substituting NO3− ions with DS− ions, the basal spacing was increased to 2.76 nm, which was almost the same as the length of an individual DS chain (2.08 nm), suggesting the formation of the intercalation of monolayer DS− ion with perpendicularly oriention to LDH sheets.30 In addition, the TEM images of the NO3−LDHs and SDS−LDHs were displayed in inset of Figure S3. The nanoscale crystal particles with irregular shapes were observed in the image of NO3−LDHs. While 8


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the SDS−LDHs exhibited the partly aggregation of particle as a result of the hydrophobic interaction of the interlayer alkyl chains.31 The effective assembly of calcein on the surface of SDS−LDHs was demonstrated by zeta potential measurements. As shown in Table S1, the zeta potential of SDS−LDHs was negatively charged (−25.3 mV), meaning that the formation of the DS bilayer bunches on the surface of SDS−LDHs via hydrophobic chain−chain interaction with hydrophilic head groups exposed to the continuous aqueous phase.32 Interestingly, the zeta potential was increased when calcein was added into SDS−LDH colloidal solution. We can infer that part of DS− ions can be replaced by negatively charged calcein with larger size (Figure S4). Moreover, the adsorption time of calcein on the surface of SDS−LDHs was optimized and found that 20 min can produce the strongest CL intensity (Figure S5). The fluorescence spectrum and photos of sediments obtained by centrifuging calcein assembled on the surface of SDS−LDHs further confirmed that calcein can be strongly absorbed on the surface of SDS−LDHs.

Calcein@SDS−Amplified ONOOH CL. ONOOH produced from the on−line reaction of nitrite with acidified H2O2 may be further transformed to an activated form of peroxynitrous acid (ONOOH*),1 which can produce a weak CL signal when it is isomerized to form nitrate.33 In the present work, we investigated the effects of calcein and SDS on the ONOOH CL under different conditions in a flow injection CL setup (Figure S1). As shown in Figure 2, there appeared to be a remarkable increase in CL emission of ONOO− system, when calcein on the surface of the centrifuged SDS−LDH colloidal solution was acidified. In comparison, a lower CL enhancement was observed if the uncentrifuged SDS−LDHs were used as absorption materials for calcein. The CL differences were attributed to the presence of a high loading amount of DS− ions on the surface of SDS−LDHs, inhibiting the replacement of DS bilayer by calcein as a result of electrostatic repulsion between calcein and SDS. Interestingly, it can be seen that a slight CL enhancement appeared when the NO3−LDHs rather than SDS−LDHs were used. These phenomena indicated that 9



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the CL enhancement in the present system was not merely ascribed to the concentrating of calcein on the surface of SDS−LDHs. Finally, three control experiments were carried out, including SDS solution, calcein−SDS mixture solution, uncentrifuged SDS−LDH solution, centrifuged SDS−LDH solution and calcein solution with the same concentrations. The results indicated that no obvious CL signals were emitted in these control experiments. On the other hand, fluorescence spectra of calcein under the same experimental conditions along with these control experiments showed the similar results (inset of Figure 2). Therefore, it was concluded that the geometrical configuration of calcein−SDS in the acidified SDS−LDH colloidal solution played an important role in enhancing the ONOO− CL emission.

Figure 2. CL intensity of ONOO− system in the presence of (a) uncentrifuged SDS−LDHs, (b) SDS−LDHs, (c) SDS, (d) the mixture solution of calcein and SDS, (e) calcein, (f) calcein on the surface of the NO3−LDHs, (g) calcein on the surface of the uncentrifuged SDS−LDHs, and (h) calcein on the surface of the centrifuged SDS−LDH colloidal solution, respectively. Inset: normalized fluorescence intensity of above−mentioned solutions. The concentrations of calcein and SDS for each solution were 1.0 mM and 0.1 M, respectively. The concentration of ONOO− was 10 µM. All the solutions were acidified with 1.0 M HCl.

Geometrical Configuration of Calcein−SDS. The geometrical configuration of 10


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calcein−SDS in the acidified SDS−LDH colloidal solution can be confirmed by their polarized luminescence properties.34 When the parallel and perpendicular directions to the excitation polarization direction (IVV vs. IVH), the average r value for calcein@SDS solution was found to be 0.11 in the range of 516−800 nm (Figure 3A), indicating that the arrangement of calcein in the SDS bilayer bunches was highly orderly and dispersive due to the fact that the SDS bilayer bunches on the surface of SDS−LDHs can isolate calcein molecules effectively. As a result, the aggregation between calcein molecules was restrained. However, the average r value was decreased to 0.03 in the same wavelength after the calcein@SDS solution was gently stirred for 60 min (Figure 3B). These results demonstrated that the ordered arrangement of calcein−SDS molecules became disordered with the increase of holding time, leading to the formation of calcein aggregates due to intermolecular π−π stacking interaction. In addition, the fluorescence lifetime of the calcein@SDS solution was decreased when the holding time was increased to 60 min (Figure 3C). The decreased fluorescence lifetime was ascribed to an increased neighborhood collision frequency arising from the formation of aggregates, resuling in an increase in the nonradiative decay rate.35

Figure 3. (A) Polarized photoluminescence profiles in the VV (blue), VH (red) modes and anisotropic value (r) for calcein@SDS solution; (B) Polarized photoluminescence profiles in the VV (blue), VH (red) modes and anisotropic value (r) for calcein@SDS solution with gentle stirring for 60 min; (C) Fluorescence decay profiles for (1) calcein@SDS solution without stirring, and (2) calcein@SDS solution with gentle stirring for 60 min. Inset: fluorescence lifetimes with monoexponential fitting. 11



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Next, the lasting time of the delay effects of calcein−SDS geometrical configuration was invesgitated by the change of the CL intensity. As shown in Figure 4, the CL intensity was almost constant within 2 min, and then gradually decreased until the baseline as the holding time was prolonged to 10 min. In addition, the fluorescence spectra of the orderly calcein@SDS solution were compared with those of the collapsed calcein@SDS solution in the presence of different concentrations of •OH. The results (Figure S6) indicated that the decomposition of calcein by •OH was much slower for the orderly calcein@SDS solution. These findings provided the compelling evidence for the rearrangement of calcein and SDS molecules from the structure gradual collapse of SDS−LDH layer. Note that the detection limit for ONOO− using the orderly calcein@SDS was 0.3 µM; while the detection limit was 100 µM using the calcein+SDS mixture. Therefore, the preparation of the orderly calcein@SDS is significant for improving the sensitivity for ONOO−.

Figure 4. CL intensity of the calcein@SDS solution with different holding time in the presence of ONOO−. The concentrations of calcein and SDS for each solution were 1.0 mM and 0.1 M, respectively. The concentration of ONOO− was 10 µM. The colloidal solution was acidified with 1.0 M HCl.

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Selective CL Response of Calcein@SDS to ONOO−. The CL reactions of the calcein@SDS solution with a series of ROS including 1O2, H2O2, •OH, •O2−, ClO−, and ONOO− were investigated in a flow injection CL setup in Figure S1. As shown in Figure 5, there were no obvious CL signals when the calcein@SDS solution was mixed with 1O2, H2O2, •OH, and •O2− of higher concentrations (each 10 mM), respectively. However, a strong CL was produced in the presence of 10 µM ONOO−. These results indicated that the calcein@SDS solution exhibited a highly selective CL response towards ONOO−. 10 mM ClO− showed a slight CL signal in the presence of the calcein@SDS solution; therefore, we added the concentrations of ClO− up to 100 mM and found that the CL intensity kept constant (Figure S7). To investigate the CL mechanism of the selective response of the calcein@SDS solution towards ONOO−, the CL spectrum of the ONOO− system in the presence of the calcein@SDS solution was measured with high−energy cutoff filters combined with a flow−injection system. Figure S8 showed that the maximum emission of the proposed CL system was about 528 nm, which was almost identical to the fluorescence spectrum of calcein solution with a peak at 525 nm. Moreover, the scavengers of various reaction oxygen species were used to confirm the emitting species. The results showed that 0.05 M NaN3 (a scavenger for 1O2),36 0.05 M NBT (a scavenger for •O2−)37 and 0.05 M thiourea (a scavenger for •OH)38 did not quench the CL intensity of the present system, indicating that the emitting species might not be from 1O2, •O2− and •OH. Furthermore, the absorption band of the calcein@SDS solution was in the wavelength range from 320 to 510 nm (Figure S9), which overlaps the emission wavelength of ONOOH*.7 Moreover, the random collisions between ONOOH* donor and calcein acceptor made calcein close enough to accept the energy from ONOOH* by dipole−dipole interaction.39 Note that the emission wavelengths of 1O2 are 633 nm, 703 nm and 1270 nm, respectively,6 and thus calcein could not accept the energy from 1O2. This result can explain why the present system exhibited high selectivity toward ONOO−. Finally, the absorption spectrum of uranine is similar with that of calcein (Figure S10), and thus 13



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it was used to replace calcein to investigate the selectivity of uranine@SDS solution towards a series of ROS. Interestingly, the results showed that the uranine@SDS solution also exhibited a highly selective CL response towards ONOO−. However, the detection limit for ONOO− using the uranine@SDS solution was 50 µM, which was much higher than the calcein@SDS solution (0.3 µM). Therefore, we used the calcein@SDS solution as energy acceptors for the CL detection of ONOO−. Analytical Performances and Potential Applications. Under the optimal experimental conditions (Figure S5 and Table S2), we investigated the relationship between the CL intensity and ONOO− concentration in the range of 1.0−70.0 µM. The results found that the calibration curve for ONOO− was found to be linear from 1.0 to 20.0 µM. The regression equation was y = 46.13x + 32.11 (R2 = 0.9957), where y is the relative CL intensity and x is the concentration of ONOO− (inset of Figure 5). The detection limit for ONOO− (S/N=3) was 0.3 µM. The relative standard deviation (RSD) for nine repeated measurements of 10 µM ONOO− was 2.3%. In comparison to the calcein+SDS mixture, the high sensitivity of calcein@SDS solution towards ONOO− was ascribed to the orderly arranged structure of calcein molecules. In 2010, the concentration of ONOO− in mouse blood plasma samples was determined to be 4.52±0.33 µM by an electrochemical method using a standard addition method.26 The content of ONOO− is gradually increased as the development of cancer animal from early to late stage. The sensitivity of the present method cannot be used for monitoring ONOO− in plasma samples from early stage cancer animals. However, ONOO− can be detected in the plasma samples at different stages by performing spiked experiments. The effects of the typical interferences including some important reductants present in mouse plasma were investigated (Table S3). The results showed that cationic ions, anionic ions, glucose, glutamine, homocysteine, and uric acid had no influence on the determination of 10 µM ONOO−. The tolerance limits of Fe3+, Cu2+, PO43-, ascorbic acid and cysteine were 0.1 mM. Note that the common concentration ranges of these compounds in mouse blood plasma are lower than 0.1 mM.40-42 In conclusion, the proposed method showed high 14


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selectivity towards ONOO− in mouse plasma samples. In order to support the potential of the proposed method for clinical use, the concentrations of ONOO− in the normal and cancer mouse plasma samples were detected. The results were summarized in Table 1. The average concentration of ONOO− from the cancer mouse plasma samples was determined to be 3.92±0.18 µM, which was consistent with the reported method26. However, the concentration of ONOO− in normal mouse plasma samples cannot be detected. These results were agreed with the reports that the production of ONOO− can greatly increase under pathological conditions (e.g., cancer)43. These results indicated that proposed method is capable of sensing ONOO− in real samples.

Table 1. Assay Results of ONOO− in Normal Mouse Plasma and Cancer Mouse Plasma Samples

a

plasma samples

proposed method (µM )

normal mouse

no found

cancer mouse

3.92±0.18

added (µM)

found (µM )

recovery (%)

2.00

2.09±0.05

105

4.00

3.87±0.03

97

2.00

5.93±0.04

101

4.00

7.74±0.02

96

Mean ± SD of three measurements.

Generally, SIN−1 is a biological generator of ONOO−.44 Herein, the proposed approach was applied to monitor ONOO− from SIN−1. Figure S11 showed the plot of the estimated ONOO− concentration over time by the proposed CL method. It can be seen that the concentration of ONOO− increased with time and reached a plateau (14 µM) in approximately one hour, indicating that SIN−1 can decompose completely within one hour. These results coincided with the other reports in which the decomposition of SIN−1 conducted in different media.45 Note that the generated concentration of ONOO− was lower than the expected generation (50 µM) due to the possible reactivity with CO2 in the ambient air.46

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Figure 5. CL intensity from the reaction of calcein@SDS solution with different ROS. Inset: (A) CL intensity from the reaction of calcein@SDS solution with different concentration of ONOO−. (B) Calibration curve for ONOO− in the range of 1.0−20.0 µM. The concentration of ONOO− was 10 µM. The concentrations of other ROS solutions were 10 mM. The concentrations of calcein and SDS for each solution were 1.0 mM and 0.1 M, respectively.

CONCLUSIONS In summary, we have introduced a highly selective CL probe for ONOO− through a highly efficient CRET process between ONOOH* donor and fluorescence dye acceptor. The design was based on our new finding that the arrangement of calcein in the SDS bilayer bunches exhibited the delay effects with highly orderly and dispersive structure when the structure of LDH was collapsed in acidic solutions. The as−prepared calcein@SDS solution could improve the sensitivity for ONOO− detection. In addition, the remarkable selectivity of the proposed probe towards ONOO− was ascribed to the energy−matching rules in CRET process between ONOOH* donor and calcein acceptor. The proposed approach has been successfully used for the detection of ONOO− in cancer mouse plasma samples and decomposition reaction of SIN−1. We anticipate that this strategy may have great potential in a high−performance platform for real−time CL monitoring of intracellular 16


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ROS signaling. Of course, the proposed method cannot be used for the monitoring of ONOO− in vivo. Currently, we are studying the orderly arrangement of fluorescent materials in the galleries of LDHs so that the fabricated material−based LDHs can be used for the detection of ONOO− in biological environments. In addition, work is currently ongoing within our laboratories to extend the scope of this design with respect to the measurement of a broad range of ROS (e.g., 1O2) with a suitable selection of the dye, which can match with a specific ROS.

ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Fax/Tel.: +86 10 64411957. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21275016 and 21375006), and National Basic Research Program of China (973 Program, 2014CB932103).

Supporting Information Available Schematic diagram of the CL setup; Powder XRD patterns of NO3−LDHs and SDS−LDHs, TEM images of NO3−LDHs and SDS−LDHs, normalized fluorescence spectrum of sediments obtained by centrifuging calcein on the surface of SDS−LDHs, pictures of sediments obtained by centrifuging calcein on the surface of SDS−LDHs under visible light and ultraviolet light at 254 nm; structures of anionic surfactant SDS molecule and calcein fluorescence dye molecule; the adsorption time of calcein on the surface of SDS−LDHs; fluorescence spectra of calcein@SDS solution mixed with different concentrations of •OH, fluorescence spectra of the collapsed calcein@SDS solution (a holding time of 60 min) mixed with different concentrations of •OH; CL 17



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intensity from the reaction of calcein@SDS solution with different concentrations of ClO−; normalized CL spectrum ONOO− system mixed with calcein@SDS solution, and normalized fluorescence spectrum of calcein solution; absorption spectrum of calcein@SDS solution; CL intensity from the reaction of uranine@SDS solution with different ROS; normalized CL spectrum of ONOOH*, absorption spectrum of uranine@SDS solution; detection of ONOO− from SIN−1; comparison of zeta potential values for SDS−LDHs and calcein assembled SDS−LDHs; optimum experimental conditions for the CL determination of 10 µM ONOO−; tolerance limit of various coexistent substances on the determination of 10 µM ONOO−. This material is available free of charge via the Internet at http://pubs.acs.org.

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