Fluorescence Turn-On Detection of Gaseous Nitric Oxide Using Ferric

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Fluorescence Turn-On Detection of Gaseous Nitric Oxide Using Ferric Dithiocarbamate Complex Functionalized Quantum Dots Jian Sun,†,‡ Yehan Yan,†,‡ Mingtai Sun,‡ Huan Yu,‡ Kui Zhang,‡ Dejian Huang,*,§ and Suhua Wang*,†,‡ †

Department of Chemistry, University of Science & Technology of China, Hefei, Anhui 230026, China Institute of Intelligent Machines, Chinese Academy of Sciences, Hefei, Anhui 230031, China § Food Science and Technology Programme, Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore ‡

S Supporting Information *

ABSTRACT: Functional quantum dots (QDs) grafted with ferric dithiocarbamate complex layers (QDs-Fe(III)(DTC)3) were fabricated and demonstrated to be selectively reactive to nitric oxide. The dithiocarbamate (DTC) was covalently conjugated to the amine-coated QDs by a condensation reaction of the carboxyl in DTC and the amino polymer in surface of QDs. The weak fluorescence of QDs-Fe(III)(DTC)3 was attributed to the energy transfer between CdSe/ZnS and Fe(III)(DTC)3 complex at the surface of the functionalized quantum dots. Nitric oxide could greatly switch on the fluorescence of QDs-Fe(III)(DTC)3 by displacing the DTC in the Fe(III)(DTC)3 accompanied by reducing Fe(III) to Fe(II), thus shutting off the energy transfer way. The limit of detection for nitric oxide was estimated to be 3.3 μM and the specific detection was not interfered with other reactive oxygen species. Moreover, the probe was demonstrated for the sensing of gaseous nitric oxide, and the visual detection limit was as low as 10 ppm, showing the potential for sensing nitric oxide by the naked eye.

A

photobleaching, broad absorption, narrow and symmetric emission spectra, size-dependent and large Stokes shift,13,14 hence, the advantages as novel fluorophores.15−17 Indeed, there has been considerable scientific and substantial success stemming from the application of free radical detection based on surface coated QDs in the past few years.16,18 Amphiphilic coating of QDs through chemical modifications or functionalization can improve their dispersibility in a given solvent and modulate their reactivity to specific molecules.19 Herein, we report the functional QDs which are coated by amphiphilic polymer modified with the complex of ferric (Fe(III)) and dithiocarbamate groups (Fe(III)(DTC)3), which can be reactive sites to nitric oxide. As illustrated in Scheme S1 in the Supporting Information, the functional QDs were prepared by encapsulating the hydrophobic QDs (CdSe/ZnSTOPO) cores in amphiphilic shells made from poly(maleic anhydride-alt-1-octadecene). The polymer chains were grafted with ammonium N-(dithiocarbaxy) sarcosine (DTC) through amide bonding formation, producing dithiocarbamate groups on the surface (QDs-DTC). The surface dithiocarbamate moieties reacted with ferric ions to form dark brown ferric dithiocarbamate complexes Fe(III)(DTC)3 on the polymer shells. The fluorescence of the QDs was greatly decreased due

ir pollution has been of great attention because of the potential to cause acute and chronic respiratory illness.1,2 Nitric oxide (NO), an uncharged reactive free radical molecule, is one of the most important primary pollutants directly emitted from combustion processes. It has been well documented that inhaling a little gaseous NO can harm the respiratory tract.1 Moreover, nitric oxide can be converted to nitrogen dioxide (NO2), which is one of the important sources of acid rain and atmospheric particulate matter (such as PM 2.5).3 Since NO is very reactive and short-lived, its on-site sensitive and selective detection still remains a challenge. The most commonly used methods for the detection of NO include electron spin resonance spectroscopy, colorimetry, fluorometry, chemiluminescence, and electrochemical methods.4−6 Of all these conventional methods, fluorometry is one of the most promising methods for visual detection of gaseous NO. The Lippard laboratory reported several NO-selective sensors based on fluorescein derivatives.7,8 Diaminonaphthalene was also used for monitoring intracellular NO formed in neuronal cells.9 Besides, numerous near-infrared fluorescent probes were also used for free radical detection and vivo imaging.10−12 These fluorescence probes were designed on the basis of organic fluorophores which generally exhibit broad emission spectra and a narrow excitation wavelength band. Compared with organic dyes, semiconductor quantum dots (QDs) have been shown with superior photophysical properties such as high fluorescence quantum yield, resistance to © 2014 American Chemical Society

Received: April 11, 2014 Accepted: June 2, 2014 Published: June 2, 2014 5628

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the proper concentration of Fe(III). Therefore, a 30 μM of Fe(III) mixed with 8 nM QDs-DTC was obtained to make the probe (QDs-Fe(III)(DTC)3) for further experiments. The fluorescence quenching mechanism of QDs by Fe(III) can be proved by the UV−vis spectrum of Fe(III)(DTC)3. The Fe(III)(DTC)3 complex was freshly synthesized by mixing Fe(III) and DTC solution in water at a molar ratio of 1 to 3. Figure S5 in the Supporting Information shows the absorption spectra of Fe(III)(DTC)3, and it clearly indicates that Fe(III)(DTC)3 shows two broad absorption bands at 510 and 590 nm, respectively. Since the emission maximum of CdSe/ZnS QDs was at 586 nm, the broad absorption band at 590 nm completely overlaps the orange emission of QDs, which allows possible FRET from excited QDs to Fe(III)(DTC)3. Therefore, FRET mechanism could be the most probability for the emission deactivation of the QDs grafting with ferric (III) dithiocarbamate. Other mechanism such as photoinduced electron transfer could be possible or contributes to the emission deactivation of the QDs because of the ferric’s high oxidation state. However, nitric oxide can rapidly react with the dark brown Fe(III)(DTC)3 and forms a nearly colorless adduct, Fe(II)NO(DTC)2, which shows a great blueshift of the absorption maximum (Figure S5 in the Supporting Information). It is expected that the fluorescence of the probe can be turned on if the surface ligand Fe(III)(DTC)3 reacts to nitric oxide because the FRET efficiency between QDs and the adduct Fe(II)NO(DTC)2 greatly decreased, thus shutting down the FRET pathway. The QDs-Fe(III)(DTC)3 probe was also confirmed to be highly resistant to photobleaching (Figure S6 in the Supporting Information). Further we examined the responses of QDsFe(III)(DTC)3 to NO solution (prepared from DEA/NO (NO donor, diethylamine NONOate diethylammonium salt)), and the results were presented in Figure 1. The QDs-Fe(III)-

to the Förster resonance energy transfer (FRET) between the CdSe/ZnS QDs and the ferric complex. Because of the specific rapid reactivity between NO and Fe(III)(DTC)3 to form colorless nitric oxide adduct Fe(II)NO(DTC)2, the energy transfer pathway was shut down. The fluorescence of the QDs cores was thus turned on by nitric oxide. The analytical performance of the functional ferric dithiocarbamate coated QDs was thoroughly examined for detecting gaseous nitric oxide. (For the detailed synthesis procedure, refer to the Supporting Information.) First, the dithiocarbamate functionalized quantum dots QDsDTC was characterized with Fourier transform-infrared spectroscopy using the KBr pellet method (Figure S1 in the Supporting Information). The strong vibration bands at 2850 and 2923 cm−1 are assigned as the symmetric and asymmetric stretch of methylene (−CH2−) groups in the TOPO molecules.20 The band at 1467 cm−1 can be ascribed to the deformation of −CH2− next to the phosphorus and P → O stretching.20,21 The two bands at 1492 and 1580 cm−1 are assigned to the CO vibration of the amido bond (OC− N−). The broad bands at 1635, 1720, and 3430 cm−1 are assigned to the −NH− stretching of the amido bond (OC− N−). Other bands at 710, 990, 1025, and 1110 cm−1 can be associated with the CS2 vibrations of the dithiocarbamate moieties20 (refer to the chemical structure in Scheme S1 in the Supporting Information). These FT-IR results clearly show that the dithiocarbamate moieties were covalently linked to the polymer shells of the quantum dots. We further evaluated the optical properties of the QDs after the surface coating, which was shown by comparing the PL spectra of the TOPO-QDs dispersed in chloroform and QDsDTC in water. It can be seen that the QDs-DTC have similar emission peaks as that of TOPO-QDs, suggesting the surface coating process do not affect the particle sizes of the QDs. The relative fluorescence quantum yield measured for the asprepared QDs-DTC was ∼50% as compared to the native TOPO-QDs (Figure S2 in the Supporting Information), which was probably due to the formation of surface defects during the surface coating process. Photostability of the as-prepared QDsDTC was undertaken by flashing UV light through an aqueous QDs-DTC solution. After 20 consecutive illuminations at 365 nm (3 min for each time), there was almost no apparent change in fluorescence intensity, which was clearly displayed in Figure S3 in the Supporting Information, implying the probe is resistant to photobleaching in aqueous solution. It has been well documented that dithiocarbamates have a good binding ability with ferric ion to form complex with high extinction coefficient in the visible light range. The fluorescent QDs-DTC initially emitted an emission peak at 586 nm under a 365 nm wavelength excitation. Upon the addition of Fe(III), the fluorescence intensity at 586 nm of the QDs was gradually decreased (Figure S4 in the Supporting Information). This could be attributed to the coordination of DTC with Fe(III) ion, forming the ferric dithiocarbamate complex on the surface of the QDs shell (refer to the chemical structure in Scheme S1 in the Supporting Information). The complex has absorption spectrum overlapping with the emission of QDs. To prepare the NO turn-on probe, an optimal ratio of ferric and QDs-DTC was obtained by carefully examining the fluorescence response of the QDs-DTC to Fe(III). The fluorescence intensity of the system was closely linearly proportional to the amount of Fe(III) with a correlation coefficient of 0.998, ranging from 5 × 10−6 to 3 × 10−5 M, which can be used for the determination of

Figure 1. Fluorescence enhancing responses of the QDs-Fe(III)(DTC)3 (dot line) upon the exposure to DEA/NO (30 μM, 1−15 min, solid line). The inset shows the fluorescence images of QDsFe(III)(DTC)3 before (left) and after (right) DEA/NO addition under the irradiation of a 365 nm UV lamp.

(DTC)3 showed good sensitivity to NO. With the addition of DEA/NO, the fluorescence of the QDs was increased by about 3 times within 1 min. With further increasing of reaction time, the fluorescence was gradually enhanced and finally reached maximum (∼6 times) after 15 min, which is in consistent with the NO releasing kinetic of DEA/NO.22 Meanwhile, the 5629

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determined to be 3.3 and 11 μM, respectively. As illustrated in Scheme 1, the fluorescence enhancement of the QDs could be attributed to the NO-induced displacement of DTC in the surface bound Fe(III)(DTC)3 complex, which has been confirmed by Wang et al.,17 who demonstrated that NO broke the structure of Fe(III)(DTC)3 and shut off the FRET pathway. Finally, we examined the selectivity of the QDs-Fe(III)(DTC)3 for nitric oxide. The spectroscopic responses of the probe to other reactive species including nitrogen dioxide (NO2), hydrogen peroxide (H2O2), superoxide anion (O2−), hypochlorous acid (HClO), tert-butylhydroperoxide (TBHP), hydroxyl radical (•OH), peroxynitrite anion (ONOO−), nitrite anion (NO2−), nitrate anion (NO3−), hydrogen sulfide (H2S), and pi-acid ligand carbon monoxide (CO) were carefully examined at the same conditions as NO (Supporting Information, Experimental Section). It can be seen in Figure 3, the fluorescence of the probe solution was sharply turned on by NO and only slightly enhanced by superoxide anion and peroxynitrite anion. No apparent interferences were observed in fluorescence intensity after adding CO and other common reactive oxygen species in the probe solution. The results indicate that the QDs-Fe(III)(DTC)3 do not react with other ROS or the reaction rate is very slow. As demonstrated by the selectivity experiments, the probe QDs-Fe(III)(DTC)3 exhibits good selectivity for identification of NO over other reactive oxygen species (ROS). The interferences of some common transition metal cations such as Cu2+, Hg2+, and Cd2+ have been examined in aqueous solution (Figures S8 and S9 in the Supporting Information). It is noted that the presence of Cu2+, Hg2+, and Cd2+ degraded the sensitivity of the method because they quenched the fluorescence turned on by NO. Fortunately, these transition metal cations can be readily removed from the aqueous samples by simple pretreatment or preprecipitation. It is rational to assume that, in a gas sample or air sample, the content of these transition metal cations is very low and can be ignored. Therefore, the QDs-Fe(III)(DTC)3 can be developed for the detection of gaseous NO. To demonstrate this application, three different gas mixtures containing nitrogen plus NO, air plus NO, and nitrogen plus NO plus NO2 were chosen to examine the analytical performance for detection of gaseous samples. It was found that, under the same experimental conditions, injecting the mixture of NO and air exhibited weak fluorescence enhancement (Figure 4, red), this was because NO was rapidly oxidized by oxygen to NO2 which has been proved to have no fluorescence enhancing effect on QDs-Fe(III)(DTC)3. As we have proved, the fluorescence of QDs-Fe(III)(DTC)3 was enhanced ∼4 times after injecting the mixture of NO and N2. When the isometric mixture of NO− NO2−N2 was added, the fluorescence was turned on to a slightly larger extent (Figure 4, green) than the mixture of NO−N2 (Figure 4, blue). We assume it may be caused by the trace amount of air in N2 gas with a purity of 99%. The small amount of air in nitrogen gas could reduce the real content of NO. In contrast, the presence of NO2 in the gas mixture of NO−NO2−N2 could inhibit the oxidation of NO by air, thus induce a slightly larger fluorescence turn-on effect. These results indicate that the presently developed sensor based on dithiocarbamate functionalized quantum dots has practical utility in gaseous NO sensing. We further demonstrated that the quantum dots-probe could be used for the visual monitoring of gaseous NO. The gaseous

solution changed from colorless to bright orange under a UV lamp which can be seen with the naked eye (inset of Figure 1). Because the nitric oxide donor solution of DEA/NO was alkaline, a control experiment was conducted to exclude the effect of hydroxide ions on the fluorescence properties of the detection system (Figure S7 in the Supporting Information). The result showed that excess amount of hydroxide ions slightly enhanced the fluorescence, but the enhancement could be ignored compared with the increment induced by adding DEA/NO solution. To evaluate the dose dependent kinetic response of the QDsFe(III)(DTC)3 sensor to DEA/NO, the fluorescence intensity was recorded after the addition of various concentrations of nitric oxide with different reaction time. Also, the results are shown in Figure 2A. The dependence of fluorescence increase

Figure 2. (A) Increments of fluorescence intensity of QDsFe(III)(DTC)3 against time (0−15 min) after the addition of DEA/ NO at different concentrations in degassed water at room temperature. (B) Plot of fluorescence enhancing efficiency of the QDs-Fe(III)(DTC)3 as a function of the DEA/NO concentration. F0 and F were the fluorescence intensity of QDs-Fe(III)(DTC)3 in the absence and in the presence of different concentrations of DEA/NO, respectively. The fluorescence intensity were recorded 15 min after addition of DEA/NO with excitation at 365 nm.

percentage vs the amount of DEA/NO were plotted as shown in Figure 2B. When the NO concentration was as low as 5 μM, the fluorescence intensity was enhanced by 50%. Unambiguously, the fluorescence enhancement is closely related to the amount of DEA/NO added to the QDs-Fe(III)(DTC)3 solution with a correlation coefficient of 0.998, which can be used for the quantification of NO. The limit of detection (LOD) and limit of quantification (LOQ) for NO were 5630

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Scheme 1. Schematic Illustration of Fluorescence Turn-On Detection of Nitric Oxidea

a

Reacts with the surface grafting Fe(III)(DTC)3 to form Fe(II)NO(DTC)2 decreasing the FRET efficiency from QDs to the surface grafting molecules and subsequently turning on the fluorescence of QDs. For the chemical structure, refer to Scheme S1 in the Supporting Information.

Figure 4. Fluorescence responses of the QDs-Fe(III)(DTC)3 by injecting different compounded gas mixtures. The volume of gaseous NO was kept the same in the three comparison experiments, and the concentration of NO was 20 ppm. Figure 3. (A) Selectivity of the QDs-Fe(III)(DTC)3 for NO detection. The graph was obtained by adding ROS (5 equiv) into the QD solution. Pure CO gas was bubbled into the probe solution for selectivity research. The fluorescence intensities were recorded at 1, 5, and 15 min after the ROS addition. For control experiments, degassed DI water was added with the same volume as ROS solution. (B) Digital photos showing that NO turns on the fluorescence but other ROS do not under illumination by a 365 nm UV lamp.

samples with different content of NO diluted in nitrogen were first prepared for future use. A volume of 2 μL of the probe QDs-Fe(III)(DTC)3 solution was then dropped on a piece of test paper to prepare the NO fluorescence test paper. These test paper strips were exposed to the gaseous samples with different concentrations of NO for 1 min. They were then transferred to be illuminated under a UV lamp (365 nm). Clearly, as can be seen in Figure 5, exposing to different amounts of NO led to distinguishable tiny dots of orange color on the test strips, showing a dose-responsive brightness to the concentrations of NO in the gas samples. The visual detection

Figure 5. Visual detection of gaseous NO. The NO concentrations of 0, 10, 20, 50, and 100 ppm were used. The photos were taken on the test strips which were exposed to gas samples with different concentrations of NO. The left photos were obtained under illumination of a 365 nm UV lamp in the dark. The right photos were obtained under daylight.

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limit of ∼10 ppm was estimated as the least concentration of NO capable of producing pink dots that can be noted by the independent observers. These results suggest that the test strips immobilized with the QDs-Fe(III)(DTC)3 probe can be used for on-site and rapid detection of gaseous NO. The sensitivity of the method could be improved by accumulating the exposure time to the sample or the sample volume because the fluorescent product is stable and the reaction between ferric(III) dithiocarbamate and NO is irreversible. In summary, we have developed a fluorescence turn-on probe capable of monitoring gaseous nitric oxide using quantum dots as the fluorophore and the surface grafting ferric dithiocarbamate complex as the recognition site for nitric oxide molecules. The fluorescence of the probe was initially weak due to the FRET between quantum dots and the surface grafting ferric dithiocarbamate, it was then turned on by NO, which reacted with the surface ferric dithiocarbamate to turn off the FRET. This fluorescence switch was demonstrated to be selective to nitric oxide and affords a limit of detection of 3.3 μM for NO in aqueous solution. Simple fluorescence test strips were prepared and demonstrated for the visual monitoring of gaseous NO with a visual detection limit of about 10 ppm.

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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 Authors

*E-mail: [email protected]. Phone: (65)-6516-8821. Fax: (65)-6775-7895. *E-mail: [email protected]. Phone: 86-551-65591812. Fax: 86551-65591156. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of China (Grant Nos. 21228702, 21075123, and 21302187), and the Innovation Project of Chinese Academy of Sciences (Grant KJCX2-YW-H29).

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