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Quantum Dot-Based OFF/ON Probe for Detection of Glutathione Subhash Banerjee,† Soumitra Kar,*,† J. Manuel Perez,†,‡,§ and Swadeshmukul Santra*,†,‡,§ NanoScience Technology Center, Department of Chemistry, and Biomolecular Science Center, UniVersity of Central Florida, Orlando, Florida 32826 ReceiVed: March 3, 2009; ReVised Manuscript ReceiVed: April 13, 2009
A simple strategy for detection of glutathione (GSH) in physiologically relevant concentrations is reported by manipulating the electron transfer pathways of surface-modified quantum dots. The probe consisted of fluorescent CdS:Mn/ZnS core/shell quantum dots (Qdot) properly conjugated to dopamine through CS2-assisted zero-length covalent coupling. Qdots exhibited bright yellow-orange emission at ∼ 592 nm, which could be attributed to the 4T1 f 6A1 transition of Mn2+ ions in Qdot core. The fluorescently dark Qdots (OFF state) was obtained by modifying the Qdot surface with an electron rich dopamine molecule. The fluorescence of the probe was restored (ON state) when the disulfide bond was cleaved by the reducing GSH. Introduction Activatable probes which are reactive to the intracellular environment are useful for the rapid and accurate detection of homeostasis-relevant elements.1 In this context, thiol-containing amino acids play crucial roles in protein structures and stability within cells. For example, cysteines in their reduced form take part in regulation, structure, and function of proteins. The most abundant cellular thiol is the cysteine-containing tripeptide, glutathione (GSH). The intracellular concentration of GSH ranges from 1 to 15 mM.2 Any alteration of optimum cellular ratios of reduced (GSH) to oxidized (GSSG) glutathione can lead to a number of human pathologies such as heart disease, cancer, stroke, and many neurological disorders.3 Again, several forms of drug resistance have been associated with high levels of GSH.4 This demands immediate development of suitable probes for detecting intracellular levels of GSH, preferably in trace level (e.g., nanomolar to micromolar concentration range) to effectively address these problems at the early stage. A number of detection methods for thiols and thiol-containing peptides have been described in the literature for in vitro studies.5-9 However, most of the available probes are based on organic dyes that are less sensitive and prone to photobleaching.10 Because of these difficulties, continuous and sensitive detection of GSH is practically challenging. Furthermore, cellular autofluorescence decreases the signal-to-noise ratio, hindering sensitive detection. The development of highly sensitive and photostable fluorescent probes for detection of GSH is thus highly desirable. In recent years, semiconductor quantum dots have attracted considerable interest because of their widespread applications including electronic devices, solar cells, and quantum computing to biological markers.10-18 Current studies have demonstrated that Qdot fluorescence can be tuned via electron transfer process.19 Surface-bound electron-rich small organic molecules can transfer electrons efficiently to Qdots, thereby interfering with the excitonic (i.e., electron-hole pair) recombination process. Successful application of this fundamental electron * Corresponding author. Fax: 1 407 882 2819; tel: 1 407 882 2848; e-mail:
[email protected] (S.K.) and
[email protected] (S.S.). † NanoScience Technology Center. ‡ Department of Chemistry. § Biomolecular Science Center.
transfer process has been utilized for the detection of small organic molecules (e.g., benzylamine,20 benzyl alcohol21) and metal ions22-24 and anions25 (CN-) via luminescence quenching. As part of a continuing interest in developing Qdot-based sensors, we have previously reported a Qdot-based probe for selective detection of cadmium ions26 via fluorescence enhancement. The principle of the sensing mechanism was based on the electron transfer process. It is expected that proper surface conjugation of Qdots by an appropriate small ligand capable of transferring electrons can cause Qdot luminescence quenching. In the present study, this principle was applied to design an activatable Qdot probe for detection of GSH in physiologically relevant concentrations. The probe by design consists of CdS: Mn/ZnS Qdots coated with dopamine (QDL) which is capable of transferring electrons to Qdots. Thus, a fluorescently quenched probe (OFF state) was obtained. Upon displacement of dopamine ligand, the Qdot native luminescence is restored (ON state). On the basis of this OFF/ON switching mechanism, we developed an activatable Qdot probe for GSH detection. In QDL, dopamine is covalently linked to the Qdot surface via a disulfide linkage. GSH cleaves this disulfide bond, freeing up Qdots from dopamine and thus restoring Qdot luminescence. The background signal is expected to be minimal because of the OFF/ ON sensing mechanism. Experimental Procedure Qdot Synthesis. Qdots used in this study are composed of dopant-based core-shell CdS:Mn/ZnS nanocrystals, synthesized by a water-in-oil (W/O) microemulson method following a published protocol.27 In short, Qdots were synthesized using an AOT/heptane/water microemulsion system. Typically, Cd(CH3COO)2 · 2H2O, Mn(CH3COO)2, Na2S, and Zn(CH3COO)2 were used for the preparation of (Cd2+ + Mn2+)-, S2--, and Zn2+-containing standard aqueous solutions. Each aqueous solution was stirred with an AOT/heptane solution, forming the micellar solution. Mn-doped CdS core nanocrystals were formed by mixing (Cd2+ + Mn2+)- and S2--containing micellar solutions rapidly for 10-15 min. The molar ratio W0 of water-to-surfactant of the W/O microemulsion was maintained at 10. For the growth of a shell layer, a Zn2+-containing micellar solution was added at a very slow rate (1.5 mL/min) into the CdS:Mn nanocrystal micellar solution. The nucleation and growth of a separate ZnS
10.1021/jp9019574 CCC: $40.75 2009 American Chemical Society Published on Web 05/11/2009
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SCHEME 1: Synthesis of Dopamine Conjugated Qdots (QDL)
Figure 1. (a) HRTEM image and (b) PL spectrum of the CdS:Mn/ ZnS Qdots.
phase were suppressed by the very slow addition of the Zn2+containing micellar solution. The [Zn2+] to [Cd2+] molar ratio (X0) was 8. QDL Synthesis. Dopamine was attached to the surface of Qdots via a simple “zero-length” covalent coupling. Dopamine conjugation was accomplished by using carbon disulfide (CS2) as a linker. Scheme 1 outlines the synthesis of QDL. The attachment of dopamine to the Qdot surface is carried out by using a simple experimental procedure. Briefly, a mixture of CS2 (40 µL, 0.549 mmol), dopamine (104.1 mg, 0.549 mmol), and triethylamine (5 µL) was sonicated for 5 min at room temperature. In the next step, 25 mL of microemulsion containing the Qdots was added to the above reaction mixture followed by 15 min stirring at room temperature. The final product was washed with ethanol to remove unreacted CS2, dopamine and unbound CS2-dopamine complex (ligand, L). Thus, it is expected that the final product (QDL) is free of any unbound ligands since they are highly soluble in ethanol. Results and Discussions The as-synthesized CdS:Mn/ZnS quantum dots were characterized by high-resolution transmission electron microscopy (HRTEM) to determine the particle size of the Qdots. The HRTEM image depicted in Figure 1a reveals the formation of nearly monodispersed Qdots with average particle size ∼4 nm. Photoluminescence (PL) properties of the Qdots were also measured at room temperature with 350 nm excitation to evaluate the bright fluorescent nature of the Qdots. Figure 1b shows the PL spectrum of the Qdots exhibiting bright yelloworange emission at ∼592 nm. This emission was attributed to the 4T1 f 6A1 transition of Mn2+ ions in the Qdot core.28-32
The attachment of dopamine to Qdots was confirmed by the spectroscopic (1H NMR and FTIR) data (see ESI Figures 1 and 2). The 1H NMR spectra were recorded on a Varian 500 MHz NMR instrument using D2O as solvent. The IR spectra were recorded on a Perkin-Elmer FTIR instrument. Comparative studies of the 1H NMR spectra of free dopamine, QDL, and isolated CS2-dopamine complex revealed identical spectral features for free dopamine and isolated CS2-dopamine complex. On the other hand, an upfield shift (∼0.35 ppm) of methylene (-CH2CH2NH2) protons of dopamine was observed in QDL compared to that of free dopamine. This confirmed the attachment of the ligand to the Qdot surface. Comparative analysis of the FTIR spectra showed a new peak at 1732 cm-1 for QDL compared to free dopamine (see ESI Figure 2). This new peak in the IR spectra of QDL is characteristic of the oxidized aromatic conjugated ketones (CdCdO). This result indicated a possible conversion of dopamine to its oxidized form (quinone). Both forms of dopamine can coexist in equilibrium under physiological conditions in solution. To further verify the oxidation of dopamine, QDL and the isolated CS2-dopamine complex were characterized by fluorescence spectroscopy. The photoluminescence (PL) spectra recorded at 270 nm excitation showed a blue emission peak at ∼410 nm for both the QDL and isolated ligand as shown in Figure 2a. PL Intensity of the QDL is much higher compared to the isolated ligand. This could be attributed to the higher absorption efficiency of the incident photons by the Qdots over the isolated ligand. Higher absorption of the incident photon leads to the excitation of more electrons to the excited state followed by transfer to the ligand, which in turn produced intense blue emission. The peak at 410 nm is indicative of oxidation of aromatic ring of dopamine to quinone form.33 Since the environment of the amine group did not change
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Figure 3. PL spectra showing the effects of GSH on the fluorescence (excitation wavelength is 350 nm) of QDL. Plot in the inset shows the linear restoration of the PL in different GSH molar solutions.
Figure 2. PL spectra of the (a) QDL and isolated ligand (CS2-dopamine complex) recorded with 270 nm excitation wavelength and (b) QDL and bare Qdots recorded with 350 nm excitation wavelength.
because of this oxidation, the expected electron transfer between the ligand and Qdot should remain unchanged. The CS2 formed a stable carbodithioate linkage26 with the primary amine groups of dopamine which directly formed a disulfide (S-S) bond onto the ZnS surface of Qdots. Improved stability of the probe is expected over any mercapto-based monodentate ligands, because of the bidentate nature of carbodithioate linkage. It is expected that this coupling will allow an efficient electron transfer process from the electron rich molecule dopamine to Qdots. To confirm the efficient electron transfer process, the PL spectrum of the QDL is recorded, revealing drastic quenching (∼53 times) compared to the bare Qdots as shown in Figure 2b. Control experiments showed that fluorescence intensity of bare Qdots remained unaffected in the presence of (CS2) whereas it decreased slightly (2.5 times) by addition of dopamine (see SCHEME 2: Working Principle of Our “OFF”/“ON” Probe
ESI Figure 3). These experiments established the fact that the dopamine attachment provided an alternative annihilation pathway to the excited electrons causing the fluorescence quenching. The luminescence quenching of Qdots in QDL is indicative of photoinduced electron transfer from oxidized dopamine (electron rich amine) to Qdots through the carbodithioate linkage. The slight quenching of the fluorescence intensity of the Qdot in the presence of dopamine in solution is expected, as the dopamine molecule can reach the Qdot surface providing few electron transfer routes, but effective quenching is only possible through a proper linkage. Recently, Nadeau et al.33 have reported the photophysics of dopamine-modified Qdots (CdSe/ZnS) and their effects on biological systems. They also reported that the fluorescence quenching (electron transfer) of these Qdots depended on their sizes. In contrast, our study is focused on sensitive detection of GSH using Mn dopant-based Qdots (where the 4T1 )> 6A1 Mn2+ ion transition is independent of Qdot size28-32). Moreover, direct attachment of dopamine to Qdots could possibly have enhanced the efficiency of the electron transfer process because of “zero-length” coupling. To evaluate the efficacy of our activatable probe, we have performed GSH sensing experiments in DI water. Since GSH is a disulfide reducing agent, it is expected that upon addition of GSH to the solution of QDL, it will detach the ligand from the Qdot surface. This will restore the PL as the electron transfer pathways will be eliminated. As expected, a significant restoration (25 fold) of the fluorescence intensity was observed upon treatment with GSH (Figure 3), thus validating the proof-ofconcept. The fluorescence intensity was found to increase linearly with the GSH concentration [0-10 mM solution of
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Figure 4. Photoluminescence of QDL in the presence and absence of a 10 mM solution of GSH in phosphate buffer solution with different pH values.
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Figure 6. Effect of DTT on the luminescence of QDL.
Conclusions
Figure 5. PL of Qdots in the presence and absence of a 10 mM solution of GSH in DI water.
GSH] varying in the physiologically relevant limit (Figure 3). The plot in the inset of Figure 3 shows the linear restoration of the PL upon treatment with GSH solution in a range of physiological concentrations. The increase of fluorescence intensity is certainly due to the reduction of disulfide linkage by GSH, which results the detachment of the ligand from the Qdot surface. As soon as the ligand is detached from Qdots, the electron transfer process from dopamine to Qdots is inhibited. The working principle of our activatable probe is presented in Scheme 2. The PL restoration experiments were carried out by adding different amounts of GSH to phosphate buffer solution having pH 5.8, 7.4, and 8.2, respectively (Figure 4). PL restoration was observed in buffer solution similar to that observed in DI water. It was also observed that the variation in pH did not affect the PL restoration capability. This shows that our activatable probe is quite stable and robust to different physiological environments. In order to rule out the possibility of fluorescence intensity increase of Qdots due to further surface passivation by the thiol (GSH), PL of the bare Qdots was also measured in the presence of GSH (Figure 5). The results showed that the PL intensity of the Qdots remained unaffected upon addition of GSH. In order to consolidate our claim that the disulfide bond cleavage was responsible for the PL restoration, PL measurements were carried out with the QDL in the presence of another reagent, dithiothreitol (DTT). DTT has a disulfide bond reducing property similar to that of GSH. DTT also showed an effect similar to that of GSH on the PL of QDL (Figure 6). These studies confirmed our conclusion that detachment of the ligand is responsible for the PL restoration.
We have developed a highly sensitive activatable Qdot probe for the detection of glutathione at physiologically relevant levels. The basic idea is to modify the Qdot surface with an electron rich organic molecule via zero-length coupling chemistry. The fluorescently dark Qdots become highly fluorescent when reduced by the GSH. It is established that an electron transfer process took place between the Qdots and their surface-bound ligand, resulting in fluorescence quenching. Furthermore, the present probe is highly photostable compared to traditional fluorescent organic dye-based similar probes. To the best of our knowledge, this is the first report of Qdot-assisted detection of GSH. Taken together, the present probe could be useful to maintain the GSH level inside the cell. Acknowledgment. This work has been partly supported by the National Science Foundation (NSF CBET-63016011and NSF-NIRT Grant EEC-0506560). Supporting Information Available: Spectroscopic characterization details of the dopamine conjugated Qdots. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Zhang, J.; Campbell, R. E.; Ting, A. Y.; Tsien, R. Y. Nat. ReV. Mol. Cell Biol. 2002, 3, 906. (2) Hou, Y. C.; Guo, Z. M.; Li, J.; Wang, P. G. Biochem. Biophys. Res. Commun. 1996, 228, 88. (3) Deneke, S. M. Thiol-based antioxidants. Curr. Top. Cell. Regul. 2000, 36, 151. (4) Balendiran, G. K.; Dabur, R.; Fraser, D. Cell Biochem. Funct. 2004, 22, 343. (5) Lee, K.; Dzubeck, V.; Latshaw, L.; Schneider, J. P. J. Am. Chem. Soc. 2004, 126, 13616. (6) Maeda, H.; Matsuno, H.; Ushida, M.; Katayama, K.; Saeki, K.; Itoh, N. Angew. Chem., Int. Ed. 2005, 44, 2922. (7) Shao, N.; Jin, J. Y.; Cheung, S. M.; Yang, R. H.; Chan, W. H.; Mo, T. Angew. Chem., Int. Ed. 2006, 45, 4944. (8) Sudeep, P. K.; Joseph, S. T. S.; Thomas, K. G. J. Am. Chem. Soc. 2005, 127, 6516. (9) Zhu, J. G.; Dhimitruka, I.; Pei, D. Org. Lett. 2004, 6, 3809. (10) Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013. (11) Chan, W. C. W.; Nie, S. M. Science 1998, 281, 2016. (12) Gao, X. H.; Cui, Y. Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. M. Nat. Biotechnol. 2004, 22, 969. (13) Han, M. Y.; Gao, X. H.; Su, J. Z.; Nie, S. Nat. Biotechnol. 2001, 19, 631. (14) Kongkanand, A.; Tvrdy, K.; Takechi, K.; Kuno, M.; Kamat, P. V. J. Am. Chem. Soc. 2008, 130, 4007. (15) Larson, D. R.; Zipfel, W. R.; Williams, R. M.; Clark, S. W.; Bruchez, M. P.; Wise, F. W.; Webb, W. W. Science 2003, 300, 1434. (16) Loss, D.; DiVincenzo, D. P. Phys. ReV. A 1998, 57, 120.
Quantum Dot-Based OFF/ON Probe (17) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538. (18) Robel, I.; Subramanian, V.; Kuno, M.; Kamat, P. V. J. Am. Chem. Soc. 2006, 128, 2385. (19) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nat. Mater. 2005, 4, 435. (20) Landes, C.; Burda, C.; Braun, M.; El-Sayed, M. A. J. Phys. Chem. B 2001, 105, 2981. (21) Bavykin, D. V.; Savinov, E. N.; Parmon, V. N. Langmuir 1999, 15, 4722. (22) Chen, Y. F.; Rosenzweig, Z. Anal. Chem. 2002, 74, 5132. (23) Gattas-Asfura, K. A.; Leblanc, R. M. Chem. Commun. 2003, 2684. (24) Isarov, A. V.; Chrysochoos, J. Langmuir 1997, 13, 3142. (25) Jin, W. J.; Fernandez-Arguelles, M. T.; Costa-Fernandez, J. M.; Pereiro, R.; Sanz-Medel, A. Chem. Commun. 2005, 883. (26) Banerjee, S.; Kar, S.; Santra, S. Chem. Commun. 2008, 3037.
J. Phys. Chem. C, Vol. 113, No. 22, 2009 9663 (27) Santra, S.; Yang, H. S.; Holloway, P. H.; Stanley, J. T.; Mericle, R. A. J. Am. Chem. Soc. 2005, 127, 1656. (28) Biswas, S.; Kar, S.; Chaudhuri, S. J. Phys. Chem. B 2005, 109, 17526. (29) Biswas, S.; Kar, S.; Chaudhuri, S.; Nambissan, P. M. G. J. Phys. Cond. Matter 2008, 20, 235226. (30) Kar, S.; Biswas, S. J. Phys. Chem. C 2008, 112, 11144. (31) Kar, S.; Santra, S.; Heinrich, H. J. Phys. Chem. C 2008, 112, 4036. (32) Sarkar, I.; Sanyal, M. K.; Kar, S.; Biswas, S.; Banerjee, S.; Chaudhuri, S.; Takeyama, S.; Mino, H.; Komori, F. Phys. ReV. B 2007, 75, 224409. (33) Clarke, S. J.; Hollmann, C. A.; Zhang, Z. J.; Suffern, D.; Bradforth, S. E.; Dimitrijevic, N. M.; Minarik, W. G.; Nadeau, J. L. Nat. Mater. 2006, 5, 409.
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