Ion-Based Ensemble for Fluorescence Turn on Detection

Aug 31, 2010 - Solutions of TO/DNA/Hg2+ or Cu2+ ([TO] = 5 × 10−7 M, [DNA] = 5 ... (32, 33) In previous work, the quenching of fluorescence of .... ...
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Anal. Chem. 2010, 82, 8211–8216

DNA/Ligand/Ion-Based Ensemble for Fluorescence Turn on Detection of Cysteine and Histidine with Tunable Dynamic Range Fang Pu,† Zhenzhen Huang,†,‡ Jinsong Ren,*,† and Xiaogang Qu† Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resources Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China, and Graduate School of the Chinese Academy of Sciences, Beijing 100039, P. R. China A new type of rapid, highly sensitive, and selective fluorescence turn-on assay for detection of cysteine and histidine using a DNA/ligand/ion ensemble is developed. This assay is based on the highly specific interaction between the amino acids and the metal ions and the strong fluorescence thiazole orange (TO)/DNA probe in a competition assay format. The resulting high sensitivity and selectivity for cysteine and histidine was achieved by changing the metal ions. The system is simple in design and fast in operation and is more convenient and promising than other methods. The novel strategy eliminated the need of organic cosolvents, enzymatic reactions, separation processes, chemical modifications, and sophisticated instrumentations. The detection and discrimination process can be seen with the naked eye under a hand-held UV lamp and can be easily adapted to automated highthroughput screening. The detection limit of this method is lower than or at least comparable to previous fluorescence-based methods. The dynamic range of the sensor can be tuned simply by adjusting the concentration of metal ions. Importantly, the protocol offers high selectivity for the determination of cysteine among amino acids found in proteins and in serum samples. The assay shows great potential for practical application as a diseaseassociated biomarker and will be needed to satisfy the great demand of amino acid determination in fields such as food processing, biochemistry, pharmaceuticals, and clinical analysis. Among various naturally occurring amino acids, cysteine (Cys) and histidine (His) have attracted much attention in recent years due to their vital biological functions. For example, cysteine deficiency is involved in many syndromes, such as edema, lethargy, liver damage, muscle and fat loss, slowed growth, and skin lesion.1 Additionally, cysteine-induced hypoglycemic brain damage has been studied as an alternative mechanism to excitotoxicity.2 An abnormal level of the thiol-containing amino acid also has been proved to be a risk factor for cancer and Alzheimer’s * To whom correspondence should be addressed. E-mail: [email protected]. Fax: (+86) 0431-85262625. † Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. ‡ Graduate School of the Chinese Academy of Sciences. (1) Shahrokhian, S. Anal. Chem. 2001, 73, 5972–5978. 10.1021/ac101647k  2010 American Chemical Society Published on Web 08/31/2010

and cardiovascular diseases.3,4 Histidine (His) is also essential for the growth and repair of tissue as well as for the control of transmission of metal elements in biological bases.5 Histidine is the precursor amino acid of histamine, which is an important neurotransmitter and involved in cellular growth and differentiation, regulation of nucleic acid and protein synthesis, and stabilization of lipids.6-9 Recent studies have shown that a deficiency of histidine in plasma may lead to an impaired nutritional state in patients with chronic kidney disease.10 Several methods have been developed for the determination of cysteine or histidine, including liquid chromatography,11-15 flow injection,16 voltammetry,17 spectroflourimetry,18-20 and capillary zone electrophoresis.21 Due to the structural similarity of amino acids, incorporating both carboxylic and amino groups, and their spectroscopic inertness, many of the analytical methods are based on redox chemistry or labeling with chromophores or fluorophores and a combination (2) Gazit, V.; Ben-Abraham, R.; Coleman, R.; Weizman, A.; Katz, Y. Amino Acids 2004, 26, 163–168. (3) Seshadri, S.; Beiser, A.; Selhub, J.; Jacques, P. F.; Rosenberg, I. H.; D’Agostino, R. B.; Wilson, P. W.; Wolf, P. A. N. Engl. J. Med. 2002, 346, 476–483. (4) Refsum, H.; Ueland, P. M.; Nygard, O.; Vollset, S. E. Annu. Rev. Med. 1998, 49, 31–62. (5) Chen, G. N.; Wu, X. P.; Duan, J. P.; Chen, H. Q. Talanta 1999, 49, 319– 330. (6) Su, S. C.; Chou, S. S.; Chang, P. C.; Hwang, D. F. J. Chromatogr., B 2000, 749, 163–169. (7) Paproski, R. E.; Roy, K. I.; Lucy, C. A. J. Chromatogr., A 2002, 946, 265– 273. (8) Zhang, L. Y.; Sun, M. X. J. Chromatogr., A 2004, 1040, 133–140. (9) Zeng, Q.; Jim, C. K. W.; Lam, J. W. Y.; Dong, Y. Q.; Li, Z.; Qin, J. U.; Tang, B. Z. Macromol. Rapid Commun. 2009, 30, 170–175. (10) Watanabe, M.; Suliman, M. E.; Qureshi, A. R.; Garcia-Lopez, E.; Barany, P.; Heimburger, O.; Stenvinkel, P.; Lindholm, B. Am. J. Clin. Nutr. 2008, 87, 1860–1866. (11) Potesil, D.; Petrlova, J.; Adam, V.; Vacek, J.; Klejdus, B.; Zehnalek, J.; Trnkova, L.; Havel, L.; Kizek, R. J. Chromatogr., A 2005, 1084, 134–144. (12) Amarnath, K.; Amarnath, V.; Amarnath, K.; Valentine, H. L.; Valentine, W. M. Talanta 2003, 60, 1229–1238. (13) Gegg, M. E.; Clark, J. B.; Heales, S. J. Anal. Biochem. 2002, 304, 26–32. (14) Katrusiak, A. E.; Paterson, P. G.; Kamencic, H.; Shoker, A.; Lyon, A. W. J. Chromatogr., B 2001, 758, 207–212. (15) Chwatko, G.; Bald, E. Talanta 2000, 52, 509–515. (16) Zhao, C.; Zhang, J.; Song, J. Anal. Biochem. 2001, 297, 170–176. (17) Amini, M. K.; Khorasani, J. H.; Khaloo, S. S.; Tangestaninejad, S. Anal. Biochem. 2003, 320, 32–38. (18) Chen, Y. H.; Cai, R. X. Spectrochim. Acta, Part A 2003, 59, 3357–3362. (19) Liang, S. C.; Wang, H.; Zhang, Z. M.; Zhang, X.; Zhang, H. S. Spectrochim. Acta, Part A 2002, 58, 2605–2611. (20) Wang, H.; Wang, W. S.; Zhang, H. S. Talanta 2001, 53, 1015–1019. (21) Jin, W.; Wang, Y. J. Chromatogr., A 1997, 769, 307–314.

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of separation techniques.22 In most cases, these approaches require sophisticated instrumentation, involve cumbersome laboratory procedures, and have low throughput, which limits the scope of their practical applications. Moreover, the main disadvantage is their low sensitivities, in which only micromolar concentrations of amino acid is detectable. With the development in nanotechnology, new methods have been used for the detection of amino acids.23-25 Sudeep et al. reported a strategy for the selective detection of micromolar concentrations of cysteine by exploiting the interplasmon coupling in Au nanorods.23 However, low sensitivity limited the effectiveness of such strategy. Recently, Mirkin and his co-workers used oligonucleotide-modified gold nanoparticles (AuNPs) as a probe for colorimetric detection of cysteine.24 Although promising, this method needs steps such as modifying the oligonucleotide onto AuNPs and separating the modified AuNPs from the unmodified AuNPs or surplus oligonucleotide, which add to the complexity, cost, and overall assay time. Furthermore, the colorimetric response was only detectable at temperature above 50 °C. Recently, highly selective probes for cysteine, as well as a thiol-quantification enzyme assay, were developed on the basis of the covalent interaction between the probe molecule and the analyte.26-28 In addition, sensing assemblies for cysteine based on metal receptor were also developed.29,30 Here, we present a new strategy for the highly sensitive and selective detention of cysteine and histidine with tunable dynamic range by exploring a ligand/DNA/ion ensemble. EXPERIMENTAL SECTION Materials and Measurement. Thiazole orange (TO) was purchased from Aldrich and used without further purification. The extinction coefficient of TO is 63 000 M-1 cm-1 at 500 nm. DNA (5′-CACTG TGGTT GGTGT GGTTG G-3′) was purchased from Sangon Biotechnology Inc. (Shanghai, China). The concentration of DNA and TO was determined using a JASCO V-550 UV/vis spectrophotometer, equipped with a temperaturecontrolled cuvette holder. All other reagents were all of analytical reagent grade and used as received. The amino acid solutions were prepared freshly on the day of use. Water was purified by a Millipore filtration system. Fluorescence Quenching Measurements. Solutions of TO/ DNA ([TO] ) 5 × 10-7 M, [DNA] ) 5 × 10-8 M) in 400 µL of Tris-HCl (10 mM, pH7.0) buffer were titrated with divalent metal ions, including Hg2+, Cu2+, Ni2+, and Zn2+. In all cases, (22) Nekrassova, O.; Lawrence, N. S.; Compton, R. G. Talanta 2003, 60, 1085– 1095. (23) Sudeep, P. K.; Joseph, S. T.; Thomas, K. G. J. Am. Chem. Soc. 2005, 127, 6516–6517. (24) Lee, J. S.; Ulmann, P. A.; Han, M. S.; Mirkin, C. A. Nano Lett. 2008, 8, 529–533. (25) Li, Z. P.; Duan, X. R.; Liu, C. H.; Du, B. A. Anal. Biochem. 2006, 351, 18–25. (26) Rusin, O.; St Luce, N. N.; Agbaria, R. A.; Escobedo, J. O.; Jiang, S.; Warner, I. M.; Dawan, F. B.; Lian, K.; Strongin, R. M. J. Am. Chem. Soc. 2004, 126, 438–439. (27) Wang, W.; Rusin, O.; Xu, X.; Kim, K. K.; Escobedo, J. O.; Fakayode, S. O.; Fletcher, K. A.; Lowry, M.; Schowalter, C. M.; Lawrence, C. M.; Fronczek, F. R.; Warner, I. M.; Strongin, R. M. J. Am. Chem. Soc. 2005, 127, 15949– 15958. (28) Maeda, H.; Matsuno, H.; Ushida, M.; Katayama, K.; Saeki, K.; Itoh, N. Angew. Chem., Int. Ed. 2005, 44, 2922–2925. (29) Han, M. S.; Kim, D. H. Tetrahedron 2004, 60, 11251–11257. (30) Shao, N.; Jin, J. Y.; Cheung, S. M.; Yang, R. H.; Chan, W. H.; Mo, T. Angew. Chem., Int. Ed. 2006, 45, 4944–4948.

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Scheme 1. Schematic Illustration of the TO/DNA/Metal Ion-Based Amino Acid Sensora

a Chemical structures of cysteine, histidine, and thiazole orange (TO) are shown.

samples were excited at 490 nm, and emission spectra were collected from 500 to 650 at a 1000 nm/min scan rate using a JASCO FP-6500 spectrofluorimeter. Fluorescence Titration of TO/DNA/Metal Ion System with Amino Acids. Solutions of TO/DNA/Hg2+ or Cu2+ ([TO] ) 5 × 10-7 M, [DNA] ) 5 × 10-8 M, [Hg2+] ) 6.25 × 10-7 M, or [Cu2+] ) 1.25 × 10-6 M) were prepared in 400 µL of buffer and placed in quartz cell. The solutions of amino acids were prepared in distilled water and were added in portions. Samples were excited at 490 nm, and emission spectra were collected from 500 to 650 nm. Visual Detection of Amino Acids. Solutions of TO/DNA/ Hg2+ or [Cu2+] ([TO] ) 5 × 10-6 M, [DNA] ) 2.5 × 10-6 M, [Hg2+] ) 2.5 × 10-5 M, or [Cu2+] ) 5.0 × 10-5 M) were prepared in 50 µL of buffer. Amino acids [2.5 × 10-5 M (for Hg2+) or 5.0 × 10-5 M (for Cu2+)] were added to react for 5 min at room temperature. Photographs of solutions were taken using an UV transilluminator. RESULTS AND DISCUSSION As demonstrated in Scheme 1, the fluorescence turn on strategy is based on the analyte competing for a metal reporter with a chromogenic indicator: (1) Thiazole orange (TO) was chosen as a fluorescence indicator in the ensemble. The asymmetric cyanine dye TO is a very popular fluorescent marker consisting of two aromatic ring systems connected by a exocyclic vinyl bond. It is virtually nonfluorescent in aqueous solution but shows intense fluorescence upon interaction with nucleic acids without regard to base composition.31 (2) Many divalent metal ions are well-known as a fluorescence quencher and work via numerous mechanisms, including ground-state complexation, collisional conversion of electronic to kinetic energy, heavy atom effects, magnetic perturbations, charge-transfer phenomena, electronic energy transfer, and fluorescence resonance energy transfer.32,33 In previous work, the quenching of fluorescence of DNA-binding ethidium bromide by some transition metal ions has (31) Nygren, J.; Svanvik, N.; Kubista, M. Biopolymers 1998, 46, 39–51. (32) Rupcich, N.; Chiuman, W.; Nutiu, R.; Mei, S.; Flora, K. K.; Li, Y.; Brennan, J. D. J. Am. Chem. Soc. 2006, 128, 780–790. (33) Atherton, S. J.; Beaumont, P. C. Photochem. Photobiol. 1993, 57, 460–464.

Figure 1. (a) Fluorescence emission spectra of TO/DNA containing Hg2+ ([TO] ) 5 × 10-7 M, [DNA] ) 5 × 10-8 M, [Hg2+] ) 6.25 × 10-7 M) in the absence and presence of cysteine (2.5 × 10-6 M). For comparison, the sensor response of other amino acids (AAs, 2.5 × 10-6 M) is also presented. (b) Fluorescence emission spectra of TO/DNA containing Hg2+ ([TO] ) 5 × 10-7 M, [DNA] ) 5 × 10-8 M, [Hg2+] ) 6.25 × 10-7 M) with increasing amounts of cysteine. The inset displays the linear plots.

been reported.34 (3) It was known that R-amino acids could form stable complexes with some metal ions, i.e., mercury, copper, and zinc ions.35 Therefore, it was expected that the addition of some R-amino acids to the solution of the TO/DNA/metal ion complex could turn on the fluorescence of the TO, if R-amino acids could snatch the metal ions from the complex. On the other hand, no restoration of fluorescence could be observed if the amino acid could not remove the metal ions from the ensemble. The feasibility of strategy was first confirmed using Hg2+ ion as quencher and cysteine as analyte. Cysteine can react with Hg2+ to form a stable complex. The structure and energetics of the complex have been reported in previous studies,36 which demonstrated that Hg2+ is coordinated to both the carboxyl oxygen and sulfur atoms of cysteine. In the experiment, the fluorescence generated from the interaction of TO and DNA was first evaluated. As can be seen in Figure 1a, TO has negligible fluorescence in solution. Upon adding 5 × 10-8 M single-stranded DNA (ssDNA), the fluorescent spectrum of TO displayed an emission maximum at 540 nm upon excitation at 490 nm (Figure 1a). Therefore, the strong fluorescence signals obtained here ensure a good signal-to-noise ratio. The sensitive response of TO/DNA complex to the added metal ion was then carried out. Figure S1 in the Supporting Information displays the fluorescent spectra of the TO/DNA complex in the presence of Hg2+ with varying concentration. As shown, with increasing the concentration of Hg2+, the fluorescent intensity at 540 nm decreased gradually. The quenching was analyzed using the Stern-Volmer equation: F0/F ) 1 + Ksv[Q], where F and F0 are the fluorescent intensity at 540 nm in the presence and absence of Hg2+ ions, respectively, Ksv is the Stern-Volmer quenching constant, and [Q] is the quencher concentration. The quenching of TO/DNA by Hg2+ ions showed a good linear relationship in the Stern-Volmer plot (F0/F versus [Hg2+]) with the Ksv value of 5.3 × 107 M-1 (Figure S1 inset, Supporting Information). The Ksv suggested that Hg2+ exhibited a strong quenching ability toward the fluorescence of the TO/DNA complex. The high quenching efficiency imparts the assay high sensitivity.

In order to assess the application of this method, cysteine was used for testing. Upon the addition of cysteine to the solution of the TO/DNA/Hg2+ ensemble, the quenched fluorescence of TO/DNA turned on immediately (Figure 1a). The enhancement of fluorescence was time dependent and reached a constant value only after 5 min, which showed one of the advantages of the quick response of the system (Figure S2, Supporting Information). In addition, the intensity at 540 nm increased rapidly with increasing the concentration of cysteine (Figure S3, Supporting Information). The fluorescent intensity could recover to about 70% of the original one of TO/DNA, when the concentration of cysteine was about 2.5 × 10-6 M. These changes demonstrated that cysteine could snatch successfully the Hg2+ ion from the TO/DNA/Hg2+ system. Thus, the obtained results clearly demonstrated that the TO/DNA/Hg2+ ion system could report the presence of cysteine, and the fluorescence recovery could be used to develop a calibration curve to afford a quantitative measurement. Figure 1b presents the fluorescence data as a function of fluorescent intensities versus the concentration of cysteine. The enhanced fluorescence intensity is proportional to the cysteine concentration in the range from 2.5 × 10-9 to 1.1 × 10-7 M, and the detection limit of cysteine is 5.1 × 10-9 M. This detection limit is comparable to or even better than previous reports for cysteine detection.25,30,37-39 More importantly, the label-free and turn-on sensing mode offered additional advantages here. A major limitation of turn-off probes is that variations in sample environment may be problematic for utilization in quantization measurement, while the turn-on response is able to efficiently reduce background noise and increase detection sensitivity. For an excellent chemosensor, high selectivity is a matter of necessity. To study the selectivity for cysteine determination with current approach, the fluorescent signal of various amino acids found in proteins were measured and compared with that of cysteine at the same concentration (2.5 × 10-6 M). The results are shown in Figure 2a. It can be seen that only cysteine exhibited a significant perturbation on the system of TO/DNA/Hg2+. The Hg2+-quenched fluorescence was recovered to the greatest extent by cysteine, in contrast to virtually no recovery or a much smaller recovery induced by other amino acids. Achiev-

(34) Atherton, S. J.; Beaumont, P. C. J. Phys. Chem. 1986, 90, 2252–2259. (35) Severin, K.; Bergs, R.; Beck, W. Angew. Chem., Int. Ed. 1998, 37, 1635– 1654. (36) Belcastro, M.; Marino, T.; Russo, N.; Toscano, M. J. Mass Spectrom. 2005, 40, 300–306.

(37) Nie, L.; Ma, H.; Sun, M.; Li, X.; Su, M.; Liang, S. Talanta 2003, 59, 959– 964. (38) Shang, L.; Qin, C. J.; Wang, T.; Wang, M.; Wang, L. X.; Dong, S. J. J. Phys. Chem. C 2007, 111, 13414–13417. (39) Shang, L.; Dong, S. J. Biosens. Bioelectron. 2009, 24, 1569–1573.

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Table 1. Determination of Cysteine in Some Samples Containing Fetal Bovine Seruma sample

spiked (µM)

found (µM)

recovery (%)

1 2 3

0 1.0 4.0

0.39 1.40 4.44

101 101.3

a Fetal bovine serum was diluted 500-fold with Tris-HCl (10 mM, pH 7.0) buffer before detection. [TO] ) 5 × 10-7 M, [DNA] ) 5 × 10-8 M, and [Hg2+] ) 5 × 10-6 M.

Figure 2. (a) Fluorescence emission response profiles of TO/DNA/ Hg2+ ([TO] ) 5 × 10-7 M, [DNA] ) 5 × 10-8 M, [Hg2+] ) 6.25 × 10-7 M) toward amino acids (2.5 × 10-6 M). The sensor response to Cys in the presence of a mixture of other amino acids is also presented. (b) Photographs of solutions of TO (A), TO/DNA (B), TO/DNA/Hg2+ (C), and TO/DNA/Hg2+ in the presence of Ala (D), Gly (E), Val (F), Leu (G), Ile (H), Ser (I), Thr (J), Asp (K), Asn (L), Glu (M), Gln (N), Arg (O), Lys (P), His (Q), Cys (R), Met (S), Phe (T), Tyr (U), and Trp (V), taken using an UV transilluminator.

ing high selectivity for the analyst of interest over a complex background of potentially competing species is a challenge in sensor development. Thus, the competition experiments were conducted in the presence of mixture of other amino acids. Significantly, the fluorescence intensity change of cysteine in the mixture of other amino acids was less than 5%, relative to cysteine alone at same concentration. The result suggested that the coexistence of other amino acids in the system of TO/ DNA/Hg2+ did not affect the detection of cysteine. Therefore, the proposed method is practical for the determination of cysteine in the mixture of amino acids found in protein without separation. In addition to fluorescence analysis, the remarkable selectivity toward cysteine over other amine acids can be observed by the naked eye, with the aid of an UV transilluminator. Figure 2b shows the photographs taken from the solutions of TO/DNA and TO/ DNA/Hg2+ in the absence and presence of amino acids excited under an UV transilluminator. The difference of the solutions of TO/DNA/Hg2+ before and after the addition of cysteine and other amino acids was apparent. The solution containing cysteine exhibited bright yellow light, and the solutions containing other amino acids exhibited no emission. These observations strongly support the sensing mechanism of our method, and it provides an additional simple approach for detecting cysteine. The development of a selective sensor in the physiological condition for cysteine is crucial. However, many methods could not be performed at optimal conditions since special organic cosolvents19,30,40 or pH25 are required to modulate the stability of the system. For example, the effect of amino acids on Au nanorods (40) Zeng, Y.; Zhang, G.; Zhang, D. Anal. Chim. Acta 2008, 627, 254–257.

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was investigated in a mixture (4:1) of acetonitrile and water. The pH of a Au nanorod solution is ∼5.6, in which cysteine can assist self-assembly of Au nanrods.23 Considering the physiological link between cysteine and a variety of diseases and disease status, our assay is particularly attractive. Unlike some organic fluorophores or Au nanorods, the high water solubility of the TO/DNA/metal ion system allows this sensor to be used in aqueous media without the need for organic cosolvents or special pH. As in the case for monitoring cysteine in biological samples, it is important to consider the possible interference from physiological species. We subsequently investigated the fluorescence assay of cysteine in the presence of serum. Serum is plasma without fibrinogen or other clotting factor, which contains proteins, glucose, mineral ions, hormones, and other biological substances. In the experiment, fetal bovine serum (FBS) was diluted 500-fold with buffer before detection to mimic a biological sample. Before addition of cysteine, the nonspecific interaction between TO and the components in serum induced some background emission. However, addition of cysteine into the solution led to continuous fluorescence increase in emission. The method had good recoveries, as given in Table 1. These data show that the proposed method can be satisfactorily applied to detect serum cysteine. Jacobsen et al. reported the total concentration of cysteine in human serum was 266.90 ± 34.23 µM for men and 230.50 ± 32.70 µM for women.41 The detection limit for cysteine is much lower than that in human serum (µM), which suggests that this method has great potential for diagnostic purpose. A tunable dynamic range is important for practical applications, as the desirable concentrations for the same target analyte can be different for various applications. For example, besides the application in food supplements and pharmaceuticals, thanks to its biological function, cysteine is widely used in other industries, such as flavor, cosmetics, and function food, due to its characteristic sulfhydryl group. In order to tune the dynamic range and fit the sensor for different detection requirements, we further investigated if the concentration of metal ions can be used as a tunable parameter. Given that the calibration curve can be obtained at various concentrations of metal ions, accurate quantification of cysteine over different concentration ranges can be achieved by our sensor. As shown in Figure 3, the dynamic range was from 2.5 × 10-9 to 1.1 × 10-6 M, in the presence of 6.25 × 10-7 M Hg2+. When the experiment was carried out using 1.25 × 10-6 M Hg2+, the dynamic range was shifted to 5 × 10-7-2 × 10-6 M. With the increase of Hg2+ concentration to 2.5 × 10-6 M, the dynamic range was from 2.1 × 10-6 to 3.3 × 10-6 M. When more mercury ions were present, the added cysteine (41) Jacobsen, D. W.; Gatautis, V. J.; Green, R.; Robinson, K.; Savon, S. R.; Secic, M.; Ji, J.; Otto, J. M.; Taylor, L. M., Jr. Clin. Chem. 1994, 40, 873–881.

Figure 3. Calibration curves of the label-free sensor. The complex TO/DNA ([TO] ) 5 × 10-7 M, [DNA] ) 5 × 10-8 M) was treated with different Hg2+ concentrations and then treated with increasing amounts of cysteine.

would react first with free Hg2+ in solution, which then decreases the enhancement effect. Thus, the dynamic range of the sensor can be tuned simply by adjusting the concentration of metal ions while other assay procedures are the same. Moreover, our system could be extended to a multimicrowell plate-based assay to achieve different sample detection at one time without the need to develop new sensors. Serial dilutions are less necessary to achieve an accurate measurement. For unknown samples, this simplifies range finding and eliminates errors introduced by dilution. There are two factors responsible for the high sensitivity and selectivity of this method. First, the quenching of fluorescence of TO/DNA allows one to monitor the efficient recovery by addition of cysteine, and this effect provides the basis for the “turn on” assay. The fluorescence spectrum of TO/DNA was also measured in the presence of Zn2+ ion. Little fluorescence signal change at 540 nm was observed after addition of Zn2+ ion (Figure S4a, Supporting Information). Thus, Zn2+ ion could not be used in this method, though Zn2+ could form a stable complex with cysteine.36 Second, the high selectivity and tight binding of cysteine for the Hg2+ compared with the other amino acids lead to an assay with high specificity. Hg2+ is known to have an affinity for certain N-type ligands, potentially including basic amino acids such as histidine or lysine.24 However, in this system, the TO/DNA appears to be a strong enough Hg2+ binder to effectively compete against all of the amino acids other than cysteine. Ni2+ ion could quench the fluorescence of TO/DNA with Ksv of 2.4 × 106 M-1 (Figure S4b, Supporting Information). However, the amino acids were not able to dislodge the Ni2+ ion from the system of the TO/DNA/Ni2+ ion, with no restoration of fluorescence (Figure S4c, Supporting Information). Therefore, Ni2+ ion could not be employed as a selective probe for amino acids. Another unique characteristic of the novel system is that it could detect cysteine and histidine simultaneously by only changing the metal ion in the ensemble. Cu2+ is a well-known and highly efficient fluorescence quencher. Meanwhile, Cu2+ could form a stable complex with many amino acids. Then, we used Cu2+ instead of Hg2+ to detect amino acids. First of all, the fluorescence quenching of TO/DNA in the presence of Cu2+ ions was examined. Ksv was determined to be 1.3 × 107 M-1 according to Stern-Volmer equation (Figure S5, Supporting Information). The Ksv suggested that Cu2+ exhibited a strong quenching ability toward the fluorescence of the TO/

Figure 4. (a) Fluorescence emission response profiles of TO/DNA/ Cu2+ ([TO] ) 5 × 10-7 M, [DNA] ) 5 × 10-8 M, [Cu2+] ) 1.25 × 10-6 M) toward amino acids (5.0 × 10-6 M). The sensor response to Cys or His in the presence of a mixture of other amino acids is also presented. (b) Photographs of solutions of TO (A), TO/DNA (B), TO/ DNA/Cu2+ (C), and TO/DNA/Cu2+ in the presence of Ala (D), Gly (E), Val (F), Leu (G), Ile (H), Ser (I), Thr (J), Asp (K), Asn (L), Glu (M), Gln (N), Arg (O), Lys (P), His (Q), Cys (R), Met (S), Phe (T), Tyr (U), and Trp (V), taken using an UV transilluminator.

DNA. Then, the solutions of amino acids were added, respectively, and fluorescence intensity changes were recorded. In our system, both histidine and cysteine could be discriminated from other amino acids. As shown in Figure 4a, other amino acids produced negligible influence on the Cu2+-quenched fluorescence of TO/DNA at a concentration up to 5 × 10-6 M. Histidine and cysteine dislodged Cu2+ and restored the fluorescence of TO/DNA. The photographs taken using an UV transilluminator support these results (Figure 4b). We added histidine or cysteine into a solution of TO/DNA/Cu2+ in the presence of other amino acids except for cysteine or histidine. It can be seen that no significant variation in fluorescence intensity was found in comparison to that containing only histidine or cysteine (Figure 4a). That means most amino acids did not interfere with the fluorescence enhancement induced by histidine or cysteine. These results are essentially determined by the binding affinity between the metal ions and the amino acids. Both histidine and cysteine could be discriminated from other amino acids since the side chains of histidine and cysteine play an important role in the complex of Cu2+ and amino acids. Cu2+ could interact with both the carbonyl oxygen and the sulfur atom of cysteine.36 The X-ray structure of physiological Cu2+-Lhistidine complex has been reported.42 The imidazole side chain of histidine is imperative for copper chelation.43 Histidine and cysteine appear to be much stronger than other amino acids to compete for Cu2+ with the TO/DNA system. However, (42) Deschamps, P.; Kulkarni, P. P.; Sarkar, B. Inorg. Chem. 2004, 43, 3338– 3340. (43) Sarkar, B.; Wigfield, Y. J. Biol. Chem. 1967, 242, 5572–5577.

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Figure 5. (a) Fluorescence emission spectra of TO/DNA ([TO] ) 5 × 10-7 M, [DNA] ) 5 × 10-8 M) containing 1.25 × 10-6 M Cu2+ in the absence and presence of amino acids. For comparison, the sensor response of other amino acids except for cysteine (AAs, 5.0 × 10-6 M) is also presented. (b) Fluorescence emission spectra of TO/DNA containing Cu2+ ([TO] ) 5 × 10-7 M, [DNA] ) 5 × 10-8 M, [Cu2+] ) 1.25 × 10-6 M) with increasing amounts of histidine. The inset displays the linear plots. Table 2. Determination of Binary Mixtures of Cysteine and Histidinea add (µM)

found (µM)

recovery (%)

sample

Cys

His

Cys

His

Cys

His

1 2

8.0 12.0

42.0 38.0

7.8 12.4

43.1 37.1

97.5 103.3

102.6 97.6

a [TO] ) 5 × 10-7 M, [DNA] ) 5 × 10-8 M, [Hg2+] ) 5 × 10-6 M, and [Cu2+] ) 1.25 × 10-5 M.

the situation is more favorable to histidine in our system. The detection of histidine demonstrated even higher sensitivity than cysteine, as shown in Figure S6 in Supporting Information. The detection limit is 1.0 × 10-8 M for histidine. It is noteworthy that this detection limit is much lower than the previously reported spectroscopic methods for histidine detection.9 The linear range is from 0 to 4.4 × 10-6 M (Figure 5, Figure S7 in Supporting Information). The fluorescence intensity could recover to about 70% at a concentration as low as 5.6 × 10-6 M (Figure 5a). The normal concentrations of histidine in urine and serum are in the range of 1.8 × 10-4 to 1.2 × 10-3 and 8.6 × 10-5 to 1.1× 10-4 M, respectively.44,45 Thus, by changing the metal ion, the off/on fluorescence sensor is suitable for selective detection of histidine in the physiological condition. We performed experiments for the detection of binary mixtures using the proposed method. First, cysteine could be determined in the system of TO/DNA/Hg2+ due to high selectivity. Then, we changed the metal ion to determinate histidine. As shown in Figure S8 in the Supporting Information, the sum of the emission of TO/DNA/Cu2+ in the presence of cysteine and histidine is equal to the emission of TO/DNA/Cu2+ with the binary mixture. Thus, histidine could be determined in the system of TO/DNA/Cu2+. The results shown in Table 2 demonstrate that the proposed method had good recoveries, which validate the reliability and practicality of this method. CONCLUSION In summary, we have demonstrated a new type of rapid, highly sensitive, and selective fluorescence turn-on assay for detection (44) Ye, J. N.; Baldwin, R. P. Anal. Chem. 1994, 66, 2669–2674. (45) Pitkanen, H. T.; Oja, S. S.; Kemppainen, K.; Seppa, J. M.; Mero, A. A. Amino Acids 2003, 24, 413–421.

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of cysteine and histidine using a DNA/ligand/ion ensemble. This assay is based on the highly specific interaction between the amino acids and the metal ions and the strong fluorescence DNA/ TO probe in a competition assay format. The resulting high sensitivity and selectivity for cysteine and histidine was achieved by changing the metal ions. The system is simple in design and fast in operation and is more convenient and promising than other methods. The novel strategy eliminated the need of organic cosolvents, enzymatic reactions, separation processes, chemical modifications, and sophisticated instrumentations. In addition, the detection and discrimination process can be seen with the naked eye under a hand-held UV lamp and can be easily adapted to automated high-throughput screening. The detection limit of this method is lower or at least comparable to the previous fluorescencebased method. The dynamic range of the sensor can be tuned simply by adjusting the concentration of metal ions. Importantly, the protocol offers high selectivity for the determination of cysteine among amino acids found in proteins, as well as detection in serum samples. Thus, the assay showed great potential for practical application as a disease-associated biomarker and would be needed to satisfy the great demand of amino acid determination in fields such as food processing, biochemistry, pharmaceuticals, and clinical analysis. Significantly, as many biomolecules could form stable complexes with metal ions selectively, these findings offer a potential approach to the detection of a wild spectrum of analytes. ACKNOWLEDGMENT The authors thank the National Natural Science Foundation of China (Grants 20831003, 90813001, 90913007 and 20833006) and the fund from CAS for financial support. SUPPORTING INFORMATION AVAILABLE Fluorescence spectra of TO/DNA in the presence of Hg2+, 2+ Zn , Ni2+, and Cu2+; time-dependence fluorescence change of TO/DNA/Hg2+ upon adding cysteine; fluorescence spectra of TO/DNA containing metal ions with increasing amounts of amino acids. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review June 22, 2010. Accepted August 23, 2010. AC101647K