“Molecular Beacon”-Based Fluorescent Assay for ... - ACS Publications

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“Molecular Beacon”-Based Fluorescent Assay for Selective Detection of Glutathione and Cysteine Hui Xu† and Maria Hepel* Department of Chemistry, State University of New York at Potsdam, Potsdam, New York 13676, United States ABSTRACT: We report on the development of a fluorescence turn-on “molecular beacon” probe for the detection of glutathione (GSH) and cysteine (Cys). The method is based on a competitive ligation of Hg2þ ions by GSH/Cys and thyminethymine (T-T) mismatches in a DNA strand of the selfhybridizing beacon strand. The assay relies on the distancedependent optical properties of the fluorophore/quencher pair attached to the ends of the molecular beacon DNA strand. In a very selective coordination of Hg2þ to GSH/Cys, the fluorophore/quencher distance increases concomitantly with the dehybridization and dissociation of the beacon stem T-Hg2þ-T due to the extraction of Hg2þ ions. This process results in switching the molecular beacon to the “on” state. The concentration range of the probe is 4-200 nM with the limit of detection (LOD) of 4.1 nM for GSH and 4.2 nM Cys. The probe tested satisfactorily against interference for a range of amino acids including sulfurcontaining methionine.

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range of sensitive and highly selective biosensors utilizing the exceptional affinity and specificity of biorecognition schemes,1-10 including antibody-antigen, receptor-protein, and DNA-protein interactions, has been developed for largesize biomolecules.1,11,12 Since the biorecognition principle based on weak multiple-point interactions becomes less viable for smaller analyte molecules, other mechanisms leading to high sensor selectivity have to be explored. These are based either on strong affinity interactions, e.g., in immunosensors for atrazine and polychlorinated biphenyls,13-15 or on a highly specific covalent binding,16 intercalation,17,18 coordination complexes,5 or other supramolecular binding.19 Recently, Lee et al.5 reported on the development of a colorimetric assay for cysteine (Cys), based on the DNA-Cys competition for Hg2þ ions. Another type of strong interaction, provided by gold nanoparticles (AuNP) and homocysteine (Hcys), can be utilized for Hcys detection by resonance elastic light scattering (RELS).20,21 On the other hand, a small biomolecule, glutathione (GSH), can be efficiently analyzed using highly specific covalent binding to monochlorobimane (MCB), even in the absence of an enzyme GSH-transferase, in a fluorescence “turn-on” kinetic assay.16 In this work, we have developed a novel molecular beacon method for very sensitive detection of GSH and Cys, based on a competitive mercury ligation and fluorescence resonance energy transfer (FRET). Glutathione is a thiol group containing tripeptide (γ-Glu-CysGly) which plays an essential role in the health of organisms, particularly aerobic organisms.22 It participates in the main redox potential maintaining system in eukaryotic cell homeostasis and prevents the oxidative stress and helps to trap free radicals that can damage DNA and RNA. The oxidative stress has been implicated in many diseases and accelerates the aging process. r 2011 American Chemical Society

There is also evidence that oxidative stress contributes to the development of autism in children.23 Hence, the analysis of biomarkers of oxidative stress, such as GSH, becomes the key factor for preventive treatments. Due to the strong interactions with metal ions, GSH plays an important role as the potential antidote for heavy metals.24-26 Cysteine is an essential amino acid in natural proteins and is biologically active. It plays a crucial biological role in the human body including protein synthesis, detoxification, and metabolism. Elevated levels of Cys have been associated with neurotoxicity.27,28 Additionally, Cys has been proven to act as the physiological regulator29 in various diseases, and hence, it is used in medicine and in special nutrition preparations. The concentration of Cys in vivo is highly correlated to the physiological functions helping in diagnosing the underlying disease. On the basis of these applications, considerable attention has been drawn to the determination of these biological compounds. The concentration of Cys in vivo is highly correlated with physiological functions, and the diagnosis of disease is often dependent on the determination of Cys. The analysis of GSH level is critical in cases of oxidative stress to prevent serious damage to DNA, proteins, and lipid membranes. There is evidence that a low GSH level contributes to the development of autism in children. Therefore, the analysis of Cys and GSH is important, and so the development of new simple and accurate techniques is desired. Currently, the determination of GSH and Cys is carried out using various detection techniques, including optical spectroscopy,30-32 electrochemical pulse voltammetric Received: September 3, 2010 Accepted: December 12, 2010 Published: January 6, 2011 813

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Scheme 1. Mechanism of Turning “On” the Molecular Beacon by Addition of GSH/Cys Caused by Extraction of Hg2þ Ions from the MB Stem and Separation of the Fluorophore 6-FAM (Red) from the Quencher DABCYL (Blue)

methods,33-35 high-performance liquid chromatography (HPLC),36-38 and colorimetric assays.39-44 Several drawbacks, such as the lower selectivity against other amino acids due to their structural similarity, absence of spectral groups in GSH and Cys, and the required cumbersome laboratory procedures, limit their clinical application. In this paper, we present the principles of a new sensing platform that is based on the Mirkin effect5 of Hg2þ complexation competition, but it is applied to a molecular beacon framework resulting in a highly increased detection sensitivity. Molecular beacons are composed of a single-stranded oligonucleotide with self-complementary 50 and 30 ends that can self-hybridize. In the absence of a target, it forms a stemloop structure that brings a fluorophore/quencher pair, attached to the ends of the DNA strand, into close proximity, reducing fluorescence emission. Once the single-stranded loop portion of the molecular beacon hybridizes to the target, the stem melts, and the resulting spatial separation of the fluorophore from the quencher leads to the enhancement in fluorescence emission. It is well-known that Hg2þ-GSH and Hg2þ-Cys complexes,45,46 as well as Hg2þ complexes with other biothiols and nitrogen bases, have very high stability constants.45-53 Recently, Lee et al.5 developed a highly sensitive and selective colorimetric detection method for Cys based upon oligonucleotide-functionalized AuNP probes. The DNA sequence contains a thyminethymine (T-T) mismatch which is found to complex efficiently with Hg2þ ions.47 A T-T mismatch is very selective for Hg2þ binding,47,48 and when Hg2þ binds to the DNA it can increase the Tm by ∼10 °C. When adding analyte is added, it can bind Hg2þ and remove it from thymine-Hg2þ-thymine complex thereby lowering the temperature at which the DNA duplexes dissociate and the corresponding purple to red color change is observed. Inspired by their work, we have developed a novel “molecular beacon” based detection for GSH and Cys relying on Hg2þ-induced self-hybridization of the beacon strand. Instead of two different DNA strands with one T-T mismatch and AuNP used in the Mirkin group’s study,5 we have labeled a molecular beacon designed with a fluorophore and a quencher at the ends of the stem that contained a T-T mismatch for complexation of a Hg2þ cation. By adding GSH or Cys, we were able to extract Hg2þ ions very efficiently and dehybridize the stem. The new method presented here is simple, highly sensitive, and selective.

Apparatus. The fluorescence spectra were measured using LS-55 spectrophotometer (Perkin-Elmer, Waltham, MA, U.S.A.) equipped with 20 kW xenon lamp excitation source. Both the excitation and emission slit widths were set to 5.0 nm. FAM was excited at λex = 480 nm, and its fluorescence emission was followed at λem = 518 nm. The UV-vis spectra were recorded using a Perkin-Elmer Lambda 50 spectrophotometer in the range of 220-1100 nm or Ocean Optics R4000 precision spectrometer in the range from 340 to 900 nm. Procedures. A 100 nM Hg2þ solution was incubated with 100 nM probe solution (MB) in 2.5 mL of 10 mM MOPS (3-(Nmorpholino)propanesulfonic acid) buffer containing 0.05 M NaNO3 (pH 7.45) for 1 h so that the molecular beacon could be formed. Then, freshly prepared GSH/Cys stock solution was added. The final concentration of GSH/Cys was 200 nM. The fluorescence was measured for excitation at 480 nm as a function of temperature which was increased at a rate of 1 °C/min and kept for 40 s at every temperature. For the determination of sensitivity, 100 nM Hg2þ solution was first incubated with the same concentration of probe solution for 1 h, then different amounts of Cys stock solution were added into the mixture. The concentration of GSH/Cys ranged from 5 to 200 nM. The fluorescence was measured at 52 °C after the mixture was heated at 52 °C for 15 min. The selectivity for GSH/Cys was confirmed by adding other amino acid stock solutions to the final concentration of 200 nM. (Note: due to the high toxicity of Hg2þ, the solutions after assays should be discarded following the waste disposal procedure.)

’ MATERIALS AND METHODS Chemicals. All chemicals used for investigations were of analytical grade purity. Reduced L-glutathione, minimum 99% (GSH), and L-cysteine, minimum 98.5% (Cys), were purchased from Sigma-Aldrich Chemical Co. and used as received. The DNA oligonucleotide MB (50 -6-FAM-CCTCCAAAAGGTGGDABCYL-30 ) was synthesized and purified by HPLC from MWG-Operon (Eurofins, U.S.A.). The probe was labeled at the 50 -end with a carboxyfluorescein (6-FAM) dye and at the 30 end with a quencher 4-(4-dimethylaminophenylazo) benzoic acid (DABCYL). The DNA concentration was determined by measuring the absorbance at 260 nm in a 1 cm quartz cuvette. All other chemicals were purchased from Sigma-Aldrich Chemical Co. (Atlanta, GA, U.S.A.) and used as received. Solutions were prepared using Milli-Pore Milli-Q deionized water (conductivity σ = 55 nS/cm). They were deoxygenated by bubbling with purified argon.

’ RESULTS AND DISCUSSION Sensor Operation Principle. As depicted in Scheme 1, a molecular beacon with self-complementary 50 and 30 ends, including a T-T mismatch, forms a stable stem-loop structure after adding Hg2þ. In the absence of GSH/Cys, the molecular beacon is in the “off” state due to the hybridized stem and formation of the T-Hg-T structure. In the presence of GSH/ Cys in solution, Hg2þ interacts very strongly with GSH/Cys and the structure of stem is destabilized leading eventually to its dehybridization. This results in the increase of the distance between fluorophore and quencher and an increased fluorescence emission of the fluorophore. 814

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Figure 1. Fluorescence emission spectra of MB/Hg2þ in the absence (1) and presence of GSH (2) or Cys (3) at (a) room temperature (28 °C) and (b) 52 °C for 15 min: [MB] = [Hg2þ] = 100 nM; [GSH] = [Cys] = 200 nM; buffer, 10 mM MOPS, 0.05 M MaNO3, pH 7.45.

Figure 1a shows the fluorescence spectra with GSH/Cys and without them at room temperature (28 °C). We can see that there is a large difference in fluorescence intensity for samples with GSH/Cys (200 nM) and without them. Therefore, GSH or Cys can be detected at room temperature. To improve further the detection sensitivity of the system, we heated the “molecular beacon” to a higher temperature in the presence of GSH/Cys. Under these conditions, the stem of the molecular beacon dehybridized completely (Scheme 1) and the fluorescence increased considerably. At this stage, the molecular beacon is in the “on” state. We have found that, at the same temperature, the MBs are more stable in the absence of GSH/Cys than in their presence because the melting temperature Tm of MB is higher by ∼10 °C when Hg2þ binds to the DNA, which leads to a close proximity of FAM to DABCYL. Since the fluorescence quenching is closely correlated with the distance between fluorophore and the quencher, the intensity of fluorescence in the absence of GSH/Cys is much weaker than that in the presence of the GSH/ Cys at the same temperature. As shown in Figure 1b, when the MB/Hg2þ system is heated with and without GSH/Cys at a temperature of 52 °C for 15 min, the fluorescence intensity is 195 AU in the absence of GSH/Cys, whereas the intensity of fluorescence reaches 560 (500) AU in the presence of GSH (Cys). As seen in Figure 1, the fluorescence intensity is higher in the presence of GSH than that in the presence of Cys. This indicates that GSH can bind Hg2þ more strongly than Cys in the present detection system. According to Han et al.,54 who observed more efficient fluorescence restoration by GSH than by Cys for Hg2þquenched quantum dots, the stronger effect of GSH is likely associated with more extensive steric hindrance of GSH. Optimization of Assays. To maximize the difference between fluorescence in the absence and in the presence of GSH/ Cys, we have gradually increased the temperature with and without GSH/Cys for the MB/Hg2þ system. After 100 nM Hg2þ was mixed with the same concentration of probe solution to react for 1 h, the GSH/Cys stock solution was added into the mixture (the blank without GSH/Cys). The final concentration of GSH/Cys was 200 nM due to formation of a 2:1 GSH/ Hg2þ 49 or Cys/Hg2þ adduct.45 The fluorescence was measured by excitation at 480 nm as a function of temperature which was increased at a rate of 1 °C/min and kept for 40 s at every temperature. The fluorescence increases both with and without GSH/Cys when temperature increases (Figure 2). It is because the stem gradually dehybridizes with increasing temperature and the

distance between FAM and quencher increases. The dehybridization of MB in the presence of GSH/Cys is faster than in the absence of GSH/Cys due to the very selective coordination of GSH/Cys with Hg2þ.45,46 We have also found that the melting temperature of MB is decreased in the presence of GSH/Cys. When GSH/Cys coordinate to Hg2þ, the Hg2þ cation is removed from the stem of the molecular beacon, which destabilizes the stem structure and lowers the temperature at which the stem of the molecular beacon dissociates. The melting temperature of MB depends on the analyte concentration as well. With increasing concentrations of GSH/Cys, the melting temperature decreases. Figure 3a illustrates the dependence of the relative fluorescence increase ((F - F0)/F0) versus temperature for MB/Hg2þ (F is the fluorescence intensity with GSH/Cys, F0 is the fluorescence intensity without GSH/Cys). From it, we can see that in the presence of GSH, (F - F0)/F0 increases first and then decreases with increasing temperature. In the presence of Cys, (F - F0)/F0 increases first and then levels off and remains nearly constant with increasing temperature. The relative fluorescence increase for GSH is somewhat higher than that for Cys for the same concentration. We can also deduce from these data that GSH binds to Hg2þ more strongly or quickly in our study, although the literature formation constants for Hg(GSH)2 and Hg(Cys)2 complexes are very close to each other (about 1042).45,46 We can see from Figure 3 that the maximum (F - F0)/F0 value is found at 50 °C for GSH, and when the temperature reaches 52 °C, the (F - F0)/F0 value remains constant for Cys. Therefore, we have selected 52 °C to determine the concentration of GSH/Cys using the MB/Hg2þ system. In the following experiments, Hg2þ solution (final concentration, 1  10-7 M) was mixed with the probe solution (same final concentration) to react for 1 h so that the molecular beacon can be formed. Then, the stock solution of GSH/Cys was added. Both the blank solution and GSH/Cys solutions were heated at 52 °C for different time periods. The fluorescence was recorded at different times (Figure 3b). We have found that, after the addition of GSH, the fluorescence intensity increases quickly up to 15 min and then remains nearly constant. For the Cys solution and blank, the fluorescence intensity increases quickly within 15 min and increases slowly after that. To shorten the detection time and obtain larger difference between blank and GSH/Cys, we have employed the heating at 52 °C for 15 min for all samples in following experiments. The high sensitivity of the assay is achieved for the concentration of Hg2þ ions close to the concentration of MB due to fairly 815

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Figure 3. (a) Relative fluorescence increase (F - F0)/F0 vs temperature for MB/Hg2þ/GSH or MB/Hg2þ/Cys in 10 mM MOPS þ 0.05 M NaNO3, pH 7.45; (b) time dependence of fluorescence emission intensity after heating MB/Hg2þ, MB/Hg2þ/GSH, and MB/Hg2þ/ Cys to 52 °C; [MB] = [Hg2þ] = 100 nM, [GSH] = [Cys] = 200 nM.

nonlinear Levenberg-Marquardt least-squares (LSQ) fitting with a polynomial function: ð1Þ F ¼ F0 þ aC - bC2 as shown in the insets in Figure 4, parts b and d, where a and b are the coefficients, C is the analyte concentration (CGSH or CCys, mol/L), and F0 is the background fluorescence. For Cys, we have obtained a = 20.77  108 M-1, b = 0.2740  1016 M-2, F0 = 197.44, with regression coefficient R = 0.9989 and standard deviation σ = 4.19. For GSH, the fitting parameters are a = 22.90  108 M-1, b = 0.2243  1016 M-2, F0 = 197.53, with R = 0.9995 and σ = 3.37. The limit of detection (LOD) for GSH/Cys, based on the generalized 3σ method,55 is LODCys ¼ 3σ=s0

Figure 2. Fluorescence spectra for 100 nM molecular beacon solution as a function of temperature from 28 to 58 °C (from bottom to top) scanned at a rate 1 °C/min and kept for 40 s at each temperature: (a) blank, (b) with 200 nM Cys, (c) with 200 nM GSH.

where s is the slope of the calibration curve:   DF s ¼ ¼ a - 2bC DC

strong complexation of Hg2þ to a T-T mismatch in MB, requiring virtually no mercury excess. Sensitivity. The limit of detection and concentration range of the assay were determined by adding different concentrations of GSH/Cys. Three parallel trials were run for the sensitivity tests. The fluorescence intensity was dependent on the concentration of GSH/Cys over a range of 5 to 200 nM when we set the concentrations of MB and Hg2þ both at 100 nM (Figure 4). The experimental data can be fitted using a

ð2Þ

For Cys, the initial slope at CCys = 0 is s0 = 20.77  108 M-1. Therefore, LODCys = 6.0  10-9 M and the concentration range is from 6  10-9 to 2  10-7 M. In the same way, for GSH, we obtain s0 = 22.90  108 M-1, σ = 3.37, LODGSH = 4.4  10-9 M and the concentration range is from 5 10-9 to 2 10-7 M. For the trace analysis, a calibration curve limited to a lower concentration range, from 0 to 4  10-8 M, can be used. In this 816

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Figure 4. Fluorescence emission spectra for different concentrations of GSH (a) and Cys (c) and concentration dependence of fluorescence intensity at λmax (b and d) after heating MB/Hg2þ, MB/Hg2þ/GSH, and MB/Hg2þ/Cys at 52 °C for 15 min; [MB] = [Hg2þ] = 100 nM; error bars represent (3σ; insets show a wider concentration range (see text); (e) fluorescence emission intensity of MB/Hg2þ in the absence and presence of different amino acids after heating them at 52 °C for 15 min, [DNA] = [Hg2þ] = 100 nM, [amino acid] = 200 nM.

see that 4  10-9 M GSH/Cys can induce measurable fluorescence increase, indicating that the present method can successfully detect the GSH/Cys with high sensitivity. Selectivity. To determine the selectivity of the assay, we have investigated the fluorescence response to the other amino acids at a concentration of 200 nM under the optimum experimental conditions. The experimental results are shown in Figure 4e, with the error bars indicating the measurement error in triplicate measurements. It is clear that only GSH/Cys showed significantly higher fluorescence intensity. In contrast to significant

range, the experimental data can be approximated with a linear LSQ fit, as shown in Figure 4, parts b and d: F ¼ F0 þ aC

ð3Þ

For Cys, we have obtained a = 21.77  108 M-1, F0 = 195.65, with R = 0.9972 and σ = 3.01. For GSH, the fitting parameters are a = 21.96  108 M-1, F0 = 198.10, with R = 0.9971 and σ = 3.04. Thus, the limits of detection for GSH/Cys are as follows: LODCys = 4.1  10-9 M, LODGSH = 4.2  10-9 M. We can 817

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fluorescence enhancement observed for GSH/Cys, very little change of the fluorescence intensity was observed upon addition of other amino acids, although Hg2þ is known to have an affinity to certain N-type ligands.50 The binding affinity of Hg2þ to a T-T mismatch site appears to be stronger than that to all amino acids studied except for GSH/Cys. The binding of GSH/Cys to Hg2þ at a T-T mismatch site is highly selective due to the high values of the formation constants for the resulting complexes which leads to an assay with high specificity. We have also found that the addition of methionine did not result in a significant fluorescence enhancement although it is a sulfur-containing amino acid. The formation constant for GSH/Cys to Hg2þ is ca. 1042, and that for methionine to Hg2þ is 1017.6,45,51-53 indicating that the binding affinity of Hg2þ for the thiol in GSH/Cys is much greater than that for the thioether in methionine.

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’ CONCLUSION We have demonstrated that a “molecular beacon” method can be applied for the detection of GSH and Cys. In the absence of GSH/Cys, the molecular beacon developed is shown to hybridize in the stem area owing to Hg2þ binding to T-T mismatches in the DNA sequence to form a T-Hg-T structure. In this state, fluorescence of a dye attached to one end of the DNA strand is effectively quenched by the quencher molecule attached to the other end of the DNA strand due to their close proximity. We have found that by adding GSH/Cys and heating the molecular beacon, Hg2þ can be efficiently extracted from the stem due to strong binding in HgL2 complexes (where L is either GSH or Cys). The molecular beacon platform thus developed shows very high sensitivity (LOD ≈ 4 nM, based on the 3σ method) and excellent selectivity with respect to other amino acids including sulfur-containing methionine. The novel “molecular beacon” based method offers a wide concentration range of 4 to 200 nM with an LOD of 4.1 nM for GSH and 4.2 nM for Cys.

’ AUTHOR INFORMATION Corresponding Author

*Phone: þ1-315-267-2264. Fax: þ1-315-267-3170. E-mail: [email protected]. Notes †

On leave from School of Chemistry and Materials Science, Ludong University, China.

’ ACKNOWLEDGMENT This work was supported by the U.S. DoD Research Program, Grant No. AS-073218. ’ REFERENCES (1) Skl
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