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Enzymatic Product-Mediated Stabilization of CdS Quantum Dots Produced In Situ: Application for Detection of Reduced Glutathione, NADPH, and Glutathione Reductase Activity Gaizka Garai-Ibabe, Laura Saa, and Valeri Pavlov* CICbiomaGUNE, Parque Tecnológico de San Sebastián, Paseo Miramón 182, 20009, Donostia-San Sebastián, Spain S Supporting Information *

ABSTRACT: Glutathione is the most abundant nonprotein molecule in the cell and plays an important role in many biological processes, including the maintenance of intracellular redox states, detoxification, and metabolism. Furthermore, glutathione levels have been linked to several human diseases, such as AIDS, Alzheimer disease, alcoholic liver disease, cardiovascular disease, diabetes mellitus, and cancer. A novel concept in bioanalysis is introduced and applied to the highly sensitive and inexpensive detection of reduced glutathione (GSH), over its oxidized form (GSSG), and glutathione reductase (GR) in human serum. This new fluorogenic bioanalytical system is based on the GSH-mediated stabilization of growing CdS nanoparticles. The sensitivity of this new assay is 5 pM of GR, which is 3 orders of magnitude better than other fluorogenic methods previously reported.

fluorogenic assay based on the in situ production of inorganic fluorescent nanoparticles (NPs) in response to the presence of the stabilizing agents (GSH) in the medium. Metallic and semiconductor NPs are extensively used as labels for the optical and electrical sensing of biorecognition elements .20 Semiconductor inorganic NPs can be photoexcited to generate electron/hole couples that recombine to yield fluorescent emission of light.21 The stable and bright emission of semiconductor NPs arises from quantum confinement effects that occur in nanometer-sized semiconductors; hence, such NPs are called quantum dots (QDs). The advantages of QDs over traditional organic fluorophores include higher quantum yield, reduced photobleaching, and higher extinction coefficient. Until now QD-based assays relied on presynthesized semiconductor NPs, which could be split into two subsets of methods: assays based on QDs decorated with recognition elements, for detection of affinity interactions, and assays for enzymatic activities, employing donor/quencher fluorescence resonance energy transfer (FRET) pairs in which one of the elements is a QD. However, such assay systems are frequently limited by high background signals caused by nonspecific adsorption of decorated QDs on surfaces or poor quenching of donor couples. We believe that the generation of QDs in situ can address these drawbacks of relevant analytical systems by decreasing the background signal. Our group introduced different unconventional approaches to achieve biocatalytic growth of QDs.22 The first approach is based on enzymatic

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lutathione is a tripeptide synthesized endogenously from the precursor amino acids L-cysteine, L-glutamic acid, and glycine. It is the most abundant nonprotein molecule in the cell and plays a key role in the maintenance of intracellular redox states, detoxification of xenobiotics, intracellular signal transduction, and gene regulation.1−4 Under normal conditions, the majority of the glutathione exists in the reduced form (GSH), but it can be rapidly oxidized to glutathione disulfide (GSSG) in response to the oxidative stress of the cell. However, the highly efficient NADPH-mediated reduction of GSSG to GSH by glutathione reductase (GR) maintains the intracellular GSH/GSSG ratio above 99%.5 Due to the important role of GSH in many cellular processes, GSH levels and the GSH/ GSSG ratio have been linked to several human diseases, such as AIDS, Alzheimer disease, alcoholic liver disease, cardiovascular disease, diabetes mellitus, and cancer.6−11 Blood GSH concentration may reflect the redox status of other less accessible tissues and has been considered as a useful indicator of a whole subject’s oxidative status.12−14 Several methods have been developed to quantify GSH in human samples, including high-performance liquid chromatography and mass spectrometry techniques.15−17 The disadvantage of those methods is that they require nonportable expensive equipment that need to be operated by well-trained personnel. On the other hand, fluorescent probes have also been developed for biological thiol determination.18,19 However, fluorescence probes are expensive, and their production is time consuming as they have to be synthesized and purified before use. Therefore, it would be of interested to develop a simple and low-cost fluorimetric assay system for a rapid, highly sensitive, and selective detection of GSH over GSSG. We believe this goal could be met through the development of a © XXXX American Chemical Society

Received: March 14, 2013 Accepted: May 8, 2013

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Scheme 1. Detection of GR Activity through Enhancing the Growth of Fluorescent CdS QDs with the Enzymatic Product, GSH

with 100 μM GSSG and varying concentrations of NADPH (from 2.5 to 30 μM) in 87.5 μL of citrate−phosphate buffer pH 7.5 at RT for 30 min. Afterward, 10 μL of 1 mM Na2S and 2.5 μL of 50 mM Cd(NO3)2 were added to the mixtures (87.5 μL), and the fluorescence emission spectra of the resulting suspensions were measured after 5 min at λex = 300 nm. CdS QD-Mediated Determination of GSSG. The GSSG concentration in aqueous media was also determined by the in situ modulation of CdS QDs. GR (100 pM) was incubated with 17.5 μM NADPH and varying concentrations of GSSG (from 10 to 200 μM) in 87.5 μL of citrate−phosphate buffer pH 7.5 at RT for 30 min. Afterward, 10 μL of 1 mM Na2S and 2.5 μL of 50 mM Cd(NO3)2 were added to the mixtures (87.5 μL), and the fluorescence emission spectra of the resulting suspensions were measured after 5 min at λex = 300 nm. CdS QD-Mediated Determination of GR in Human Serum. The GR concentration was determined in 1000-times diluted human serum to evaluate the sensitivity of the assay in complex biological media. Different concentrations of GR (from 5 to 50 pM) were incubated with 17.5 μM NADPH and 100 μM GSSG in 87.5 μL of citrate−phosphate buffer pH 7.5 at RT for 30 min. Afterward, 10 μL of 1 mM Na2S and 2.5 μL of 50 mM Cd(NO3)2 were added to the mixtures (87.5 μL), and the fluorescence emission spectra of the resulting suspensions were measured after 5 min at λex = 300 nm.

generation of S2− anions followed by interaction with Cd2+ cations to yield fluorescent CdS QDs.22,23,25 Unfortunately, a relatively high concentration of enzymatically produced S2− is required to give CdS QDs; hence, this method is not so sensitive. The second method is more sensitive, because it employs inhibition of the formation of CdS QDs from Cd2+ and S2− ions by enzymatic products such as thiophenol, formed by the hydrolysis of S-phenyl acetate by PON1.24 In this case, the increase in enzymatic activity is related with the decrease in fluorescence intensity. However, the system in which enzymatic activity is directly related with the intensity of fluorescent signal will be much more sensitive. The assay system we have developed is based on a new bioanalytical concept, consisting of the enzymatic product mediated stabilization of in situ produced QDs. To demonstrate the potential of the reported assay system to be applied in real samples as a point of care test, it was validated by the detection of GSH and GR activity in complex biological media (human serum). Furthermore, we believe that this system could be used in the development of portable devices for determination of GSH status.

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MATERIAL AND METHODS Materials. GSH, GR (from Baker’s yeast), β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH), human serum, and other chemicals were obtained from Sigma−Aldrich (Spain) and used as supplied. Enzymatic activity assays were performed in black flat-well (330 μL) NUNC 96 wells microtiter plates, and the fluorescence spectra were recorded with a Varioskan flash fluorimeter (Thermo Scientific). All water used was Milli-Q ultrapure grade (EMD Millipore). CdS QD-Mediated Determination of GSH. The determination of GSH in aqueous media was performed by the in situ modulation of CdS QDs. The assay system was composed of citrate−phosphate buffer (pH 7.5) containing varying amounts of GSH (from 2 to 20 μM), Na2S (0.1 mM), and Cd(NO3)2 (1.25 mM). The mixtures were incubated for 5 min at room temperature (RT), and the fluorescence emission spectra of the resulting suspensions were recorded at λex = 300 nm. Furthermore, the validation of the technique for the quantification of GSH on real samples was performed by the standard addition method. Diluted human serum (80, 100, and 250 times) was spiked with different concentrations of GSH. Afterward, the GSH concentration was measured as described previously. CdS QD-Mediated Determination of NADPH. To determine the NADPH concentration in aqueous media, by the in situ modulation of CdS QDs, 100 pM GR was incubated

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RESULTS AND DISCUSSION The proposed operating mechanism of our assay system is represented in Scheme 1. The enzyme GR breaks down GSSG, using NADPH as a cofactor, to two molecules of GSH. The latter has three functional groups, that is, a mercapto group, an amino moiety, and two carboxyl groups. The GSH molecule binds to the surface of the growing CdS crystals through the mercapto group by a thiol linkage, while the amino and the carboxylic groups confer hydrophilicity to the structure. Stabilization of in situ growing CdS crystals with the enzymatic product (GSH), leads to the generation of fluorescent CdS QDs in the aqueous media. Figure 1A shows UV−Visible (UV−Vis) absorption and emission spectra (solid and dashed lines, respectively) of the GSH-stabilized CdS QDs in buffer solution. It should be noted that the presence of citrate ions in the reaction mixture was essential for the formation of GSH-capped QDs. From the UV−Vis spectrum, we observed an increased absorption below 500 nm and a shoulder at about 300 nm. The presence of this shoulder is explained by the excitonic transition between the electron 1S state and the hole 1S state, in semiconductor QDs with a diameter about 2 nm.26,27 On the other hand, the emission spectrum demonstrates a well-shaped peak at 550 nm, B

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components of blood. Due to this extremely high concentration of GSH, all other potential thiol molecules can be ignored.29,30 Indeed, the emission of formed QDs is highly sensitive to the concentration of the stabilizing molecule GSH. The sensitivity of this simple assay for GSH measurement was similar to that based on the Tb(III)-cyclen maleimide fluorogenic probe recently reported.18 Having confirmed the distinguished influence of GSH concentration on the rate of QDs generation and before validating the system for its use in real samples, we studied the effect of several interfering agents present in serum, such as thiol-based biomolecules and amino acids, on the behavior of our system. As expected, thiol-based amino acids, cysteine and homocysteine, were able to stabilize fluorescent QDs. However, naturally occurring amino acids (1 mM) showed no capacity to stabilize CdS QDs and did not produce a significant change in the fluorescence emission. Furthermore, the occurrence of such amino acids (1 mM) in the assay system containing GSH (100 μM) did not induce a significant variation in the fluorescent response of the system to GSH (see Figure 3S, Supporting Information). In addition, we also tested the ability of the system to selectively detect GSH (100 μM) in the presence of its oxidized form GSSG (1 mM), which lacks the nucleophilic thiol moiety. The excess of GSSG in the reaction mixture did not show any influence in the response of the system to GSH. Furthermore, we validated the usefulness of our technique for detection of GSH in human serum by the standard addition method. This method consists of the addition of known varying standard amounts of GSH to a serum sample divided into several aliquots of equal volume. The results were represented as a plot with the concentration standard added on the x axis and the fluorescence intensity on the y axis (Figure 3). Taking

Figure 1. (A) UV−Vis absorption (solid line) and emission (dashed line) spectra of CdS QDs produced in the presence of GSH (100 μM), Na2S (0.1 mM), and Cd(NO3)2 (1.25 mM) in citrate−phosphate buffer pH 7.5. (B) TEM image of the GSH-stabilized CdS QDs.

which arises from excitonic emission of CdS QDs, indicating a homogeneous distribution of sizes among CdS QDs.28 Transmission Electron Microscopy (TEM) was used to confirm the existence of stable CdS QDs in the reaction mixture (Figure 1 B). This technique revealed the presence of spheroidal QDs with a medium diameter of 1.82 ± 0.2 nm. (Figure 1S, Supporting Information). A number of control experiments were carried out to confirm the suggested mechanism of operation of the enzymatic analytical system presented in Scheme 1. A significant growth in the fluorescence signal was only observed when all components of the assay were present in the mixture (GR, GSSG, Cd(NO3)2, Na2S, and NADPH) (Figure 2S, Supporting Information). Whereas, if any of the components was absent, an insignificant increase of fluorescence was observed. The formation of CdS QDs was followed by fluorescence spectroscopy. We first investigated the effect of GSH concentration on the fluorescent emission of QDs (Figure 2).

Figure 3. (A) Fluorescence intensities (λem = 550 nm) of the formed CdS QDs in the system containing different concentrations of added standard solution of GSH, Na2S (0.1 mM), Cd(NO3)2 (1.25 mM), and varying volumes of human serum: (●) 0 μL; (▼) 0.4 μL; (▲) 1 μL; (■) 1.25 μL. (B) Dependence of the calculated GSH concentration on human serum volume.

Figure 2. (A) Emission spectra of CdS QDs formed in the system containing Na2S (0.1 mM), Cd(NO3)2 (1.25 mM), and different concentrations of GSH: (a) 0 μM; (b) 2 μM; (c) 8 μM; (d) 10 μM; (e) 15 μM; (f) 20 μM, (g) 25 μM. (B) Calibration curve of GSH at λem = 550 nm.

into consideration all dilutions of the samples, we determined that the concentration of GSH in the commercial human serum was equal to 618 μM. This value is within the range previously reported for healthy subjects.14,31−33 These results demonstrate that our method allows quantification of GSH in human serum with high sensitivity and selectivity. Furthermore, we estimate that our method requires only 0.4 μL of human serum per well to carry out the GSH test.

As could be noted, the response of the fluorescence intensity signal to the increase in GSH concentration shows a linear relation up to 25 μM GSH. On the basis of this phenomenon, a very simple method for quantification of this important antioxidant can be developed, as it has been reported that the GSH content makes up 90% of all the thiol-containing C

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gives an additional indication that the rate of QDs formation depends on the GSSG concentration according to the Michaelis−Menten kinetics of the enzymatic reaction. The value of apparent KM for GSSG obtained by the nonlinear regression method was 19.31 ± 6.93 μM similar to that reported in the bibliography.35 The fact that both NADPH and GSSG yield realistic Michaelis−Menten constants further confirms the reaction mechanism depicted in Scheme 1. Having found the optimal concentrations of GSSG and NADPH for GR activity determination, we proceeded with the evaluation of the sensitivity of the assay in a complex biological medium. Figure 6 shows the emission spectra of the assay

It was also demonstrated that the QD-based assay is able to monitor the enzymatic reduction of GSSG to GSH by GR, which is of great interest for determination of the GSH/GSSG ratio, an important indicator of the cellular redox environment.34 In order to optimize the assay shown in Scheme 1, we studied changes in the response of the system, containing fixed amounts of GR and GSSG, to varying concentrations of NADPH (Figure 4). As can be observed, the fluorescence signal

Figure 4. (A) Emission spectra of CdS QDs formed in the system containing GR (100 pM), GSSG (100 μM), Na2S (0.1 mM), Cd(NO3)2 (1.25 mM), and different concentrations of NADPH: (a) 0 μM; (b) 2.5 μM; (c) 4 μM; (d) 8 μM; (e) 12 μM; (f) 20 μM; (g) 30 μM. (B) Calibration curve of NADPH at λem = 550 nm.

Figure 6. (A) Emission spectra of CdS QDs formed in diluted human serum (1:1000) containing NADPH (17.5 μM), Na2S (0.1 mM), Cd(NO3)2 (1.25 mM), and different concentrations of GR: (a) 0 pM; (b) 5 pM; (c) 10 pM; (d) 25 pM; (e) 50 pM. (B) Calibration curve of GR at λem = 550 nm.

and NADPH concentration show a linear dependence up to 8 μM NADPH, followed by a section approaching asymptotically the maximum response, starting from 15 μM NADPH. This trait is typical for an enzymatic system obeying the Michaelis− Menten kinetic model. The apparent Michaelis−Menten constant (KM) computed by a nonlinear regression method was 7.12 ± 1.85 μM. This value is in concordance with that previously reported.35 According to these results, a concentration of cofactor of 17.5 μM was used in subsequent experiments. We also studied the response of the system to different concentrations of GSSH (Figure 5). The relation between the fluorescence emission and the GSSG concentration demonstrates a linear behavior up to 70 μM GSSG and a saturated signal above 100 μM GSSG. The shape of the calibration curve

mixtures in diluted human serum (1:1000) containing different concentrations of GR. As could be noted, a detection limit of 5 pM was achieved. This detection limit is lower by 1000 times than that reported for the recently published fluorogenic test employing the interaction of GSH with a Tb(III)-cyclen maleimide.18

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CONCLUSIONS We demonstrated a new class of enzymatic assays in which a product of enzymatic reaction facilitates the stabilization and growth of fluorescent semiconductor NPs. This concept can be readily applied to quantification of GSH, GSSG, and GR in human serum resulting in a very robust, simple, and inexpensive method with an unprecedented sensitivity and selectivity for a fluorogenic assay. This system can open a new direction for the development of numerous analytical and bioanalytical techniques using optical, photochemical, and electrochemical methods to follow the readout signal.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

Figure 5. (A) Emission spectra of CdS QDs formed in the system containing GR (100 pM), NADPH (17.5 μM), Na2S (0.1 mM), Cd(NO3)2 (1.25 mM), and different concentrations of GSSG: (a) 0 μM; (b) 10 μM; (c) 25 μM; (d) 50 μM; (e) 100 μM; (f) 200 μM. (B) Calibration curve of GSSG at λem = 550 nm.

*Phone: +34-943005308; fax: +34-943005314; e-mail: [email protected]. Notes

The authors declare no competing financial interest. D

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(32) Kand’ar, R.; Zakova, P.; Lotkova, H.; Kucera, O.; Cervinkova, Z. J. Pharm. Biomed. Anal. 2007, 43, 1382−1387. (33) Zunic, G.; Spasic, S. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2008, 873, 70−76. (34) Schafer, F. Q.; Buettner, G. R. Free Radical Biol. Med. 2001, 30, 1191−1212. (35) Mavis, R. D.; Stellwagen, E. J. Biol. Chem. 1968, 243, 809−814.

ACKNOWLEDGMENTS This work was supported by the Spanish Ministry of Science and Innovation (project BIO2011-26356). V.P. acknowledges the contract Ramon y Cajal from the Spanish Ministry of Science and Innovation.

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