Research Article pubs.acs.org/journal/ascecg
Gold Nanoparticle-Catalyzed Clock Reaction of Methylene Blue and Hydrazine for Visual Chronometric Detection of Glutathione and Cysteine Yi He* and Lei Zheng School of National Defence Science & Technology, Southwest University of Science and Technology, 59 Qinglong Road, Mianyang, Sichuan 621010, P. R. China S Supporting Information *
ABSTRACT: A visual chronometric assay for glutathione (GSH) and cysteine (Cy) is reported based on a gold nanoparticle (AuNP)-catalyzed clock reaction of methylene blue and hydrazine. The blue MB is reduced to colorless leucomethylene blue (LMB) by hydrazine in the presence of AuNPs as the nanocatalysts, and it is again oxidized back to MB by oxygen under shaking. Therefore, a periodic oscillation is observed between blue MB and colorless LMB. However, when GSH or Cy is added to the clock reaction system, the catalytic activity of AuNPs decreases because of the inhibition of the formation of gold-hydride species, leading to a long reaction time from MB to LMB. The chronometric assay displays a good linear range of 10 to 330 μM for GSH and 20 to 400 μM for Cy, respectively. The limit of detection is estimated to be 8 and 10 μM at a 3σ, respectively. Different concentrations of GSH/Cy can be visually detected by observing the height of the blue solution under different reaction time points. In addition, the present assay shows good selectivity, and it is successfully applied for detecting GSH/Cy in fetal bovine serum. KEYWORDS: Clock reaction, Methylene blue, Visualization, Chronometric detection
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INTRODUCTION Biothiols such as glutathione (GSH) and cysteine (Cy) play important roles in physiological metabolism.1 For instance, GSH is one of the most important antioxidants in cells, which neutralizes reactive free-radical species to maintain the cell’s redox state.2 The deficiency of Cy causes many diseases, including depigmentation of hair, lethargy, psoriasis, edema, liver damage, etc.3,4 Hence, the detection of Cy and GSH is of interest to biological monitoring and disease diagnosis. So far, various approaches such as capillary electrophoresis, highperformance liquid chromatography, fluorescence spectroscopy, electrochemical assay, and colorimetric methods have been established for the measurement of GSH and Cy.4−17 Although these methods can effectively determine GSH and Cy, they require complicated and expensive instruments and tedious sample treatment processes, limiting their further application. To this end, it is highly desirable to design and develop rapid and simple assays for GSH and Cy without the help of advanced instruments. Clock reactions are a special type of chemical kinetics phenomena which are characterized by an abrupt concentration change of some chemical species after a well-defined, sharp induction time.18 The clock reaction provides a fascinating and visually reversible color change, which is usually based on redox © 2017 American Chemical Society
systems. Many clock reactions are reported spanning from inorganic iodate-bisulfite reaction in the presence of HgCl2 to organic molecules (methylene blue).19 Conventional clock reactions occur at relatively high concentrations of chemical species to visually observe a reversible color change, making them unsuitable for the development of various assays for GSH and Cy. Recently, Pal et al., as well as several others, demonstrated that Cu2O nanoparticles, CuO-based nanocomposites, and Cu7S4 nanocages are able to catalyze the methylene blue (MB)-hydrazine clock reactions, in which the rate of the reaction is greatly enhanced at a low concentration of MB and hydrazine.19−21 MB is easily reduced to the colorless leucomethylene blue (LMB) by hydrazine in the presence of these nanocatalysts, and it is again oxidized back to MB by the oxygen under shaking. Therefore, an oscillation is observed between blue MB and colorless LMB. However, the application of nanoparticle-catalyzed clock reactions for the detection of GSH and Cy has not been reported, to the best of our knowledge. Received: July 17, 2017 Revised: August 22, 2017 Published: September 5, 2017 9355
DOI: 10.1021/acssuschemeng.7b02391 ACS Sustainable Chem. Eng. 2017, 5, 9355−9359
Research Article
ACS Sustainable Chemistry & Engineering In this work, we demonstrate that gold nanoparticles (AuNPs) have a capacity for catalyzing the MB-hydrazine clock reaction. When GSH or Cy is introduced, the catalytic activity of AuNPs is inhibited because of the inhibition of the formation of gold-hydride species induced by the analytes. On the basis of the reaction time change from blue MB to colorless LMB, a visual chronometric assay is fabricated for the detection of GSH and Cy.
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EXPERIMENTAL SECTION
Materials and Instruments. GSH, Cy, MB, hydrazine hydrate, glycine, serine, alanine, tyrosine, and threonine were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. Chloroauric acid, sodium hydrate, phosphoric acid, and acetic acid were obtained from Sinopharm Chemical Reagent Co., Ltd. All the chemicals were at least analytical grade and used without further purification. Deionized water was applicable to prepare various solutions. UV−vis absorption spectra were conducted on a spectrophotometer (Shimadzu UV-1800). Transmission electron microscopy (TEM) images were performed using a JEOL-2010 instrument. Gold Nanoparticles-Catalyzed Clock Reaction. The 13 nm AuNPs were synthesized by reduction of the chloroauric acid with sodium citrate aqueous solution (Figure S1 of the Supporting Information, SI) and their concentrations were estimated to be 0.9 nM.22 For the clock reaction, 0.2 mL of 0.5 mM MB solution and hydrazine hydrate (2.6 mL, 0.6 M) were injected into a 1 cm quartz cuvette. Subsequently, 0.2 mL of AuNP dispersion with different concentrations was added to the above mixture, and the timedependent UV−vis absorption spectra for each clock reaction were recorded. Assay Procedures for Glutathione and Cysteine. A mixture solution containing 60 μM MB and 0.42 M hydrazine hydrate was prepared in 2 mL of Britton-Robinson (BR) buffer in the presence of varying concentrations of Cy or GSH. After that, 1 mL AuNPs (0.09 nM) dispersion was introduced, and the color of the mixture gradually changed from blue to colorless, which was recorded with the video camera of a smartphone. The reaction time change from blue to colorless in the absence and presence of target analytes was used for the quantitative detection. In order to detect the real samples, fetal bovine serum (FBS) was diluted 20-fold with deionized water, which was spiked with GSH/Cy and analyzed by the present assay.
Figure 1. (a, b) UV−vis absorption spectra of MB and N2H4 in the absence and presence of AuNPs. (c) UV−vis spectra of the aerial oxidation of LMB. (d) Plot of ln (A) versus reaction time with variable concentrations of the AuNPs.
Figure 2. Reversible color change of MB reduced by N2H4 in the presence of AuNPs as the catalyst.
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the excess N2H4. A periodic oscillation is observed between blue MB and colorless LMB, demonstrating the clock reaction (Figure 2). The clock reaction can proceed at least 30 cycles. Different concentrations of AuNPs are employed to identify the catalyst dosage dependent kinetics as displayed in Figure 1d. With an increasing catalyst dosage, the corresponding clock reaction becomes faster. All of the results demonstrate that AuNPs can effectively catalyze the MB−N2H4 clock reaction. It has been reported that N2H4 is able to interact with AuNPs via Au−N covalents and be activated by AuNPs, giving birth to goldhydride species, and the resultant hydride has a higher reaction activity than N2H4, which catalyzes the reduction of various compounds.23 Accordingly, it is reasonable to suppose that the catalytic mechanism of the MB−N2H4 clock reaction is also ascribed to the formation of gold-hydride species which promote the reduction of MB to LMB. Principle of Visual Chronometric Detection of Cy and GSH. As is well-known, thiol groups bind to the surface of AuNPs with high affinity by formation of strong Au−sulfur bonds. Therefore, when Cy or GSH is added to the clock reaction system, the catalytic activity of AuNPs decreases because GSH or Cy inhibits the formation of gold-hydride species due to the interaction between AuNPs and GSH/Cy.
RESULTS AND DISCUSSION Catalysis of MB−N2H4 Clock Reaction. The maximum absorption wavelength of the MB is located at 664 nm and shows a blue color. Nevertheless, when MB is reduced to colorless LMB by N2H4, the solution does not exhibit any absorption in the visible region. Consequently, we can monitor the clock reaction of MB and N2H4 by the corresponding UV− vis absorption spectra. In the absence of AuNPs, the clock reaction between MB and N2H4 cannot proceed because the absorbance at 664 nm merely changes from 1.18 to 1.1 after 30 min of reaction (Figure 1a). On the contrary, the MB solution was first mixed with AuNP dispersion, followed by addition of N2H4. It is observed that the absorbance at 664 nm decreases as the reaction time increases, which decreases to 0.1 within 10 min in the presence of 0.03 nM AuNPs (Figure 1b) and generates a colorless solution (Figure 2), demonstrating that AuNPs can catalyze the reaction between MB and N2H4. The colorless mixture solution regenerates blue color by gentle shaking thanks to the ready oxidation of LMB by oxygen from the air (Figure 2), and the corresponding absorbance increases as shown in Figure 1c. The color of the solution fades to colorless again after standing for a few minutes due to the presence of 9356
DOI: 10.1021/acssuschemeng.7b02391 ACS Sustainable Chem. Eng. 2017, 5, 9355−9359
Research Article
ACS Sustainable Chemistry & Engineering
Under these optimized experimental conditions, different concentrations of GSH/Cy were added to the AuNPs-catalyzed MB−N2H4 clock reaction, and the corresponding reaction time was recorded. Figures 5a and S4a show the optical images of
Under the circumstances, the reduction reaction of blue MB and N2H4 to produce colorless LMB catalyzed by AuNPs requires a long time, which allows for visual chronometric detection of GSH/Cy (Figure 3).
Figure 3. Schematic illustration of the principle of the chronometric detection of GSH and Cy.
In order to demonstrate the feasibility of this chronometric assay, the time of the AuNP-catalyzed MB−N2H4 clock reaction was determined under different experimental conditions as depicted in Figure 4. It can be seen that the blue
Figure 5. (a) Representative optical images of the AuNP-catalyzed MB−N2H4 system in the presence of different concentrations of GSH under different reaction time. (b) Plot of the reaction time (t) versus the GSH concentration. (c) Calibration curve for detecting GSH in the range of 10−330 μM.
the clock reaction in the presence of different concentrations of GSH/Cy under different reaction times. Obviously, the clock reaction becomes slower, and the reaction time gradually increases with an increase of the GSH/Cy concentration (Figures 5b and S4b). As indicated in Figures 5c and S4c, the plots of the reaction time versus the concentration of analyte concentration exhibit a good linear relationship in the range of 10 to 330 μM for GSH and 20 to 400 μM for Cy, respectively. The limit of detection (LOD) for GSH and Cy is calculated to be 8 and 10 μM at a 3σ, respectively. As shown in Table 1, this sensitivity is better than or comparable to that of the reported methods for detection of GSH/Cy such as fluorimetry, electrochemiluminescence, and colorimetry. Moreover, the used molecules in the reported assays are easy to hydrolyze under an acid or alkaline environment, leading to an unsatisfied selectivity. Furthermore, the preparation of the molecules and nanomaterials, including Au nanorods and functionalized AuNPs, and the sample pretreatment require a long reaction time, which causes a long detection time (Table 1). On the contrary, the present AuNP-based chronometric assay for GSH/Cy not only can be finished within 2.5 h and has a good selectivity (Figure S5), but also does not involve any advanced instrument, which can be further improved using a more efficient catalyst. Importantly, different concentrations of GSH/ Cy can be visually distinguished by observing the height of the blue solution as shown in Figures 5a and S4a. The selectivity of this assay is further investigated under the same experimental conditions. The reaction time responses of the AuNP-catalyzed MB−N2H4 clock reaction to GSH, Cy, and
Figure 4. Optical images of the AuNP-catalyzed MB−N2H4 system in the absence (a) and presence of (b) GSH or (c) Cy. (d) The corresponding of the color change time.
solution of MB fades to colorless after standing for 750 s in the absence of GSH/Cy. However, the time of the color change from blue to colorless greatly increases after addition of GSH or Cy to the clock reaction system, demonstrating that the AuNPcatalyzed MB−N2H4 clock reaction is capable of determining GSH/Cy (Figure 4b−-d). Sensitivity and Selectivity of the Visual Chronometric Assay. To achieve a good performance, some experimental conditions, such as the concentrations of MB, AuNPs, and N2H4, and the pH of the buffer solution were optimized. The reaction time change from blue MB to colorless LMB in the presence and absence of GSH/Cy was employed as the criterion to examine the various detection conditions (Figures S2 and S3). It is found that the optimal experimental conditions are as follows: 0.04 mM MB, 0.28 M N2H4, 0.03 nM AuNPs, and pH 5.8 for the detection of GSH; 0. 04 mM MB, 0.28 M N2H4, 0.03 nM AuNPs, and pH 9.6 for the detection of Cy, respectively. 9357
DOI: 10.1021/acssuschemeng.7b02391 ACS Sustainable Chem. Eng. 2017, 5, 9355−9359
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ACS Sustainable Chemistry & Engineering Table 1. Overview on the Reported Methods for Determination of GSH/Cy material/method used
limit of detection (μM)
interference
detection time (h)
ref
tetraphenylethene/fluorimetry FR-thiol/fluorimetry graphene oxide/electrochemiluminescence chloro-hydroxyl merocyanine/fluorimetry chromene/colorimetry Au nanorod/colorimetry Au nanoparticles/colorimetry Au nanoparticle/chronometry
200 1.87 8.3 5 5 1.75 10 8
weak base weak acid ascorbic acid weak acid weak acid
25.3 15 24 22 72 20 12 2.5
24 25 26 27 28 29 30 this work
other amino acids including glycine, serine, alanine, tyrosine, threonine, glucose (Glu), fructose (Fru), urea (UA), Mg2+, Na+, K+, Ca2+, Cl−, and NO3− at a concentration of 330 μM are summarized in Figure S5. It is clear that only GSH/Cy causes a significant reaction time change, while other amino acids, organic and inorganic species display no apparent effects on the reaction time of the assay. The good selectivity of this assay is due to the fact that the affinity of AuNPs toward GSH/Cy is much greater than that for other substances. Compared with Cy, GSH has more thiol groups, resulting in a longer reaction time. Taken together, these results confirm that the present assay shows good selectivity for GSH/Cy. Detection of Cysteine and Glutathione in Fetal Bovine Serum. To identify the potential application, the present chronometric assay is also applied to determine GSH and Cy in fetal bovine serum (FBS). The 20-fold dilution of the FBS was carried to eliminate the matrix effect. Then, different concentrations of GSH/Cy were spiked into the diluted FBS samples, which were determined by the present assay. The detection results are indicated in Tables S1 and S2. The recoveries of these six samples are obtained from 100% to 108%, and the relative standard deviations (RSDs) below 5.0%. Such results reveal that this chronometric assay is suitable for the detection of GSH and Cy in real serum samples.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (Y.H.). ORCID
Yi He: 0000-0002-9324-3305 Author Contributions
The manuscript was written through contributions of all authors. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The support of this research by the National Natural Science Foundation of China (Grant No. 21705134), Foundation of Science and Technology Department of Sichuan Province (Grant No. 2015JY0053), and Doctoral Program of Southwest University of Science and Technology (Grant No. 14zx7165) is gratefully acknowledged.
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CONCLUSIONS We developed a visual chronometric method for the detection of GSH and Cy based on an AuNP-catalyzed MB−N2H4 clock reaction. The catalytic mechanism is attributed to the catalytic activation of N2H4 by AuNPs to generate gold-hydride species. The addition of GSH/Cy restrained the catalytic activity of the AuNPs owing to the formation of Au−sulfur bonds which prevent the interaction between AuNPs and N2H4, leading to a long time for the color change from blue to colorless. This chronometric assay has many distinct advantages. First, it does not involve expensive and complicated instruments, which ensures simple, convenient, and low-cost detection. Second, different concentrations of analytes can be visually detected by observing the height of the blue solution. Third, it works well in serum samples. The present work proposes an alternative method for the detection of GSH/Cy, which is a promising candidate for clinical diagnosis and other biological applications.
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reaction time change for detection of GSH and Cy; representative optical images of the AuNP-catalyzed MB−N 2 H 4 system in the presence of different concentrations of Cy under different reaction time; time responses of the AuNPscatalyzed MB−N2H4 system to 330 μM GSH, Cy, glycine, serine, alanine, tyrosine, and threonine; and detection results of Cy and GSH concentrations in FBS samples (PDF)
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b02391. TEM image of AuNPs; effects of AuNP concentration, MB concentration, N2H4 concentration, and pH on the 9358
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