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A sensitive and selective colorimetric sensor for reduced glutathione detection based on silver triangular nanoplates conjugated with gallic acid
Colloids and Surfaces A 541 (2018) 36–42
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A sensitive and selective colorimetric sensor for reduced glutathione detection based on silver triangular nanoplates conjugated with gallic acid
T
⁎
Ekarat Detsria,b, , Panpailin Seeharaja,b, Chaval Sriwonga a b
Department of Chemistry, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand Advanced Materials Research Unit, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand
G RA P H I C A L AB S T R A C T
A R T I C L E I N F O
A B S T R A C T
Keywords: Silver triangular nanoplates Gallic acid Colorimetric sensor Reduced glutathione Dietary supplements
Silver triangular nanoplates conjugated with gallic acid (AgTNPs⋅GA) was designed and synthesized for colorimetric detection of reduced glutathione (GSH). The surface plasmon resonance (SPR) properties of AgTNPs⋅GA were strongly influenced by the addition of GSH. The detection principle was based on the aggregation of AgTNPs⋅GA, which leads to the significant bathochromic shift of the SPR spectra from 602 nm to 650 nm. The dramatic color changes from the initial dark blue to light blue can be observed, which allowed simple monitoring of GSH ether by naked eye and UV–vis spectrophotometer. With UV–vis spectrophotometer measurements, a quantitative linearity was established in the range of 0.5–5.0 nM (R2 = 0.9919) and with a limit of detection (LOD) of 0.12 ± 0.02 nM. Relative standard deviation (RSD) of 3.46% and 1.82% (n = 10) were achieved for the determination of 1.0 and 3.0 nM, respectively. No interfering substances such as ascorbic acid, glucose, sucrose, citric acid, cysteine, Ca2+, Mg2+, Fe3+ and K+ were revealed in the AgTNPs⋅GA based assay. The proposed colorimetric strategy could be extended as the general platform for detection of GSH in dietary supplements and no significant differences in accuracy and precision were observed compared to HPLC standard method.
⁎ Corresponding author at: Advanced Materials Research Unit and Department of Chemistry, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand. E-mail address: [email protected] (E. Detsri).
https://doi.org/10.1016/j.colsurfa.2018.01.016 Received 21 November 2017; Received in revised form 8 January 2018; Accepted 10 January 2018 Available online 30 January 2018 0927-7757/ © 2018 Elsevier B.V. All rights reserved.
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1. Introduction
2. Experimental
Nowadays, reduced glutathione (GSH) supplements are attracting much attention from the teenagers in Bangkok of Thailand. Many teenagers have come to believe that, GSH is one of the great ways to whiten the skin tone in short span of time. GSH has a skin-whitening effect in humans through its tyrosinase inhibitory activity. The skin whitening benefits of GSH are a result from its ability to significantly lower the melanin index [1]. Therefore, there is a large array of GSH containing supplements currently on the drugstore or even the market. GSH supplements are available in several forms such as powdered, capsules, pills and liquid with varying the amount of GSH. The recommended dosage of GSH that should be obtained is between 60 and 250 mg/day [2]. If the dosage is greater than the recommended dose, can cause allergic to shock and death [3]. Therefore, the improvement of sensitive, selective, rapid, simple and inexpensive detection method of GSH in dietary supplements is highly desired. Although some analytical techniques such as capillary electrophoresis (CE) [4], spectrofluorometry [5], hi-performance liquid chromatography (HPLC) [6] and gas chromatography with flame photometric detection [7] have also been used for quantitative analysis of GSH, some problems still occur due to labor-intensive, time consuming and sophisticated instrumentation [8,9]. Recently, colorimetric sensor based on silver nanoparticles (AgNPs) have been attracting much attention because they can provide convenience of visual observation, simple operations, rapid and inexpensive [10–12]. Due to their fascinating optical properties of AgNPs, construction of perceptual devices for colorimetric detection based on AgNPs have been extensively utilized for both biological and chemical analyzes [13,14]. The differentiation of various analyses detection based on AgNPs is dependent upon the phenomenon of size [15], morphology [16], dielectric constant of the surrounding medium [17] as well as interparticles distance of AgNPs [18]. Among colorimetric sensor, AgNPs have the excellent SPR properties in the visible region (400–700 nm) of the spectrum, which makes the detection of biological and chemical easier [19]. In this sense, the surface modifications of AgNPs were played the crucial roles in improving of the optical properties of AgNPs and enhance its sensitivity as a sensor. For examples, He et al. [20] developed a colorimetric detection of Mn2+ using AgNPs cofunctionalized with 4-mercaptobenzoic acid and melamine. The detection limit for Mn2+ ion was approximately to 5 × 10−8 mol L−1. Khayatian et al. [21] used chitosan to modify AgNPs for detection of cysteine in urine and plasma. Under optimum conditions, the calibration curve was linear over a concentration range of 0.1–10 μM with detection limit of 15.0 nM. Zhang et al. [22] developed a colorimetric detection of uric acid in human serum using uricase-stimulated etching of silver nanoprism. The detection limit of uric acid was 0.7 μM. Consequently, the quantitative analysis of GSH in dietary supplements using gallic acid modified the surface of silver triangular nanoplates (AgTNPs) was developed. The dark blue color of AgTNPs⋅GA solution changed to light blue in accordance with the GSH level added as the aggregation of AgTNPs⋅GA induced by GSH lead to a bathochromic shift of the SPR spectra from 602 nm to 650 nm. The effect of various parameters, including such as concentration of AgTNPs⋅GA, incubation times and pH have been explored to establish the optimized conditions. Under the optimal conditions, the colorimetric detection of GSH using AgTNPs⋅GA was found in the concentration range of 0.5–5.0 nM with the limit of detection of 0.12 ± 0.02 nM. The results confirmed that our colorimetric method is sensitive, selective, simple, rapid, and quantitative for colorimetric detection of GSH in dietary supplements.
2.1. Chemicals Silver nitrate (AgNO3, 99.99%), sodium borohydride (NaBH4, 99%), gallic acid (C6H2(OH)3COOH), cysteine (C3H7NO2S) and reduced glutathione (C10H17N3O6S) were purchased from Sigma–Aldrich, Co., Ltd USA. Hydrogen peroxide (H2O2, 30 wt%.), acetic acid (CH3COOH), sodium acetate (CH3COONa), potassium chloride (KCl), magnesium sulfate (MgSO4), calcium carbonate (CaCO3), sodium chloride (NaCl) and potassium nitrate (KNO3) were purchased from Carlo Erba Co., Ltd USA. All reagents obtained commercially were of analytical reagent grade (AR grade) and used without purification. All solutions were prepared using ultrapure water with a resistivity of 18.2 MΩ cm at 25 °C (Milli-Q®, Millipore system). 2.2. Synthesis of AgTNPs⋅GA AgTNPs⋅GA was prepared following the literature [23] with some modifications, in which sodium citrate was removed from the preparation. Briefly, 50 mL of 1 mM silver nitrate was mixed with 3 mL of 20 mM gallic acid in the presence of 0.12 mL of 30 wt.% H2O2. Then, an aliquot of 0.33 mL NaBH4 (100 mmol/L) was rapidly added to a mixture solution under stirring for 10 min at 25 °C. During this time, the initial colorless of the mixture solution was changed gradually to yellow, orange, red, purple and blue, respectively. Blue color of the colloidal solution was indicated the formation of silver triangular nanoplates. Finally, the blue solution of AgTNPs⋅GA was stored at 4 °C in the refrigerator for further use. To estimate the concentration of AgTNPs⋅GA, the stock colloidal AgTNPs⋅GA solution was diluted 3 times using ultrapure water. The SPR spectra of AgTNPs⋅GA were recorded by UV–vis spectrophotometer for five repetitive measurements. According to Beer’s law [24], the concentration of AgTNPs⋅GA was estimated to be 0.014 ± 0.001 nM according to extinction coefficient on particle diameter. For references, the extinction coefficient (ε) and mean diameter of AgTNPs⋅GA were calculated to 5.56 × 1010 M−1 cm−1 and 55.4 ± 1.2 nm, respectively. In addition, the stability of the as-synthesized AgTNPs stabilized with GA was evaluated by keeping them in 4 °C refrigerator over 2 months (Fig. S1, Supporting material). The SPR spectra shows the minimal peak shift of AgTNPs in the presence of GA stabilizer, indicating that the as-synthesized AgTNPs⋅GA were intact and more stable than those prepared in the previous works [23,25]. 2.3. Characterizations The SPR spectra of AgTNPs⋅GA were recorded on UV1800 Ultraviolet–visible (UV–vis) spectrophotometer (Shimadzu Co. Ltd., China) with a matched pair of 10 mm quartz cuvette. The zeta potential (surface charges) and size distribution of colloidal solutions were acquired using Zeta-sizer Nano, ZS with 633 nm HeliumNeon lazer (Malvern instrument, England). Transmission electron microscope (TEM, JEM-2001 model, JEOL Co., Ltd Japan) was used to evaluate the morphology of AgTNPs⋅GA. For TEM analysis, samples have been prepared by spotting diluted solution of AgTNPs⋅GA onto carbon coated 200 mesh copper grids and it were allowed to dry before imaging. 2.4. Colorimetric detection of GSH To detection of GSH, 2 mL of different concentrations from 0.5 nM to 6.0 nM of GSH were mixed with 2 mL of AgTNPs⋅GA. The mixture solutions were incubated for 10 min at room temperature (25 °C) until instant coloration. Following this, UV–vis spectrophotometer was used to record the SPR of the mixture solutions. From the spectral results, calibration curve was constructed. The quantification of GSH in the 37
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chelate formation through the phenolic hydroxyl groups (eOH). Upon the addition of NaBH4, Ag ions/GA complexes are quickly reduced to AgNPs as evidenced by the color change of solution from colorless to light yellowish. The resulting light yellowish solution, which contains spherical particles were then used as the templates for TNPs formation. The shape transformation of spherical nanoparticles (NPs) to TNPs was accomplished simply by adding H2O2. Upon the addition of H2O2, a gradual change in color of silver colloid was observed. The color changed gradually from light yellowish to orange, red, purple and eventually blue. Blue color of colloidal solution displayed the AgTNPs characteristic which has the SPR of 602 nm (Fig. S2, Supporting material). As suggested in previously published work [27], an early development of plate structure is noticed by three SPR bands located at approximately 333, 430 and 680 nm, which are ascribed to the out-ofplane quadrupole, the in-plane quadrupole and the in-plan dipole of the AgTNPs, respectively. These values are similar to those already reported for AgTNPs [27,28]. To examine the size and particle morphology, TEM analyses were performed. As shown in Fig. 1, the particles were found to be mostly triangular in shaped with an average particle size of 55.4 ± 1.2 nm. Well dispersion was observed without the formation of agglomerated or aggregated NPs. Zeta potential analyzer has been used to clarify the surface charges of AgTNPs⋅GA. Zeta potential analysis (Fig. S3-A, Supporting material) demonstrated that the as-synthesized AgTNPs⋅GA had negative charges of −34.3 ± 2.1 mV. This result evidenced that the AgTNPs⋅GA are electronegative charged and can be dispersed from each other in water by electrostatic repulsion. This observation suggested that GA also played a role as a stabilizing agent to provide a good dispersion state of the AgTNPs.
solution was based on the SPR at central maximum wavelength (λmax) 602 nm. 2.5. GSH detection in dietary supplements Three bands of dietary supplements were weighed and ground to fine powder. Thereafter, an amount of powder equivalent to 10 mg of GSH dietary supplement was mixed with 1000 mL of ultrapure water. The mixture was then sonicated for 10 min using probe sonicator (130 watts, VCX 130, Sonics USA). Finally, to detect GSH in dietary supplement, 2 mL of GSH sample was added into 2 mL of AgTNPs⋅GA solution. GSH detection and quantification in dietary supplements were done using the calibration curve. In order to explore the potential of the AgTNPs⋅GA colorimetric sensor, standard addition method was applied to detect GSH. 2.6. Method validation In order to ensure the applicability and reliability of the proposed method, HPLC [26] was used as the validation technique. Orthopthaldehyde (C6H4(CHO)2) was used as a derivatizing agent for GSH assay. Chromatography of GSH was accomplished using Variance, Agilent 1100 series equipped with a LC-10AD HPLC pump. C18 column (Hector, 5 μm, 10 mm × 4.6 mm) and fluorimetric detector at the excitation and emission wavelength of 350 and 420 nm were used for chromatographic experiments. The mobile phase consisted of 20% v/v methanol (CH3OH) in pH 6 of 20 mM sodium hydrogenphosphate (Na2HPO4). The flow rate was maintained at 0.5 mL/min at 25 °C. 3. Results and discussion
3.2. Detection principle of GSH using AgTNPs⋅GA as colorimetric probe 3.1. Characterization of AgTNPs⋅GA To further demonstrate the assay for the direct colorimetric visualization of GSH, different concentrations of GSH were added to the colloidal solution of AgTNPs⋅GA. Fig. 2a shows the UV–vis absorption spectra and photographs of AgTNPs⋅GA with and without GSH. The assynthesized AgTNPs⋅GA in the current study were dark blue in color and exhibited a characteristic and intense absorption peak at 602 nm, which was attributed to the SPR of the polydispersed TNPs. After
In the direct chemical reduction procedure, AgTNPs⋅GA were prepared by the reduction of AgNO3 in aqueous solution by NaBH4 in the presence of gallic acid (GA). H2O2 has been employed as an efficient etchant for the triangular nanoplates (TNPs) formations. The reaction mechanism is illustrated in Scheme Scheme 1a. Herein, GA acts as a stabilizing agent. GA can easily bind to the surface of Ag via tridentate
Scheme 1. (a) Schematic illustration of AgTNPs⋅GA formation and (b) Illustration mechanism of AgTNPs⋅GA for colorimetric sensing of GSH.
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Fig. 1. (a1-2) TEM images of AgTNPs⋅GA at scale 100 nm and 0.2 μm and (b) the corresponding size distribution histogram.
a decrease from −34.3 to −27.9 and −18.2 mV upon the addition of 0.5 and 2.5 nM GSH, respectively. The electrostatic interaction between the deprotonated carboxylate (COO−) of GA functionalized AgTNPs and protonated amine (NH3+) of GSH results in surface charge neutralization and this event is often associated with the formation of NPs aggregates.
addition of an appropriate amount of GSH, a distinct color change of AgTNPs⋅GA solution from dark blue to light blue was observed, resulting in the formation of the new absorption band at approximately 650 nm (red shift). New SPR band of AgTNPs⋅GA appeared at longer wavelength upon the addition of GSH, suggesting that GA stabilized AgTNPs were ready to bind GSH with rapid aggregation process, which was verified by the TEM images (Fig. 2b1-2). GSH was induced AgTNPs⋅GA aggregation to the formation of zwitterionic networks involving head-to-head interaction of the deprotonated carboxylate (COO−) of GA functionalized AgTNPs and protonated amine (NH3+) of GSH (Scheme Scheme 1b). To confirm the electrostatic interaction between GSH and GA coated AgTNPs, we employed zeta potential measurements and the obtained observations are displayed in Fig. S3-B (Supporting material). The zeta potential values of AgTNPs⋅GA showed
3.3. Optimization of experimental conditions For better evaluation of the optical characteristics of AgTNPs conjugated with GA as GSH sensor, the parameters for colorimetric detection including concentration of AgTNPs⋅GA, incubation time and pH were subsequently investigated. In this study, the absorbance at 602 nm was used to assess the degree of aggregation. Fig. 2. (a) Absorption spectra of AgTNPs⋅GA at different concentrations of GSH. [GSH]: (a1) 0, (a2) 0.5, (a3) 1.0, (a4) 2.5, (a5) 5.0 and (a6) 6.0 nM and (b1-2) representative TEM images of AgTNPs⋅GA after the addition of 2.5 nM GSH.
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Fig. 3. Effect of different parameters on the response of AgTNPs⋅GA to 1.0 nM GSH: (a) AgTNPs⋅GA concentration, (b) incubation time: where ♦ = 0.5 nM, ■ = 1.0 nM, ▲ = 2.5 nM, • = 5.0 nM and × = 6.0 nM and (c) pH of the reaction media.
Table 2 Results of accuracy and precision for GSH detection using AgTNPs⋅GA colorimetric probe.
Fig. 5. The corresponding calibration curve for the detection of GSH.
Table 1 Comparison of different methods for determination of GSH. Linear range
Detection limit
Reference
Silver triangular nanoplates conjugated with gallic acid Gold nanoparticles conjugated with water-soluble polymer Silver nanoparticles stabilized with polyethyleneimine Hg2+-quenching of fluorescent gold nanoclusters Ag Nanoparticle-decorated graphene quantum dots Carbon quantum dots and gold nanoparticles
0.5–5.0 nM
0.12 nM
This work
0–6 μM
29 nM
[30]
0.5–6 μM
380 nM
[31]
0–250 μM
9.4 nM
[32]
0.6–20 μM
0.1 μM
[33]
0.1–0.6 μM
50 nM
[34]
Found values (Mean ± SD, nM)
Accuracy (%)
Precision (% RSD)
1.0 2.0 3.0 4.0
0.98 1.94 3.24 3.98
98.4 97.2 108.0 99.7
3.46 1.08 1.82 1.85
± ± ± ±
0.03 0.02 0.06 0.07
The concentration of AgTNPs⋅GA plays an important role in the quantitative determination of GSH. To this aim, different concentrations of AgTNPs⋅GA (0.005, 0.008, 0.010, 0.012 and 0.014 nM) were investigated upon the addition of 1.0 nM GSH. As could be seen in Fig. 3a, increasing AgTNPs⋅GA concentration caused increase the absorption intensity. However, at concentration level of 0.012 nM of AgTNPs⋅GA, no significant increase in absorption intensity was observed. Therefore, 0.012 nM of AgTNPs⋅GA was chosen as optimum concentration of AgTNPs⋅GA. The effect of incubation time on the aggregation of AgTNPs⋅GA induced by GSH was also studied and optimized. Fig. 3b showed that the absorbance at the wavelength 602 nm of AgTNPs⋅GA gradually decreased and reached a relatively constant at 10 min after the addition of low concentration of GSH (0.5 nM), revealing that the aggregation of AgTNPs⋅GA was able to be completed within 10 min. Moreover, within the concentration of GSH increased to 6.0 nM, the reaction could be completed at 1 min. Based on these results, the as-mixted solution was incubated for 10 min for the following detection experiments. The pH values of the solution would affect the interaction of AgTNPs conjugated with GA and GSH. Therefore, it was necessary to investigate the effect of pH values for the detection of GSH. Fig. 3c shows the absorbance at 602 nm of AgTNPs⋅GA in the presence of 2.5 nM GSH in different pH solutions (pH 5–8). Since pH can exert a great effect on eCOOH, eNH2 and eSH of GSH [29] and AgTNPs⋅GA can be more stable in the pH from 5.0 to 8.0 (Fig. S4, Supporting material), the phosphate buffered saline (PBS buffer) pH varying 5.0 to 8.0 was investigated. The results indicated that, the highest absorbance at 602 nm was obtained at pH 6. As a result, our experiments were commonly carried out at pH 6.0.
Fig. 4. Interference study for 5.0 nM GSH in the presence of interfering species with the concentration 1000 times greater than those of analytes.
Colorimetric probe
Standard added (nM)
3.4. Selectivity of GSH detection Selectivity is another important factor in colorimetric sensing. To further investigate the selectivity of this assay, 5 μM of coexisting species including ascorbic acid, glucose, sucrose, citric acid, cysteine, Ca2+, Mg2+, Fe3+ and K+ were performed under the optimized conditions. As presented in Fig. 4, the λmax signal at 602 nm was a significant increased for GSH than those of other substances, demonstrating the high selectivity for colorimetric detection of GSH. 40
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Table 3 Determination of GSH in dietary supplements using the proposed AgTNPs⋅GA colorimetric sensor and HPLC method. Sample No
Detected (nM)
1
1.34 ± 0.14
2
2.12 ± 0.13
3
3.21 ± 0.09
Added (nM)
0.5 1.0 0.5 1.0 0.5 1.0
AgTNPs⋅GA colorimetric sensor
HPLC method
Found (nM)
Recovery (%)
RSD (%, n = 5)
Found (nM)
Recovery (%)
RSD (%, n = 5)
1.85 2.37 2.62 3.13 3.75 4.19
101.4 103.3 99.4 100.9 108.2 98.6
3.1 1.9 3.0 1.9 1.1 1.3
1.84 2.36 2.62 3.12 3.70 4.20
100.6 101.9 99.8 100.4 99.0 99.6
0.1 0.2 0.2 0.1 0.1 0.1
± ± ± ± ± ±
0.05 0.04 0.08 0.06 0.04 0.05
± ± ± ± ± ±
0.01 0.05 0.04 0.02 0.03 0.05
solution color and SPR change of AgTNPs⋅GA induced by GSH. The SPR spectra of AgTNPs⋅GA were proportionally red-shifted with GSH concentration in the range of 0.5–5.0 nM, resulting in a color change from dark blue to light blue. The proposed colorimetric method achieves high selectivity and sensitivity toward the detection of GSH with the low limit of detection (LOD) of 0.12 ± 0.02 nM. Importantly, the colorimetric sensor described here can be easily read out by naked eye and UV–vis spectrophotometry. The present method provides a new optical sensing pattern for the rapid, simple, inexpensive, sensitive and selective detection of GSH in dietary supplements.
3.5. Analytical performances of the method Under the optimized experimental conditions, the responses of colorimetric system to different concentrations of GSH were investigated. A linear relationship between the absorbance at 602 nm and the concentration of GSH was found in the range of 0.5–5.0 nM. Accordingly, linear equation of Absat λmax 602 nm = −0.0269[GSH, nM] + 0.4045 with regression coefficient of 0.9919 was obtained (Fig. 5). The limit of detection (LOD) of the method was calculated to be 0.12 ± 0.02 nM (LOD = 3SDblank/m, where SDblank and m are the standard deviation of ten blank replicate measurements and the slope of calibration curve, respectively). Such a concentration level was lower to those achieved by different GSH detection methods, as shown in Table 1. It is clearly seen that the developed AgTNPs⋅GA sensor shows good sensitivity and detection range with the acceptable for common detection. To examine the accuracy and precision of this approach, analyses of standard solutions of GSH at 1.0 nM to 4.0 nM were all performed with ten replicates. The results of accuracy and precision were summarized in Table 2. It is clearly seen that, this approach exhibited good reproducibility with relative standard deviations (RSD) of 3.46% (1.0 nM), 1.08% (2.0 nM), 1.82% (3.0 nM) and 1.85% (4.0 nM). Moreover, the accuracy for the present system were found in the range of 97.2–108.2%.
Acknowledgment We acknowledge Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, Thailand (Grant no. 2560-01-05070) for financial assistance. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfa.2018.01.016. References [1] J. Castillo, E. Handog, I. Singzon, M.S. Datuin, Glutathione as a systemic skin whitening agent via the buccal mucosa: an open-label, single-arm study, J. Am. Acad. Dermatol. 74 (2016) AB17. [2] World Health Organization (WHO), Technical Report Series 724 Energy and Protein Requirement: Report of a Joint FAO/WHO/UNU Expert Consultation, WHO, Geneva, 1985, pp. 64–69. [3] C. Chbili, A. Elouaer, N. Fathallah, M. Nouira, B.H. Jrad, L. Gaha, S. Saguem, Effects of glutathione S-transferase M1 and T1 deletions on bipolar disorder risk among a Tunisian population, Gene 607 (2017) 31–35. [4] T. Inoue, J.R. Kirchhoff, Determination of thiols by capillary electrophoresis with amperometric detection at a coenzyme pyrroloquinoline quinone modified electrode, Anal. Chem. 74 (2002) 1349–1354. [5] M.A. Raggi, L. Nobile, A.G. Giovannini, Spectrophotometric determination of glutathione and of its oxidation product in pharmaceutical dosage forms, J. Pharm. Biomed. Anal. 9 (1991) 1037–1040. [6] W. Zhang, F.L. Wan, W. Zhu, H.H. Xu, X.Y. Ye, R.Y. Cheng, L.T. Jin, Determination of glutathione and glutathione disulfide in hepatocytes by liquid chromatography with an electrode modified with functionalized carbon nanotubes, J. Chromatogr. B 818 (2005) 227–232. [7] H. Kataoka, Y. Imamura, H. Tanaka, M. Makita, Determination of cysteamine and cystamine by gas chromatography with flame photometric detection, J. Pharm. Biomed. Anal. 11 (1993) 963–969. [8] R.N. Appala, S. Chigurupati, R.V.V.S.S. Appala, K.K. Selvarajan, J.I. Mohammad, A simple HPLC-UV method for the determination of glutathione in PC-12 cells, Scientifica 2016 (2016) 1–6. [9] L.P. Yap, H. Sancheti, M.D. Ybanez, J. Garcia, E. Cadenas, D. Han, Determination of GSH, GSSG, and GSNO using HPLC with electrochemical detection, Methods Enzymol. 473 (2010) 137–147. [10] E. Detsri, J. Popanyasak, Fabrication of silver nanoparticles/polyaniline composite thin films using layer-by-layer self-assembly technique for ammonia sensing, Colloids Surf. A: Physicochem. Eng. Aspect 467 (2015) 57–65. [11] Y. Zhou, H. Zhao, Y. He, N. Ding, Q. Cao, Colorimetric detection of Cu2+ using 4mercaptobenzoic acid modified silver nanoparticles, Colloids Surf. A: Physicochem. Eng. Aspect 391 (2011) 179–183. [12] S. Chen, H. Gao, W. Shen, C. Lu, Q. Yuan, Colorimetric detection of cysteine using noncrosslinking aggregation of fluorosurfactant-capped silver nanoparticles, Sens. Actuators B: Chem. 190 (2014) 673–678. [13] K.A. Rawat, R.K. Singhal, S.K. Kailasa, One-pot synthesis of silver nanoparticles
3.6. Analysis of GSH in dietary supplements In order to evaluate the applicability of the proposed colorimetric sensor, AgTNPs⋅GA was applied for determination of GSH in dietary supplements samples. GSH tablets were taken from three different commercial bands. Several tablets from each band were grounded and homogenized. Then, a given weight of grounded tablets was dissolved in water, centrifuged and then measured the GSH content by AgTNPs⋅GA colorimetric probe. In addition, different concentrations of standard GSH were spiked into the sample solutions of the tablets to ensure the reliability of the results. The results are summarized in Table 3. As can be seen from Table 3, the recoveries can be quantitative in the range of 98.6–108.2% with the RSD less than 3.1%, indicating the potential applicability of our method in dietary supplements. To further verify the reliability and practicability of the proposed method. The HPLC method was also simultaneously used for determine GSH in dietary supplement samples and the results were summarized in Table 3. HPLC method was provided the recoveries in the range of 99.8–101.9% with the RSD lower than 0.2%. The results obtained by the two methods were compared and consistent, indicating that our proposed method possessed great potential and reliable for detecting GSH in dietary supplements. 4. Conclusions In conclusion, we have successfully demonstrated the feasibility of using AgTNPs conjugated with GA based colorimetric assay in the detection of GSH. The detection mechanism takes advantage of the 41
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Contents lists available at ScienceDirect
Colloids and Surfaces A journal homepage: www.elsevier.com/locate/colsurfa
A sensitive and selective colorimetric sensor for reduced glutathione detection based on silver triangular nanoplates conjugated with gallic acid
T
⁎
Ekarat Detsria,b, , Panpailin Seeharaja,b, Chaval Sriwonga a b
Department of Chemistry, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand Advanced Materials Research Unit, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand
G RA P H I C A L AB S T R A C T
A R T I C L E I N F O
A B S T R A C T
Keywords: Silver triangular nanoplates Gallic acid Colorimetric sensor Reduced glutathione Dietary supplements
Silver triangular nanoplates conjugated with gallic acid (AgTNPs⋅GA) was designed and synthesized for colorimetric detection of reduced glutathione (GSH). The surface plasmon resonance (SPR) properties of AgTNPs⋅GA were strongly influenced by the addition of GSH. The detection principle was based on the aggregation of AgTNPs⋅GA, which leads to the significant bathochromic shift of the SPR spectra from 602 nm to 650 nm. The dramatic color changes from the initial dark blue to light blue can be observed, which allowed simple monitoring of GSH ether by naked eye and UV–vis spectrophotometer. With UV–vis spectrophotometer measurements, a quantitative linearity was established in the range of 0.5–5.0 nM (R2 = 0.9919) and with a limit of detection (LOD) of 0.12 ± 0.02 nM. Relative standard deviation (RSD) of 3.46% and 1.82% (n = 10) were achieved for the determination of 1.0 and 3.0 nM, respectively. No interfering substances such as ascorbic acid, glucose, sucrose, citric acid, cysteine, Ca2+, Mg2+, Fe3+ and K+ were revealed in the AgTNPs⋅GA based assay. The proposed colorimetric strategy could be extended as the general platform for detection of GSH in dietary supplements and no significant differences in accuracy and precision were observed compared to HPLC standard method.
⁎ Corresponding author at: Advanced Materials Research Unit and Department of Chemistry, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand. E-mail address: [email protected] (E. Detsri).
https://doi.org/10.1016/j.colsurfa.2018.01.016 Received 21 November 2017; Received in revised form 8 January 2018; Accepted 10 January 2018 Available online 30 January 2018 0927-7757/ © 2018 Elsevier B.V. All rights reserved.
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1. Introduction
2. Experimental
Nowadays, reduced glutathione (GSH) supplements are attracting much attention from the teenagers in Bangkok of Thailand. Many teenagers have come to believe that, GSH is one of the great ways to whiten the skin tone in short span of time. GSH has a skin-whitening effect in humans through its tyrosinase inhibitory activity. The skin whitening benefits of GSH are a result from its ability to significantly lower the melanin index [1]. Therefore, there is a large array of GSH containing supplements currently on the drugstore or even the market. GSH supplements are available in several forms such as powdered, capsules, pills and liquid with varying the amount of GSH. The recommended dosage of GSH that should be obtained is between 60 and 250 mg/day [2]. If the dosage is greater than the recommended dose, can cause allergic to shock and death [3]. Therefore, the improvement of sensitive, selective, rapid, simple and inexpensive detection method of GSH in dietary supplements is highly desired. Although some analytical techniques such as capillary electrophoresis (CE) [4], spectrofluorometry [5], hi-performance liquid chromatography (HPLC) [6] and gas chromatography with flame photometric detection [7] have also been used for quantitative analysis of GSH, some problems still occur due to labor-intensive, time consuming and sophisticated instrumentation [8,9]. Recently, colorimetric sensor based on silver nanoparticles (AgNPs) have been attracting much attention because they can provide convenience of visual observation, simple operations, rapid and inexpensive [10–12]. Due to their fascinating optical properties of AgNPs, construction of perceptual devices for colorimetric detection based on AgNPs have been extensively utilized for both biological and chemical analyzes [13,14]. The differentiation of various analyses detection based on AgNPs is dependent upon the phenomenon of size [15], morphology [16], dielectric constant of the surrounding medium [17] as well as interparticles distance of AgNPs [18]. Among colorimetric sensor, AgNPs have the excellent SPR properties in the visible region (400–700 nm) of the spectrum, which makes the detection of biological and chemical easier [19]. In this sense, the surface modifications of AgNPs were played the crucial roles in improving of the optical properties of AgNPs and enhance its sensitivity as a sensor. For examples, He et al. [20] developed a colorimetric detection of Mn2+ using AgNPs cofunctionalized with 4-mercaptobenzoic acid and melamine. The detection limit for Mn2+ ion was approximately to 5 × 10−8 mol L−1. Khayatian et al. [21] used chitosan to modify AgNPs for detection of cysteine in urine and plasma. Under optimum conditions, the calibration curve was linear over a concentration range of 0.1–10 μM with detection limit of 15.0 nM. Zhang et al. [22] developed a colorimetric detection of uric acid in human serum using uricase-stimulated etching of silver nanoprism. The detection limit of uric acid was 0.7 μM. Consequently, the quantitative analysis of GSH in dietary supplements using gallic acid modified the surface of silver triangular nanoplates (AgTNPs) was developed. The dark blue color of AgTNPs⋅GA solution changed to light blue in accordance with the GSH level added as the aggregation of AgTNPs⋅GA induced by GSH lead to a bathochromic shift of the SPR spectra from 602 nm to 650 nm. The effect of various parameters, including such as concentration of AgTNPs⋅GA, incubation times and pH have been explored to establish the optimized conditions. Under the optimal conditions, the colorimetric detection of GSH using AgTNPs⋅GA was found in the concentration range of 0.5–5.0 nM with the limit of detection of 0.12 ± 0.02 nM. The results confirmed that our colorimetric method is sensitive, selective, simple, rapid, and quantitative for colorimetric detection of GSH in dietary supplements.
2.1. Chemicals Silver nitrate (AgNO3, 99.99%), sodium borohydride (NaBH4, 99%), gallic acid (C6H2(OH)3COOH), cysteine (C3H7NO2S) and reduced glutathione (C10H17N3O6S) were purchased from Sigma–Aldrich, Co., Ltd USA. Hydrogen peroxide (H2O2, 30 wt%.), acetic acid (CH3COOH), sodium acetate (CH3COONa), potassium chloride (KCl), magnesium sulfate (MgSO4), calcium carbonate (CaCO3), sodium chloride (NaCl) and potassium nitrate (KNO3) were purchased from Carlo Erba Co., Ltd USA. All reagents obtained commercially were of analytical reagent grade (AR grade) and used without purification. All solutions were prepared using ultrapure water with a resistivity of 18.2 MΩ cm at 25 °C (Milli-Q®, Millipore system). 2.2. Synthesis of AgTNPs⋅GA AgTNPs⋅GA was prepared following the literature [23] with some modifications, in which sodium citrate was removed from the preparation. Briefly, 50 mL of 1 mM silver nitrate was mixed with 3 mL of 20 mM gallic acid in the presence of 0.12 mL of 30 wt.% H2O2. Then, an aliquot of 0.33 mL NaBH4 (100 mmol/L) was rapidly added to a mixture solution under stirring for 10 min at 25 °C. During this time, the initial colorless of the mixture solution was changed gradually to yellow, orange, red, purple and blue, respectively. Blue color of the colloidal solution was indicated the formation of silver triangular nanoplates. Finally, the blue solution of AgTNPs⋅GA was stored at 4 °C in the refrigerator for further use. To estimate the concentration of AgTNPs⋅GA, the stock colloidal AgTNPs⋅GA solution was diluted 3 times using ultrapure water. The SPR spectra of AgTNPs⋅GA were recorded by UV–vis spectrophotometer for five repetitive measurements. According to Beer’s law [24], the concentration of AgTNPs⋅GA was estimated to be 0.014 ± 0.001 nM according to extinction coefficient on particle diameter. For references, the extinction coefficient (ε) and mean diameter of AgTNPs⋅GA were calculated to 5.56 × 1010 M−1 cm−1 and 55.4 ± 1.2 nm, respectively. In addition, the stability of the as-synthesized AgTNPs stabilized with GA was evaluated by keeping them in 4 °C refrigerator over 2 months (Fig. S1, Supporting material). The SPR spectra shows the minimal peak shift of AgTNPs in the presence of GA stabilizer, indicating that the as-synthesized AgTNPs⋅GA were intact and more stable than those prepared in the previous works [23,25]. 2.3. Characterizations The SPR spectra of AgTNPs⋅GA were recorded on UV1800 Ultraviolet–visible (UV–vis) spectrophotometer (Shimadzu Co. Ltd., China) with a matched pair of 10 mm quartz cuvette. The zeta potential (surface charges) and size distribution of colloidal solutions were acquired using Zeta-sizer Nano, ZS with 633 nm HeliumNeon lazer (Malvern instrument, England). Transmission electron microscope (TEM, JEM-2001 model, JEOL Co., Ltd Japan) was used to evaluate the morphology of AgTNPs⋅GA. For TEM analysis, samples have been prepared by spotting diluted solution of AgTNPs⋅GA onto carbon coated 200 mesh copper grids and it were allowed to dry before imaging. 2.4. Colorimetric detection of GSH To detection of GSH, 2 mL of different concentrations from 0.5 nM to 6.0 nM of GSH were mixed with 2 mL of AgTNPs⋅GA. The mixture solutions were incubated for 10 min at room temperature (25 °C) until instant coloration. Following this, UV–vis spectrophotometer was used to record the SPR of the mixture solutions. From the spectral results, calibration curve was constructed. The quantification of GSH in the 37
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chelate formation through the phenolic hydroxyl groups (eOH). Upon the addition of NaBH4, Ag ions/GA complexes are quickly reduced to AgNPs as evidenced by the color change of solution from colorless to light yellowish. The resulting light yellowish solution, which contains spherical particles were then used as the templates for TNPs formation. The shape transformation of spherical nanoparticles (NPs) to TNPs was accomplished simply by adding H2O2. Upon the addition of H2O2, a gradual change in color of silver colloid was observed. The color changed gradually from light yellowish to orange, red, purple and eventually blue. Blue color of colloidal solution displayed the AgTNPs characteristic which has the SPR of 602 nm (Fig. S2, Supporting material). As suggested in previously published work [27], an early development of plate structure is noticed by three SPR bands located at approximately 333, 430 and 680 nm, which are ascribed to the out-ofplane quadrupole, the in-plane quadrupole and the in-plan dipole of the AgTNPs, respectively. These values are similar to those already reported for AgTNPs [27,28]. To examine the size and particle morphology, TEM analyses were performed. As shown in Fig. 1, the particles were found to be mostly triangular in shaped with an average particle size of 55.4 ± 1.2 nm. Well dispersion was observed without the formation of agglomerated or aggregated NPs. Zeta potential analyzer has been used to clarify the surface charges of AgTNPs⋅GA. Zeta potential analysis (Fig. S3-A, Supporting material) demonstrated that the as-synthesized AgTNPs⋅GA had negative charges of −34.3 ± 2.1 mV. This result evidenced that the AgTNPs⋅GA are electronegative charged and can be dispersed from each other in water by electrostatic repulsion. This observation suggested that GA also played a role as a stabilizing agent to provide a good dispersion state of the AgTNPs.
solution was based on the SPR at central maximum wavelength (λmax) 602 nm. 2.5. GSH detection in dietary supplements Three bands of dietary supplements were weighed and ground to fine powder. Thereafter, an amount of powder equivalent to 10 mg of GSH dietary supplement was mixed with 1000 mL of ultrapure water. The mixture was then sonicated for 10 min using probe sonicator (130 watts, VCX 130, Sonics USA). Finally, to detect GSH in dietary supplement, 2 mL of GSH sample was added into 2 mL of AgTNPs⋅GA solution. GSH detection and quantification in dietary supplements were done using the calibration curve. In order to explore the potential of the AgTNPs⋅GA colorimetric sensor, standard addition method was applied to detect GSH. 2.6. Method validation In order to ensure the applicability and reliability of the proposed method, HPLC [26] was used as the validation technique. Orthopthaldehyde (C6H4(CHO)2) was used as a derivatizing agent for GSH assay. Chromatography of GSH was accomplished using Variance, Agilent 1100 series equipped with a LC-10AD HPLC pump. C18 column (Hector, 5 μm, 10 mm × 4.6 mm) and fluorimetric detector at the excitation and emission wavelength of 350 and 420 nm were used for chromatographic experiments. The mobile phase consisted of 20% v/v methanol (CH3OH) in pH 6 of 20 mM sodium hydrogenphosphate (Na2HPO4). The flow rate was maintained at 0.5 mL/min at 25 °C. 3. Results and discussion
3.2. Detection principle of GSH using AgTNPs⋅GA as colorimetric probe 3.1. Characterization of AgTNPs⋅GA To further demonstrate the assay for the direct colorimetric visualization of GSH, different concentrations of GSH were added to the colloidal solution of AgTNPs⋅GA. Fig. 2a shows the UV–vis absorption spectra and photographs of AgTNPs⋅GA with and without GSH. The assynthesized AgTNPs⋅GA in the current study were dark blue in color and exhibited a characteristic and intense absorption peak at 602 nm, which was attributed to the SPR of the polydispersed TNPs. After
In the direct chemical reduction procedure, AgTNPs⋅GA were prepared by the reduction of AgNO3 in aqueous solution by NaBH4 in the presence of gallic acid (GA). H2O2 has been employed as an efficient etchant for the triangular nanoplates (TNPs) formations. The reaction mechanism is illustrated in Scheme Scheme 1a. Herein, GA acts as a stabilizing agent. GA can easily bind to the surface of Ag via tridentate
Scheme 1. (a) Schematic illustration of AgTNPs⋅GA formation and (b) Illustration mechanism of AgTNPs⋅GA for colorimetric sensing of GSH.
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Fig. 1. (a1-2) TEM images of AgTNPs⋅GA at scale 100 nm and 0.2 μm and (b) the corresponding size distribution histogram.
a decrease from −34.3 to −27.9 and −18.2 mV upon the addition of 0.5 and 2.5 nM GSH, respectively. The electrostatic interaction between the deprotonated carboxylate (COO−) of GA functionalized AgTNPs and protonated amine (NH3+) of GSH results in surface charge neutralization and this event is often associated with the formation of NPs aggregates.
addition of an appropriate amount of GSH, a distinct color change of AgTNPs⋅GA solution from dark blue to light blue was observed, resulting in the formation of the new absorption band at approximately 650 nm (red shift). New SPR band of AgTNPs⋅GA appeared at longer wavelength upon the addition of GSH, suggesting that GA stabilized AgTNPs were ready to bind GSH with rapid aggregation process, which was verified by the TEM images (Fig. 2b1-2). GSH was induced AgTNPs⋅GA aggregation to the formation of zwitterionic networks involving head-to-head interaction of the deprotonated carboxylate (COO−) of GA functionalized AgTNPs and protonated amine (NH3+) of GSH (Scheme Scheme 1b). To confirm the electrostatic interaction between GSH and GA coated AgTNPs, we employed zeta potential measurements and the obtained observations are displayed in Fig. S3-B (Supporting material). The zeta potential values of AgTNPs⋅GA showed
3.3. Optimization of experimental conditions For better evaluation of the optical characteristics of AgTNPs conjugated with GA as GSH sensor, the parameters for colorimetric detection including concentration of AgTNPs⋅GA, incubation time and pH were subsequently investigated. In this study, the absorbance at 602 nm was used to assess the degree of aggregation. Fig. 2. (a) Absorption spectra of AgTNPs⋅GA at different concentrations of GSH. [GSH]: (a1) 0, (a2) 0.5, (a3) 1.0, (a4) 2.5, (a5) 5.0 and (a6) 6.0 nM and (b1-2) representative TEM images of AgTNPs⋅GA after the addition of 2.5 nM GSH.
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Fig. 3. Effect of different parameters on the response of AgTNPs⋅GA to 1.0 nM GSH: (a) AgTNPs⋅GA concentration, (b) incubation time: where ♦ = 0.5 nM, ■ = 1.0 nM, ▲ = 2.5 nM, • = 5.0 nM and × = 6.0 nM and (c) pH of the reaction media.
Table 2 Results of accuracy and precision for GSH detection using AgTNPs⋅GA colorimetric probe.
Fig. 5. The corresponding calibration curve for the detection of GSH.
Table 1 Comparison of different methods for determination of GSH. Linear range
Detection limit
Reference
Silver triangular nanoplates conjugated with gallic acid Gold nanoparticles conjugated with water-soluble polymer Silver nanoparticles stabilized with polyethyleneimine Hg2+-quenching of fluorescent gold nanoclusters Ag Nanoparticle-decorated graphene quantum dots Carbon quantum dots and gold nanoparticles
0.5–5.0 nM
0.12 nM
This work
0–6 μM
29 nM
[30]
0.5–6 μM
380 nM
[31]
0–250 μM
9.4 nM
[32]
0.6–20 μM
0.1 μM
[33]
0.1–0.6 μM
50 nM
[34]
Found values (Mean ± SD, nM)
Accuracy (%)
Precision (% RSD)
1.0 2.0 3.0 4.0
0.98 1.94 3.24 3.98
98.4 97.2 108.0 99.7
3.46 1.08 1.82 1.85
± ± ± ±
0.03 0.02 0.06 0.07
The concentration of AgTNPs⋅GA plays an important role in the quantitative determination of GSH. To this aim, different concentrations of AgTNPs⋅GA (0.005, 0.008, 0.010, 0.012 and 0.014 nM) were investigated upon the addition of 1.0 nM GSH. As could be seen in Fig. 3a, increasing AgTNPs⋅GA concentration caused increase the absorption intensity. However, at concentration level of 0.012 nM of AgTNPs⋅GA, no significant increase in absorption intensity was observed. Therefore, 0.012 nM of AgTNPs⋅GA was chosen as optimum concentration of AgTNPs⋅GA. The effect of incubation time on the aggregation of AgTNPs⋅GA induced by GSH was also studied and optimized. Fig. 3b showed that the absorbance at the wavelength 602 nm of AgTNPs⋅GA gradually decreased and reached a relatively constant at 10 min after the addition of low concentration of GSH (0.5 nM), revealing that the aggregation of AgTNPs⋅GA was able to be completed within 10 min. Moreover, within the concentration of GSH increased to 6.0 nM, the reaction could be completed at 1 min. Based on these results, the as-mixted solution was incubated for 10 min for the following detection experiments. The pH values of the solution would affect the interaction of AgTNPs conjugated with GA and GSH. Therefore, it was necessary to investigate the effect of pH values for the detection of GSH. Fig. 3c shows the absorbance at 602 nm of AgTNPs⋅GA in the presence of 2.5 nM GSH in different pH solutions (pH 5–8). Since pH can exert a great effect on eCOOH, eNH2 and eSH of GSH [29] and AgTNPs⋅GA can be more stable in the pH from 5.0 to 8.0 (Fig. S4, Supporting material), the phosphate buffered saline (PBS buffer) pH varying 5.0 to 8.0 was investigated. The results indicated that, the highest absorbance at 602 nm was obtained at pH 6. As a result, our experiments were commonly carried out at pH 6.0.
Fig. 4. Interference study for 5.0 nM GSH in the presence of interfering species with the concentration 1000 times greater than those of analytes.
Colorimetric probe
Standard added (nM)
3.4. Selectivity of GSH detection Selectivity is another important factor in colorimetric sensing. To further investigate the selectivity of this assay, 5 μM of coexisting species including ascorbic acid, glucose, sucrose, citric acid, cysteine, Ca2+, Mg2+, Fe3+ and K+ were performed under the optimized conditions. As presented in Fig. 4, the λmax signal at 602 nm was a significant increased for GSH than those of other substances, demonstrating the high selectivity for colorimetric detection of GSH. 40
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Table 3 Determination of GSH in dietary supplements using the proposed AgTNPs⋅GA colorimetric sensor and HPLC method. Sample No
Detected (nM)
1
1.34 ± 0.14
2
2.12 ± 0.13
3
3.21 ± 0.09
Added (nM)
0.5 1.0 0.5 1.0 0.5 1.0
AgTNPs⋅GA colorimetric sensor
HPLC method
Found (nM)
Recovery (%)
RSD (%, n = 5)
Found (nM)
Recovery (%)
RSD (%, n = 5)
1.85 2.37 2.62 3.13 3.75 4.19
101.4 103.3 99.4 100.9 108.2 98.6
3.1 1.9 3.0 1.9 1.1 1.3
1.84 2.36 2.62 3.12 3.70 4.20
100.6 101.9 99.8 100.4 99.0 99.6
0.1 0.2 0.2 0.1 0.1 0.1
± ± ± ± ± ±
0.05 0.04 0.08 0.06 0.04 0.05
± ± ± ± ± ±
0.01 0.05 0.04 0.02 0.03 0.05
solution color and SPR change of AgTNPs⋅GA induced by GSH. The SPR spectra of AgTNPs⋅GA were proportionally red-shifted with GSH concentration in the range of 0.5–5.0 nM, resulting in a color change from dark blue to light blue. The proposed colorimetric method achieves high selectivity and sensitivity toward the detection of GSH with the low limit of detection (LOD) of 0.12 ± 0.02 nM. Importantly, the colorimetric sensor described here can be easily read out by naked eye and UV–vis spectrophotometry. The present method provides a new optical sensing pattern for the rapid, simple, inexpensive, sensitive and selective detection of GSH in dietary supplements.
3.5. Analytical performances of the method Under the optimized experimental conditions, the responses of colorimetric system to different concentrations of GSH were investigated. A linear relationship between the absorbance at 602 nm and the concentration of GSH was found in the range of 0.5–5.0 nM. Accordingly, linear equation of Absat λmax 602 nm = −0.0269[GSH, nM] + 0.4045 with regression coefficient of 0.9919 was obtained (Fig. 5). The limit of detection (LOD) of the method was calculated to be 0.12 ± 0.02 nM (LOD = 3SDblank/m, where SDblank and m are the standard deviation of ten blank replicate measurements and the slope of calibration curve, respectively). Such a concentration level was lower to those achieved by different GSH detection methods, as shown in Table 1. It is clearly seen that the developed AgTNPs⋅GA sensor shows good sensitivity and detection range with the acceptable for common detection. To examine the accuracy and precision of this approach, analyses of standard solutions of GSH at 1.0 nM to 4.0 nM were all performed with ten replicates. The results of accuracy and precision were summarized in Table 2. It is clearly seen that, this approach exhibited good reproducibility with relative standard deviations (RSD) of 3.46% (1.0 nM), 1.08% (2.0 nM), 1.82% (3.0 nM) and 1.85% (4.0 nM). Moreover, the accuracy for the present system were found in the range of 97.2–108.2%.
Acknowledgment We acknowledge Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, Thailand (Grant no. 2560-01-05070) for financial assistance. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfa.2018.01.016. References [1] J. Castillo, E. Handog, I. Singzon, M.S. Datuin, Glutathione as a systemic skin whitening agent via the buccal mucosa: an open-label, single-arm study, J. Am. Acad. Dermatol. 74 (2016) AB17. [2] World Health Organization (WHO), Technical Report Series 724 Energy and Protein Requirement: Report of a Joint FAO/WHO/UNU Expert Consultation, WHO, Geneva, 1985, pp. 64–69. [3] C. Chbili, A. Elouaer, N. Fathallah, M. Nouira, B.H. Jrad, L. Gaha, S. Saguem, Effects of glutathione S-transferase M1 and T1 deletions on bipolar disorder risk among a Tunisian population, Gene 607 (2017) 31–35. [4] T. Inoue, J.R. Kirchhoff, Determination of thiols by capillary electrophoresis with amperometric detection at a coenzyme pyrroloquinoline quinone modified electrode, Anal. Chem. 74 (2002) 1349–1354. [5] M.A. Raggi, L. Nobile, A.G. Giovannini, Spectrophotometric determination of glutathione and of its oxidation product in pharmaceutical dosage forms, J. Pharm. Biomed. Anal. 9 (1991) 1037–1040. [6] W. Zhang, F.L. Wan, W. Zhu, H.H. Xu, X.Y. Ye, R.Y. Cheng, L.T. Jin, Determination of glutathione and glutathione disulfide in hepatocytes by liquid chromatography with an electrode modified with functionalized carbon nanotubes, J. Chromatogr. B 818 (2005) 227–232. [7] H. Kataoka, Y. Imamura, H. Tanaka, M. Makita, Determination of cysteamine and cystamine by gas chromatography with flame photometric detection, J. Pharm. Biomed. Anal. 11 (1993) 963–969. [8] R.N. Appala, S. Chigurupati, R.V.V.S.S. Appala, K.K. Selvarajan, J.I. Mohammad, A simple HPLC-UV method for the determination of glutathione in PC-12 cells, Scientifica 2016 (2016) 1–6. [9] L.P. Yap, H. Sancheti, M.D. Ybanez, J. Garcia, E. Cadenas, D. Han, Determination of GSH, GSSG, and GSNO using HPLC with electrochemical detection, Methods Enzymol. 473 (2010) 137–147. [10] E. Detsri, J. Popanyasak, Fabrication of silver nanoparticles/polyaniline composite thin films using layer-by-layer self-assembly technique for ammonia sensing, Colloids Surf. A: Physicochem. Eng. Aspect 467 (2015) 57–65. [11] Y. Zhou, H. Zhao, Y. He, N. Ding, Q. Cao, Colorimetric detection of Cu2+ using 4mercaptobenzoic acid modified silver nanoparticles, Colloids Surf. A: Physicochem. Eng. Aspect 391 (2011) 179–183. [12] S. Chen, H. Gao, W. Shen, C. Lu, Q. Yuan, Colorimetric detection of cysteine using noncrosslinking aggregation of fluorosurfactant-capped silver nanoparticles, Sens. Actuators B: Chem. 190 (2014) 673–678. [13] K.A. Rawat, R.K. Singhal, S.K. Kailasa, One-pot synthesis of silver nanoparticles
3.6. Analysis of GSH in dietary supplements In order to evaluate the applicability of the proposed colorimetric sensor, AgTNPs⋅GA was applied for determination of GSH in dietary supplements samples. GSH tablets were taken from three different commercial bands. Several tablets from each band were grounded and homogenized. Then, a given weight of grounded tablets was dissolved in water, centrifuged and then measured the GSH content by AgTNPs⋅GA colorimetric probe. In addition, different concentrations of standard GSH were spiked into the sample solutions of the tablets to ensure the reliability of the results. The results are summarized in Table 3. As can be seen from Table 3, the recoveries can be quantitative in the range of 98.6–108.2% with the RSD less than 3.1%, indicating the potential applicability of our method in dietary supplements. To further verify the reliability and practicability of the proposed method. The HPLC method was also simultaneously used for determine GSH in dietary supplement samples and the results were summarized in Table 3. HPLC method was provided the recoveries in the range of 99.8–101.9% with the RSD lower than 0.2%. The results obtained by the two methods were compared and consistent, indicating that our proposed method possessed great potential and reliable for detecting GSH in dietary supplements. 4. Conclusions In conclusion, we have successfully demonstrated the feasibility of using AgTNPs conjugated with GA based colorimetric assay in the detection of GSH. The detection mechanism takes advantage of the 41
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