Ultrasensitive Glutathione Detection Based on Lucigenin Cathodic

Jul 14, 2016 - To the best of our knowledge, it is the first time that MnO2 nanosheets were applied in ECL. The schematic principle of the ECL inhibit...
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Ultrasensitive Glutathione Detection Based on Lucigenin Cathodic Electrochemiluminescence in the Presence of MnO2 Nanosheets Wenyue Gao, Zhongyuan Liu, Liming Qi, Jianping Lai, Shimeles Addisu Kitte, and Guobao Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01491 • Publication Date (Web): 14 Jul 2016 Downloaded from http://pubs.acs.org on July 17, 2016

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Analytical Chemistry

Ultrasensitive Glutathione Detection Based on Lucigenin Cathodic Electrochemiluminescence in the Presence of MnO2 Nanosheets Wenyue Gao,a,b Zhongyuan Liu,a Liming Qi,a,b Jianping Lai,a,b Shimeles Addisu Kitte,a,b and Guobao Xua,* a

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, P.R. China. b University of Chinese Academy of Sciences, Beijing 100049, P.R. China. ABSTRACT: Glutathione (GSH) is a crucial antioxidant produced endogenously and plays key roles in biological systems. It is vitally important to design simple, selective and sensitive methods to sense GSH and monitor changes of GSH concentration. In this work, the cathodic electrochemiluminescence (ECL) of lucigenin in the presence of MnO2 nanosheets at glassy carbon electrode was utilized for GSH detection. GSH can reduce MnO2 nanosheets into Mn2+ which can obviously inhibit the ECL of lucigenin. The ECL inhibition efficiencies increase linearly with the concentrations of glutathione in the range of 10 to 2000 nM. The detection limit for GSH measurement is 3.7 nM. This proposed method is highly sensitive, selective, simple, fast, and cost-effective. Moreover, this approach can detect GSH in human serum samples with excellent recoveries, which indicates its promising application under physiological conditions.

Glutathione (GSH, L-γ-glutamyl-L-cysteinyl-glycine) is the most wide-spread nonprotein thiol species in living being system. It is a crucial endogenous antioxidant and plays key roles in biological systems for defense against toxins and free radicals.1 It is present almost exclusively in its reduced form under normal conditions. Upon oxidation, GSH can be transformed into glutathione disulfide (GSSG), the oxidized form of GSH. Therefore, pathophysiologic conditions causing oxidative stress can lead to an increase in GSSG concentrations. Abnormal levels of GSH or GSH/GSSG ratio are correlated with many clinical diseases, such as Alzheimer’s disease, Parkinson’s disease, liver damage, diabetes, epilepsy, atherosclerosis, arthritis, aging, and numerous types of cancers.2 Thus, it is critically important to develop simple, rapid and sensitive methods for sensing GSH and monitoring changes of GSH concentrations. Up to now, some methods have been proposed for the detection of GSH, including fluorescence spectroscopy,3-7 colorimetry,8 electrochemistry,2,9 surface-enhanced Raman scattering (SERS)10 and electrochemiluminescence.11,12 The fluorescent methods require the special synthesis of fluorescent materials and the use of external light sources. Colorimetric methods suffer from low sensitivity. The reported electrochemical methods usually need complex and tedious electrode modifications and also have low sensitivity. SERS detection requires special equipment and professional operation. Therefore, the development of simple and sensitive GSH detection methods is still highly desirable. Electrochemiluminescence (ECL) is chemiluminescence resulting from electrochemical reactions, which combines the advantages of chemiluminescent analysis with electrochemical analysis.13-15 It has attracted much attention in sensing and detecting trace amounts of samples.16,17 Although some ECL

methods for GSH detection have been reported, they usually need complicated interface modification procedures, such as electrode modification and special preparation of ECL luminophores. 18-21 Lucigenin (N,N’-dimethylbiacridinium dinitrate) has been known as a chemiluminescence (CL) luminophore since 193522 and its ECL behavior also has been studied well.23-27 Compared with other ECL luminophores, lucigenin could generate ECL signals in basic environment without the need of additional coreactants such as H2O2, tripropylamine (TPA) and S2O82− (the famous coreactants used in luminol and Ru(II) complex ECL systems). Moreover, lucigenin can emit bight light at low concentration and has high luminous efficiency, which means that it can be used in sensitive analysis. Manganese dioxide (MnO2) nanomaterials are known by its rich electrical and catalytic properties.28-31 In 2011, Deng et al. reported a new strategy to detect GSH by using MnO2nanosheet-modified upconversion nanoparticles.3 GSH can reduce MnO2 nanosheets and decrease the absorption of upconversion fluorescence by MnO2 nanosheets, and thus can be detected by enhancing fluorescence. During these few years, there were also some papers reported MnO2 nanosheets-based glutathione sensing methods using fluorescent carbon quantum dots5,7 and g-C3N4 nanosheets32. It is worth to note that these are all turn-on fluorescence probes. However, the limited quenching capability of MnO2 nanosheets and the deficiency in fluorescence resonance energy transfer between MnO2 nanosheets and fluorescent materials led to the low sensitivity of these methods. In addition, fluorescence detection needs external light source.

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In this study, the cathodic ECL of lucigenin at glassy carbon electrode was developed for GSH detection in the presence of MnO2 nanosheets. To the best of our knowledge, it is the first time that MnO2 nanosheets were applied in ECL. The schematic principle of the ECL inhibition method for GSH detection is shown in scheme 1. MnO2 nanosheets have little effect on the ECL of lucigenin when its concentration is low. GSH can reduce the MnO2 nanosheets to Mn2+, and remarkably inhibit the ECL of lucigenin, enabling the ultrasensitive detection of GSH. The method is label-free, does not need electrode modification, and fast. Besides, this method is able to sensitively detect GSH (including reduced GSH and total GSH) in biological systems.

EXPERIMENTAL SECTION Chemicals. Lucigenin, 2-(N-morpholino) ethanesulfonic acid (MES), and GSH (reduced form) were bought from TCI (Shanghai, China), J&K Scientific Ltd. (Beijing, China) and Aladdin. FeCl3, CaCl2, MgSO4 and Zn(NO3)2 were gotten from Beijing Chemical Reagent Company (Beijing, China). LHistidine dihydrochloride (His), L-Valine (Val), L-proline (Pro), L-lysine (Lys), DL-Alanine (Ala), L-Glutamic Acid (Glu) were bought from Shanghai Yuanju Biotechnology Co. Ltd. (Shanghai, China). Other chemicals were supplied by Sinopharm Chemical Reagent Co. Ltd. (Beijing, China). Lucigenin stock solution (1.0 mM) was prepared by dissolving 0.051 g lucigenin in water and diluted to 100 mL. All the other reagents were used without further purification. Doubly distilled water was used throughout all experiments. Apparatus. CHI 800B potentiostat (Shanghai, China) was employed for electrochemical experiments. All experiments were carried out with a homemade three-electrode cell. The glassy carbon electrode (GCE), the working electrode, with a diameter of 3 mm was polished by alumina and washed with distilled water before each measurement. A gold spiral wire and an Ag/AgCl (filled with saturated KCl) were used as the counter and reference electrode, respectively. ECL intensities were detected using a BPCL ultraweak luminescence analyzer (Institute of Biophysics, Chinese Academic of Sciences). Unless specifically mentioned, the photomultiplier tube (PMT) voltage was set at 1100 V. Ultraviolet−visible (UV−vis) absorption spectra were measured with a UNICO UV/vis 2802PC spectrophotometer. Transmission electron microscopy (TEM) images were obtained using a FEI Tecnai G2 F20 microscope operated at 200 kV. Scheme 1. Schematic illustration for measuring GSH using lucigenin and MnO2 nanosheets.

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ously with a minor modification.3,7 In a typical reaction, 500 µL of KMnO4 (10.0 mM) solution was added to 1.25 mL of 0.1 M pH 6.0 MES buffer solution. The final volume was adjusted to 5 mL by water. Then, the mixture was sonicated for 30 min. Subsequently, the MnO2 nanosheets were obtained by centrifugating at 12,000 rpm for 10 min and washed with water for three times to remove the excess potassium and free manganese ions. The as-prepared MnO2 nanosheets were redispersed in 5 mL doubly distilled water for the following studies. The concentration of MnO2 nanosheets in this solution can be seen as 1.0 mM, supposing the reaction completely reacted. Before ECL measurements, the MnO2 nanosheets solutions were diluted by water as needed and well dispersed by ultrasonication. ECL measurements of lucigenin. 100 µL of 1.0 mM lucigenin solution and 200 µL of doubly distilled water were pipetted into 700 µL of 0.1 M pH 10.0 phosphate buffer solutions (PBS), vortex-mixed and used for ECL measurements. The electrochemical scanning range is from 0.2 V to −1.0 V and then back to 0.2 V with a scan rate of 0.1 V/s. The ECL emission spectrum of lucigenin was measured by using various band pass filters at wavelengths of 425 nm, 440 nm, 460 nm, 490 nm, 535 nm, 555 nm, 575 nm and 620 nm. Procedures for GSH detection. Typically, 100 µL of different concentrations of GSH were pipetted into 100 µL of 200.0 µM MnO2 solutions, vortex-mixed and reacted at room temperature for 3 min. Then 700 µL of 0.1 M pH 10.0 PBS and 100 µL of 1.0 mM lucigenin solution were added into the above premixed solution, vortex-mixed and used for ECL measurements. Sensing of GSH in human serum samples. For GSH assay, 10 µL of 100-fold diluted fresh human serum sample, 90 µL of water and 100 µL of 200.0 µM MnO2 solutions were thoroughly mixed and reacted at room temperature for 3 min. Then 700 µL of 0.1 M pH 10.0 PBS and 100 µL of 1.0 mM lucigenin solution were added into the above premixed solution, vortex-mixed and used for ECL measurements. To measure recoveries, a given amount of GSH was mixed with the sample solutions and 100 µL of 200.0 µM MnO2 solutions first. ECL measurements were carried out in a similar way as mentioned above. For measurement of total GSH (tGSH, including GSH and GSSG), GSSG in the serum samples was first transformed to GSH. It was accomplished by treating 250 µL of the serum sample with 250 µL of acetic acid (1.2 M) and 20 mg of zinc powder for one hour.33 After that the resulting solution was centrifuged at 10000 rpm for 5 min and then the supernatant was diluted with water. To test recoveries, the samples were spiked with appropriate amounts of GSH. The experiments were all carried out in triplicate.

RESULTS AND DISCUSSION Synthesis and Characterization of MnO2 nanosheets. The MnO2 nanosheets were prepared according to previously reported methods with minor modifications.7 Figure 1 shows the TEM image of MnO2 nanosheets. It clearly confirms the successful synthesis of MnO2 nanosheets. Figure 2 shows that the UV-Vis absorption peak of MnO2 nanosheets is similar to that reported in the literature.3,7 Synthesis of MnO2 nanosheets. MnO2 nanosheets were synthesized according to a literature procedure reported previ-

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Analytical Chemistry crease in reduction current peak at −0.37 V and does not lead to the disappearance of reduction current peak at −0.37 V. Moreover, no reduction current peak is observed at −0.37 V in oxygen-free phosphate buffer solution (curve 7 in Figure 3B). These results show that the strong electrochemical irreversible reduction current peak appears at −0.37 V is attributed to the reduction of both O2 and lucigenin.23,34 The addition of GSH, MnO2 nanosheets, and their mixture, as well as Mn2+ has little effect on the reduction current peak. Therefore, the inhibition effect of Mn2+ on lucigenin ECL can be attributed to the reactive oxygen species scavenging property of Mn2+.35 Different from fluorescent detection based on fluorescence resonance energy transfer between MnO2 nanosheets and fluorescent materials, this ECL inhibition method relies on electrochemical reaction mechanism.

Figure 1. TEM image of MnO2 nanosheets.

2GSH + MnO2 + 2H+ → GS-SG + Mn2+ + 2H2O

(1)

Figure 2. UV-vis absorption spectrum of MnO2 nanosheets and lucigenin ECL emission spectrum. 0.1 M PBS, pH 10.0; c(lucigenin): 100.0 µM; c(MnO2): 200.0 µM.

Cathodic ECL of lucigenin for GSH detection. Figure 3A shows the ECL behaviors of lucigenin under different conditions. Lucigenin displays strong cathodic ECL intensity with a scanning range from 0.2 V to −1.0 V (curve 1). An intense ECL peak appears at −0.48 V in the cathodic potential scan and a much weaker peak at −0.32 V appeared in the backward scan. The effect of GSH on lucigenin ECL is negligible (curve 2). The ECL intensity of lucigenin decreased a little in the presence of MnO2 nanosheets (curve 3). It results from the fact that the absorption spectrum of MnO2 nanosheets has an overlap with the ECL emission spectrum of lucigenin. As shown in figure 2, the MnO2 nanosheets exhibit a broad UV-Vis absorption peak from 250 nm to 550 nm, while the maximum ECL emission spectrum of lucigenin appears near 500 nm. It means that an energy transfer occurs between lucigenin and MnO2 nanosheets. When the mixture of GSH and MnO2 nanosheets was added into lucigenin solution, the ECL intensity decreased significantly (curve 5). To further explain the ECL inhibition principle of GSH, the ECL behavior of lucigenin in the presence of Mn2+ was investigated. As shown in figure 3A (curve 4), the ECL signal was inhibited obviously by Mn2+ (figure 3A). These results suggest that the inhibition of ECL caused by GSH is attributed to the production of Mn2+ from the reduction of MnO2 nanosheets by GSH. This redox reaction between GSH and MnO2 nanosheets is shown in eq.1.3 The electrochemistry of lucigenin under various conditions was also investigated to reveal the quenching mechanism. Figure 3B shows the corresponding cyclic voltammograms (CVs). A strong electrochemical irreversible reduction current peak appears at −0.37 V in the presence of dissolved oxygen. The comparison of curve 1 and curve 6 in Figure 3B indicates that the removal of oxygen in lucigenin solution results in the de-

Figure 3. (A) The ECL intensity−potential curves and (B) the corresponding CVs at the glassy carbon electrode of lucigenin (curve 1), lucigenin and GSH (curve 2), lucigenin and MnO2 (curve 3), lucigenin and Mn2+ (curve 4), lucigenin and the mixture of MnO2 and GSH (curve 5), lucigenin in the absence of oxygen (curve 6), PBS in the absence of oxygen (curve 7). 0.1 M PBS, pH 10.0; c(lucigenin): 100.0 µM; c(GSH): 10.0 µM; c(MnO2): 20.0 µM; c(Mn2+): 10.0 µM.

Optimization of experimental conditions. Figure 4 indicates the influence of pH on the ECL inhibition efficiency. I is the ECL intensity in the presence of GSH, I0 is the ECL intensity in the absence of GSH and (I0 − I)/I0 is ECL inhibition efficiency. The ECL inhibition efficiency enhanced as the pH increase from 8.4 to 10.0, and then reduced as pH increase further. The background ECL intensities of lucigenin in the presence of MnO2 nanosheets increase greatly with increasing pH. When pH is higher than 10.0, Mn2+ produced by the reduction of MnO2 nanosheets with GSH may hydrolyze to produce Mn(OH)2, decreasing ECL inhibition efficiencies. Thus, a pH of 10.0 was used in the following experiments.

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the optimum concentration of MnO2 nanosheets in GSH sensing experiments. The reaction between GSH and MnO2 nanosheets is a timedependent process. As shown in Figure 6, the ECL intensities decreased quickly and then leveled off after two minutes. These phenomena indicate that GSH reacted with MnO2 nanosheets rapidly and the ECL inhibition method for GSH detection is fast. A reaction time of 3 minutes was employed for subsequent ECL measurements to ensure good sensitivity and reproducibility.

Figure 4. pH effects on the ECL inhibition efficiencies. 0.1 M PBS; c(lucigenin): 100.0 µM; c(GSH): 10.0 µM; c(MnO2): 10.0 µM; Voltage of PMT: 900 V.

Figure 6. The dependence of ECL intensities on reaction times between MnO2 nanosheets and GSH (immediately, 10, 20, 30, 60, 120, 180, 240, 300 and 600 seconds). 0.1 M PBS, pH 10.0; c(lucigenin): 100.0 µM; c(GSH): 10.0 µM; c(MnO2): 20.0 µM.

Figure 5. (A) ECL intensity at different concentrations of MnO2 nanosheets without (black columns) and with (red columns) GSH. (B) The dependence of ECL inhibition efficiencies on the concentrations of MnO2 nanosheets. 0.1 M PBS, pH 10.0; c(lucigenin): 100.0 µM; c(GSH): 10.0 µM.

Figure 5A presents the effects of MnO2 concentrations. The ECL intensities decrease as the concentrations of MnO2 nanosheets increase. This is attributed to the fact that the absorption spectrum of MnO2 nanosheets has an overlap with the ECL emission spectrum of lucigenin (figure 2). As shown in Figure 5B, the ECL inhibition efficiency increases with increasing MnO2 concentration to 20.0 µM, and then changes slightly when MnO2 concentration is higher than 20.0 µM. Based on the detection principle, the ECL inhibition efficiency depends on the amount of Mn2+ produced from the reduction of MnO2 nanosheets by GSH. When the concentrations of MnO2 nanosheets are much lower than GSH, the ECL inhibition efficiencies increase with the increase of MnO2 concentrations because of the generation of more Mn2+. Excess MnO2 nanosheets cannot be reduced, resulting in the level-off of ECL inhibition efficiency. Therefore, we choose 20.0 µM as

Figure 7. The ECL intensity–potential curves at various GSH concentrations (A) and GSH calibration curve (B). 0.1 M PBS, pH 10.0; c(lucigenin): 100.0 µM; c(MnO2): 20.0 µM.

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Analytical Chemistry

Table 1. Comparison of diff fferent methods for GSH sensing. ff Methods

probes

Linear range

LOD

Ref.

Fluorescence

BODIPY derivatives

Fluorescence

MnO2-Nanosheet-Modified Upconversion Nanoparticles

0−60 µM

86 nM

6

−−−

0.9 µM

3

Fluorescence

CdS nanoparticles

2−25 µM

−−−

4

Fluorescence

MnO2-CQDs nanocomposites

0−250 µM

300 nM

7

Colorimetry

Naphthalimide-capped AuNPs

0.025−2.28 µM

17 nM

8

SERS

γ-Fe2O3–Au

1pM−10 µM

1 pM

10

Electrochemistry

Nanoscale Copper Hydroxide Composite Carbon Ionic Liquid Electrode

1−50 µM and 0.1−1.8 mM

30 nM

9

ECL

graphene oxide amplified ECL of CdTe quantum dots (QDs)

24−214 µM

8.3 µM

12

ECL

ferrocyanide-ferricyanide redox couple (Fe(CN)63–/4–) induced ECL amplification of carbon dots (C-dots)

0.1−1.0 µM

54.3 nM

11

ECL

core-shell CdSe/ZnS quantum dots/Nafion composite films

10−180 µM

1.5 µM

20

ECL

Lucigenin, MnO2 nanosheets

0.01−2.0 µM

3.7 nM

This work

Detection of GSH. Figure 7 shows the ECL intensity– potential curves at various concentrations of GSH and GSH calibration curve. A good linear relationship between the ECL inhibition efficiency and the logarithm of concentration of GSH (log c) is obtained from 0.01 to 2.0 µM with a limit of detection (LOD) of 3.7 nM at a signal-to-noise ratio of 3. The linear equation is (I0 − I) /I0 = 0.35 + 0.17 log (c/µM) (R=0.998). We compared this proposed method with other reported GSH sensing methods in table 1. Our method is one of the most sensitive methods reported so far. Furthermore, this ECL inhibition method is much simple, fast and costeffective since the synthesis of MnO2 nanosheets is quite simple and the ECL measurement does not need expensive instruments and complicated operations. Compared with the reported MnO2 nanosheets-based fluorescent GSH sensors,3,5 our method does not need external light sources and use commercial available lucigenin as luminophore, which eliminate the complex synthesis of fluorescent materials.

Selectivity of the ECL inhibition method for GSH detection. To test the selectivity of this GSH detection method, the ECL inhibition efficiencies of some ions and biomolecules were measured. As shown in figure 8, the ECL inhibition efficiency of GSH is much higher than that of other amino acids, ions, and glucose. Although high concentrations of cysteine (Cys) and reducing agents (i.e. Vc) also can cause ECL decreasing, their concentrations (µM levels) is remarkably lower than the concentrations of GSH (mM levels) in biological systems.7,36 Thus, this method could be applied for selective GSH detection. GSH detection in human serum samples. The performance of this sensing approach in biological environments was investigated by detecting GSH in fresh human serum. The fresh human serum was diluted so that the GSH concentrations are in the linear range of this assay. To detect recoveries, a given amount of GSH was spiked into the serum samples. The concentration of total GSH in serum samples was measured after the reduction of GSSG to GSH using zinc powder. The corresponding recoveries were also carried out by spiking given amount of GSH (table 2). These satisfactory recoveries indicate that this proposed approach is feasible for measuring GSH in biology systems and the results can be used to reflect the biological state to some extent. Table 2. GSH detection in human serum samples.

Figure 8. The selectivity of this ECL inhibition method for GSH detection against some metal ions and biomolecules. FeCl3, CaCl2, KCl, MgSO4, Zn(NO3)2, glucose, sucrose, His, Val, Pro, Lys, Ala and Glu: 100.0 µM; cysteine (Cys) and vitamine C (Vc): 1.0 µM; GSH: 10.0 µM. 0.1 M PBS, pH 10.0; c(lucigenin): 100.0 µM; c(MnO2): 20.0 µM.

Target

Found in sample (µM)

Spiked (µM)

Total found (µM)

RSD (%, n = 3)

Recovery (%, n = 3)

GSH

0.112

0.5 1.0

0.598 1.126

4.6 3.2

97.2 101.4

tGSH

0.131

0.5 1.0

0.620 1.212

2.7 3.9

97.8 108.1

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CONCLUSIONS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

In conclusion, we developed a new ECL method for GSH detection using lucigenin as ECL luminophore and MnO2 nanosheets as mediator. GSH could reduce MnO2 nanosheets into Mn2+, and obviously inhibit the ECL of lucigenin. The ECL inhibition efficiencies increase linearly with the concentrations of GSH. This proposed approach shows a wide dynamic range and a high sensitivity for GSH. Meanwhile, satisfactory detection of GSH in human serum samples was observed, which indicates its promising application in biological environments. Owing to the unique properties of lucigenin, this ECL GSH detection method is quite simple, rapid, costeffective and environmental friendly. Besides, as far as we know this is the first time to use MnO2 nanosheets in ECL detection technique, and it will stimulate the development of other ECL detection platforms.

AUTHOR INFORMATION Corresponding Author * Guobao Xu. Address: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street Changchun, Jilin 130022, P.R. China. Tel: +86-431-85262747. Fax: +86-43185262747. E-mail: [email protected].

ACKNOWLEDGMENT

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We thanks the support from National Natural Science Foundation of China (No. 21475123 and 21505128), and the Chinese Academy of Sciences (CAS)-the Academy of Sciences for the Developing World (TWAS) President’s Fellowship Programme (2013053).

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ACS Paragon Plus Environment

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Analytical Chemistry

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7 ACS Paragon Plus Environment