Peroxidase-like Nanozyme Composite-based Colorimetric Determination of Glutathione and Cysteine

Abstract Simple, low-cost, and fast determination of cysteine and glutathione with high sensitivity is an essential problem to evaluate the physiological processes in living organisms. Artificial nanozymes have provided considerable opportunities in biosensing. In this work, using a graphene-based ternary nanozyme, we present a simple but efficient colorimetric method for the determination of cysteine and glutathione. The nanozyme exhibits excellent and stable catalytic performance toward H2O2 and catalyzes the 3,3′,5,5′-tetramethylbenzidine chromogenic reaction. However, this reaction is significantly suppressed by cysteine and glutathione. Thus, a sensitive colorimetric method is presented based upon this inhibition. Excellent performance with low detection limits, good selectivity, and rapid analysis suggests potential bioanalytical applications for the determination of cysteine and glutathione.


Introduction
Glutathione (GSH) and cysteine (Cys) are common endogenous antioxidants, which play crucial roles in maintaining the intracellular signal transduction and redox status, and thus are involved in physiological processes in living organism (Meister 1988;Finkel and Holbrook 2000).They are known to be important biomarkers.Glutathione and cysteine at deficient levels are closely associated with chronic diseases, such as slowed growth, skin lesions, edema, heart problems, cancer, and neurodegenerative disorders (Hwang, Sinskey, and Lodish 1992;Shahrokhian 2001;Rossi et al. 2002).
Currently reported sensing methods for these analytes include performance liquid chromatography (HPLC) (Vacek et al. 2006), electrochemistry (Rajaram and Mathiyarasu 2018), fluorescence (Ming et al. 2017;Cao et al. 2019), photoelectrochemistry and surface enhanced Raman scattering (SERS) (Saha and Jana 2013;Wei et al. 2018) which provide satisfactory sensitivity and selectivity in clinical analysis.Nevertheless, these methods need expensive instruments with complex operation and professional operators.Thus, they generally do not provide rapid and convenient analysis.
Colorimetric methods exhibit simple, practicability, low-cost, and fast performance for cysteine and GSH with high sensitivity (Liu et al. 2013a;Xianyu et al. 2015;Ganganboina and Doong 2018;Zhong et al. 2018;Zhou et al. 2022;Wang et al. 2021).The common colorimetric strategies employ natural enzymes, such as horseradish peroxidase (HRP).However, the catalytic activity of enzymes is easily affected by temperature and pH.In addition, the rigorous storage requirements and low stability of natural enzymes may further degrade the assay.Therefore, artificial enzyme-based colorimetric sensors have been developed in recent years.
A chiral covalent organic framework (COF) nanozyme with ultrahigh enzymatic activity and high selectivity was reported for dopa enantiomers (Zhou et al. 2020a).A polymer MoS 2 @CoFe 2 O 4 was employed to interact with H 2 O 2 and TMB for the determination of cysteine and glutathione (Xian et al. 2021).A Au@MnO 2 core-shell composite with tetramethylbenzidine (TMB) was used to determine intracellular glutathione by surface enhanced Raman scattering (Wang et al. 2022).Hemin-modified graphene quantum dots were employed for the colorimetric determination of glutathione (Li et al. 2022).However, complex syntheses of the nanozyme composites and long analysis reaction times have limited their practical applications.
In our previous work, a ternary graphene@Hemin@Au nanoflower nanozyme was shown to provide enhanced catalysis for H 2 O 2 (Liu et al. 2016).Biothiols are wellknown reducing agents for oxidized tetramethylbenzidine with a reduction in the blue color and the absorbance at 652 nm (Liu et al. 2013b;Lin et al. 2019).Here is reported a simple colorimetric method for the determination of glutathione (GSH) and cysteine (Cys), as shown in Scheme 1, based upon the inhibition of the 3,3 0 ,5,5 0 -tetramethylbenzidine chromogenic reaction in the presence of H 2 O 2 .The method displays good detection limits of 0.01 mM for glutathione and cysteine.This reported method is rapid, efficient, and robust for the determination of these analytes in biological samples.

Apparatus
All colorimetric experiments were carried out using a Cary 60 spectrophotometer (Agilent Technologies, USA).Transmission electron microscopy (TEM) was conducted by a FEI TalosF200S (Thermo Scientific) at an accelerating voltage of 200 kV equipped with energy-dispersive X-ray analysis (EDX).

Preparation of H-RGO-AuNFs
The first step is the synthesis of hemin-reduced graphene oxide (H-RGO).10 mL of GO dispersion were diluted with 40 mL of ultrapure water and sonicated.Approximately 60 lL of aqueous ammonia, 10 mg hemin, and 10 lL hydrazine solution were added in sequence and heated to 60 C in a water bath for 4 h to obtain H-RGO.
The next step is the synthesis of H-RGO@Au nanoflowers (H-RGO-AuNFs).For the preparation of the seed solution, HAuCl 4 (0.12 mM, 1 mL) and CTAB (0.048 M, 1 mL) were mixed at 25 C. 0.12 mL of 2.4 mM NaBH 4 in ice water were added, vigorously stirred, and reacted at 27 C for 2 h.For the growth solution preparation, 50 mL of 0.25 mM HAuCl 4 at pH 11.0, 2 mL of H-RGO, and 50.7 mL of 40 mM NH 2 OHÁHCl were added into 3.3 mL of seed solution with gently shaking at 25 C.The color of the solution changed from pink to blue green.The product was centrifuged at 8500 in Scheme 1. Synthesis of the H-RGO@Au nanoflower (H-RGO-AuNFs) nanozyme for the colorimetric determination of glutathione and cysteine.Definitions: H-RGO, hemin reduced graphene oxide; AuNFs, gold nanoflowers, and TMB, tetramethylbenzidine.
ultrapure water to remove the supernatant, and the precipitate was dispersed in water to obtain the H-RGO-AuNFs.

GSH/Cys determination
1 mM glutathione/Cys stock solution was prepared in phosphate buffer saline (PBS) and serially diluted to prepare working standards.The determination of glutathione and cysteine was performed as follows. 1 mL of H-RGO-AuNFs in NaH 2 PO 4 -Na 2 HPO 4 buffer at pH 7.0; 1.5 mL of 1 mM TMB, H 2 O 2 , 0.1 M citrate, and Na 2 HPO 4 ; and 500 lL of aqueous glutathione/cysteine solution were combined and incubated at 25 C for 10 min.The absorbance spectra were collected from 500 nm to 800 nm at 25 C.The absorbance value at 652 nm was used for the determination of glutathione and cysteine.

Optimization of conditions
The analytical conditions were optimized, including the pH and temperature.1 mL of H-RGO-AuNFs in NaH 2 PO 4 -Na 2 HPO 4 buffer from pH 5.0 to 9.0 were treated with 1.5 mL TMB solution and 500 lL glutathione/cysteine and the absorbance at 652 nm was monitored.
Temperature also plays a crucial role in catalytic reactions.1 mL of H-RGO-Au NFs in NaH 2 PO 4 -Na 2 HPO 4 buffer at pH 7.0, 1.5 mL TMB, and 500 lL glutathione/cysteine were reacted at various temperatures for 10 min.The absorbance at 652 nm was monitored.

Stability and selectivity
The stability of the biosensor was monitored for a period of two months.In order to characterize the selectivity, serine (Ser), arginine (Arg), lysine (Lys), histidine (His), threonine (Thr), glycine (Gly), and alanine (Ala) were introduced at concentrations higher than glutathione/cysteine concentrations employed in previous experiments.

Characterization of H-RGO-Au NFs nanozyme
Transmission electron microscopy (TEM) was used to characterize the morphology of the H-RGO-Au NFs composite.Figure 1(A) shows graphene with a uniform monolayer structure.The introduced gold nanoparticles were flower-shaped with an average size of 100 nm (Figure 1B,C).The elemental mapping images show Au and C were homogeneously distributed on the surface (Figure 1D-F).
The elemental composition of the H-RGO-Au NFs was characterized by energy-dispersive X-ray spectroscopy (EDX), as shown in Figure 1(G).The absorption spectra in Figure 1(H) characterize the synthesis of the H-RGO-Au NFs.The unmodified GO has weak peaks at 233 nm and 300 nm due to the p-p Ã transition of C ¼ C and n-p Ã transition of C ¼ O. H-RGO has a maximum at 420 nm due to the Soret band of hemin's p-p stacking on graphene.This peak shifted to 400 nm because of the attachment of the Au NFs to graphene.

Activity of H-RGO-Au NFs
The H-RGO-Au NFs catalyzed the oxidation of the peroxidase substrate (TMB) to produce a colored product (ox-TMB) in the presence of H 2 O 2 .The reaction kinetics were studied using the H 2 O 2 -mediated oxidation of TMB.Experiments were designed to characterize the time-dependent absorbance changes at 652 nm using a fixed quantity of one substrate and varying the other.The Michaelis-Menten results are shown in Figure S1(A,B).Lineweaver-Burk plots were prepared based on these results, as shown in Figure S1(C,D).The Michaelis-Menten constant K m and maximum reaction velocity V max of H-RGO-Au NFs were 0.15 mM À1 and 7.26 Â 10 À8 molÁs À1 for H 2 O 2 as the substrate and 0.66 mM À1 and 12.68 Â 10 À8 molÁs À1 for TMB.The K m and V max values of the GO control were 0.29 mM À1 and 0.14 Â 10 À8 molÁs À1 using H 2 O 2 and 0.58 mM À1 and 0.11 Â 10 À8 molÁs À1 with TMB.
The high catalytic activity was due to the high RGO surface-to-volume ratio that efficiently adsorbed the substrates.The active sites of the gold nanoparticles and hemin on the graphene sheets directly reacted with the substrates in the nanoscale region to enhance the catalytic activity.Thus, the prepared composite provided higher peroxidase-like activity due to synergistic catalyst interactions.

Mechanism and optimization of conditions for GSH and Cys
In the presence of H 2 O 2 , Au NFs catalyzed the oxidation of TMB with a blue color and strong absorbance at 652 nm.However, after introducing glutathione or cysteine into the AuNFs-H 2 O 2 -TMB system, the absorbance at 652 nm was reduced, and hence the blue color of the solution faded.Further studies showed that the discoloration was irreversible perhaps due to the decreased quantity of ox-TMB induced by the addition of glutathione or cysteine.These results confirm that both analytes may be determined by this sensing strategy (Figure 2A). Figure 2(B) shows the change in the absorbance during the reaction that reached a plateau in 60 s. 10 min was deemed to be optimum based upon the reaction stability.
The pH and reaction temperature were optimized for the biosensor.The pH was varied using NaH 2 PO 4 -Na 2 HPO 4 buffers from pH 5.0 to 9.0.Figure 3(A,C) shows the maximum absorbance was at pH 7.0 for both analytes and employed in subsequent experiments.The influence of temperature from 20 to 60 C was also evaluated.The maximum absorbance was observed at 30 C for both analytes (Figure 3C,D).Accordingly, 30 C was deemed to be the optimal temperature for the determination of cysteine and glutathione.

Determination of cysteine and glutathione
The performance of the biosensor for cysteine and glutathione was evaluated using the optimized conditions by monitoring the absorbance at 652 nm. Figure S2(A) shows the absorbance at 652 nm decreased with the cysteine concentration up to 0.1 mM.The change in absorbance (DA) at 652 nm was linear with the millimolar cysteine concentration from 0.01 to 0.1 mM (Figure S2B) described by DA ¼ 4.29c þ 0.23 (R 2 equal to 0.94).A similar linear relationship was obtained at 652 nm for glutathione from 0.01 to 0.2 mM described by DA ¼ 4.44c þ 0.26 (R 2 equal to 0.95).The performance is better or comparable to literature fluorescence (Zhou et al. 2020b;Cao et al. 2023), nuclear magnetic resonance (Wei, Jiang, and Deng 2021), and other colorimetric procedures (Lin et al. 2019(Lin et al. , 2020)).
The catalytic activity of the Au NFs-H-RGO decreased with storage and maintained 72% of its activity after two months (Figure S3).The degradation of the catalytic activity of H-RGO-Au NFs may be caused by decreased electron transfer of graphene following storage.Hence, the active sites of the gold NPs and hemin on graphene may react with the substrates more slowly.
The selectivity of the colorimetric method was investigated in Figure 4 using the similar amino acids serine (Ser), arginine (Arg), lysine (Lys), histidine (His), threonine (Thr), glycine (Gly), and alanine (Ala) in excess compared to cysteine and GSH.The responses for cysteine and glutathione are significantly higher than for the other amino acids.These results indicated good selectivity.Hence, the reported procedure was simple, sensitive, and selective for the determination of glutathione and cysteine.

Conclusion
A H-RGO-Au NFs nanozyme with excellent catalytic activities was employed the for sensitive colorimetric quantification of cysteine and glutathione.The synthesized nanozyme catalyzed the 3,3 0 ,5,5 0 -tetramethylbenzidine reaction in the presence of H 2 O 2 .Further addition of cysteine and glutathione which serve as the reducing agent leads to a decrease in the absorbance at 652 nm that served as the analytical response.This phenomenon was employed to provide a colorimetric method for cysteine and glutathione with suitable sensitivity, selectivity, and stability.This developed procedure is anticipated to have practical bioanalytical applications.

Figure 1 .
Figure 1.Transmission electron microscopy images of (A) graphene oxide and the (B and C) H-RGO-Au NFs.(D) Element mapping of the H-RGO-Au NFs: (E) Au and (F) C. (G) EDX characterization of the H-RGO-Au NFs.(H) Absorption spectra of 0.5 mg mL À1 H-RGO-Au NFs, GO, and H-RGO.

Figure 2 .
Figure 2. (A) Absorbance of the H-RGO-AuNFs in tetramethylbenzidine treated with 0.1 mM phosphate buffer saline, glutathione, and cysteine.(B) Absorbance of H-RGO-AuNFs and tetramethylbenzidine as a function of time following treatment with 0.1 mM glutathione and cysteine.

Figure 3 .
Figure 3. (A) Absorbance of H-RGO-Au NFs for 0.1 mM cysteine as a function of pH.(B) Absorbance of H-RGO-Au NFs for 0.1 mM cysteine as a function of temperature.(C) Absorbance of H-RGO-Au NFs for 0.1 mM glutathione as a function of pH.(D) Absorbance of H-RGO-Au NFs for 0.1 mM glutathione as a function of temperature.