Accelerating the Peroxidase-Like Activity of Gold Nanoclusters at

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Accelerating the peroxidase-like activity of gold nanoclusters at neutral pH for colorimetric detection of heparin and heparinase activity Lianzhe Hu, Hong Liao, Lingyan Feng, Min Wang, and Wensheng Fu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00885 • Publication Date (Web): 26 Apr 2018 Downloaded from http://pubs.acs.org on April 27, 2018

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Analytical Chemistry

Accelerating the peroxidase-like activity of gold nanoclusters at neutral pH for colorimetric detection of heparin and heparinase activity Lianzhe Hu,* † Hong Liao,† Lingyan Feng,‡ Min Wang,§ Wensheng Fu*† †

Chongqing Key Laboratory of Green Synthesis and Applications, College of Chemistry, Chongqing Normal University, Chongqing 401331, China ‡

§

Materials Genome Institute, Shanghai University, 200444 Shanghai, China

School of Pharmaceutical Sciences, Chongqing University, Chongqing 401331, China *E-mail: [email protected]; [email protected] KEYWORDS: enzyme, nanozyme, metal nanocluster, biocatalyis, bioanalysis

ABSTRACT: The peroxidase-like catalytic activity of gold nanoclusters (NCs) is quite low around physiological pH, which greatly limits their biological applications. Herein, we found heparin can greatly accelerate the peroxidase-like activity of Au-NCs at neutral pH. The catalytic activity of Au-NCs toward the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation by H2O2 was 25-fold increased in the presence of heparin at pH 7. The addition of heparin not only accelerated the initial catalytic rate of Au-NCs, but also prevented the Au-NCs

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from catalyst deactivation. This allows the sensitive colorimetric detection of heparin at neutral pH. In the presence of heparinase, heparin was hydrolyzed into small fragments, weakening the enhancement effect of catalytic activity. Based on this phenomenon, the colorimetric determination of heparinase in the range from 0.1 µg⋅mL-1 to 3 µg⋅mL-1 was developed with a detection limit of 0.06 µg⋅mL-1. Finally, the detection of heparin and heparinase activity in diluted serum samples were also demonstrated.

INTRODUCTION

Natural enzymes are powerful biocatalysts and have been widely used in the areas of clinical medicine, bioengineering, agriculture and food industry, and environmental protection.1 Nevertheless, natural enzymes have several inherent defects, such as high cost in purification, sensitivity of catalytic activity to environmental conditions, and low stability due to denaturation. As alternatives to natural enzymes, artificial enzyme mimics have attracted great attentions.2-4 In recent years, nanomaterials with enzyme-like characteristics are emerging as a new kind of artificial enzymes.5-9 Various of nanomaterials, such as metal nanoparticles (NPs), metal oxides, and carbon nanomaterials, have been found to possess enzyme-like catalytic activities.10-12 These nanozymes are promising candidates in the field of biosensors, therapeutics, and pollutant removal.13-16 However, the catalytic activity of most nanozymes is lower than that of natural enzymes. Extensive efforts have been paid to improve the catalytic activity of nanozymes.17-20 For example, the peroxidase-like activity of Fe3O4 NPs was improved by coating with ligands of different charges, and surface charge was found to be vital for the peroxidase activity;21 the oxidase-like activity of nanoceria was significantly increased after the addition of fluoride, as the


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adsorption of highly electronegative fluoride can reduce surface energy of nanoceria and facilitate the electron transfer.22 Au nanoclusters (NCs) are ultra-small NPs consisting of several to tens of Au atoms.23-25 Because of the strong quantum confinement effects, Au-NCs show discrete electronic states and exhibit size-dependent fluorescence. Fluorescent Au-NCs have shown promising applications in the fields of biological analysis, imaging, and nanomedicine.26-28 Compared with their optical properties, the studies on the catalytic properties of Au-NCs are relatively rare. It has been reported that bovine serum albumin (BSA)-stabilized Au-NCs possess intrinsic peroxidase-like activity.29 Similar with most of the peroxidase-like nanozymes, the optimum catalytic activity of Au-NCs occurs in strong acidic solution (around pH 4), which limits their biological applications. Heparin is a highly sulfated glycosaminoglycan and widely used as an anticoagulant. Herein, we found the peroxidase-like activity of BSA-stabilized Au-NCs at neutral pH can be significantly accelerated by the addition of heparin. The catalytic activity of Au-NCs toward the oxidation of peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) by H2O2 is very low at pH 7. However, the catalytic activity is dramatically accelerated after the addition of heparin to the system. Heparin can be adsorbed onto the surface of Au-NCs through the interaction with BSA. The negative charge of heparin can facilitate the adsorption of TMB, which is attributed to the main reason of the catalytic enhancement effect. In the presence of heparinase, heparin was hydrolyzed into small fragments that could not bind with Au-NCs effectively, thus resulting in the decrease of the catalytic activity enhancement (Scheme 1). Based on this findings, the sensitive and selective colorimetric detection of heparin and heparinase activity in biological samples was developed.


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EXPERIMENTAL SECTION

Materials. HAuCl4·3H2O, BSA, heparinase I from flavobacterium heparinum,

lysozyme,

glutathione (GSH), exonuclease I (ExoI), chondroitin sulfate (CS), and hyaluronic acid (HA) were purchased from Sigma. TMB, 30% H2O2 solution, 2,2′-azino-bis(3-ethylbenzothiazoline-6sulfonic acid) (ABTS), heparin sodium, trypsin, and xanthine oxidase (XOD) were obtained from Sinopharm Chemical Reagent Co., Ltd. All the solutions were prepared with ultrapure water purified by a Milli-Q system. Instrumentation.

Absorption

spectra

were

measured

using

a

UV2550

UV-Vis

Spectrophotometer (Shimadzu, Japan). Fluorescence measurements were carried out on the PerkinElmer LS55 Spectrophotometer. Transmission electron microscopy (TEM) images were obtained on the JEM-2100 transmission electron microscope (JEOL, Japan). Dynamic light scattering (DLS) and zeta potential measurements were carried out on a Zetasizer Nano ZS (Malvern Instruments, UK). Preparation of Au NCs. BSA-stabilized Au-NCs were synthesized following previous report.30 Typically, 5 mL HAuCl4 solution (10 mM, 37 ◦C) was mixed with 5 mL BSA solution (50 mg/mL, 37 ◦C) under vigorous stirring. After 5 min incubation, the pH of the solution was adjusted to pH 12 using 1 M NaOH solution. The mixture solution was incubated at 37 ◦C overnight. The obtained Au-NCs were further purified using ultrafiltration. Procedure for colorimetric assays. A typical colorimetric assay for heparin was carried out as follows. Firstly, 50 µL heparin with different concentrations were added into 450 µL 10 mM phosphate buffer (pH 7.0) containing 0.2 mM TMB, 100 mM H2O2, and 3 µM Au-NCs. Secondly, the mixed solutions were incubated for 30 min at room temperature. Finally, the solutions were used for absorption spectra measurements.


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For heparinase activity assay, heparinase with different concentrations were firstly incubated with heparin at 30 ◦C for 1 h. Then the solutions were added into the phosphate buffer (pH 7.0) containing 0.2 mM TMB, 100 mM H2O2, and 3 µM Au-NCs. After reaction at room temperature for 30 min, the absorption spectra were measured. Kinetic studies were carried out by monitoring the absorbance changes at 652 nm. The Michaelis-Menten constant was calculated using Lineweaver-Burk plots of the double reciprocal of the Michaelis-Menten equation. For the detection of heparin and heparinase activity in diluted fetal bovine serum samples, the fresh 10% fetal bovine serum samples were diluted 5 times with phosphate buffer before determinations.

RESULTS AND DISCUSSION

Scheme 1 Schematic illustration of the method for heparin and heparinase detection.

The fluorescence spectra of the Au-NCs are shown in Fig. S1. The Au-NCs showed a strong red emission upon being excited at 365 nm, demonstrating the successful preparation of the


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ultra-small fluorescent Au-NCs. The formation of Au-NCs was further confirmed by TEM data. As showed in Fig. S2, the Au-NCs are well-dispersed and the morphology of Au-NCs is consisted with previous report.30 As shown in Fig. 1A, the peroxidase-like catalytic activity of Au NCs at pH 7 is investigated with and without the addition of heparin. In the absence of heparin, the Au-NCs showed negligible catalytic activity toward the oxidation of TMB in the presence of H2O2 (curve b). Interestingly, the catalytic activity of Au-NCs was greatly accelerated after the addition of heparin into the mixture (curve c), and an obvious blue color of the solution was observed (Inset of Fig. 1A). The control experiment was carried out by the addition of heparin to the mixture of TMB and H2O2. Without of Au-NCs, heparin itself showed no catalytic activity toward the oxidation of TMB in the presence of H2O2 (curve d), and no typical blue color of the solution was observed. The kinetic curves of the peroxidase-like activity of Au-NCs with and without heparin are shown in Fig. 1B. In the absence of heparin, the Au-NCs showed a very slow catalytic rate at pH 7, and the Au-NCs were quickly deactivated at a reaction time of 10 min. In the presence of heparin, dramatic enhancement of the initial catalytic rate was observed. Surprisingly, the AuNCs were not deactivated until at a reaction time of 25 min (curve c in Fig. 1B, as the arrow indicated). This means that the addition of heparin not only can accelerate the initial catalytic rate of Au-NCs, but also can prevent the Au-NCs from catalytic poisoning. The pH dependence of the peroxidase-like activity of Au-NCs in the absence and presence of heparin was investigated (Fig. 2). In the absence of heparin, the Au-NCs showed negligible catalytic activity at neutral pH. In the presence of heparin, the catalytic activity of Au-NCs was increased over a broad pH range. Fig. 2B shows the absorbance ratio changes in the presence and


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absence of heparin with different pH values. At pH 7, the absorbance value increased 25-fold in the presence of heparin. The low background of peroxidase-like activity of Au-NCs at pH 7 will greatly facilitate the potential biological applications.

Fig. 1 Typical absorption spectra (A) and kinetic curves (B) of 200 µM TMB in the presence of (a) 100 mM H2O2, (b) 100 mM H2O2 and 3 µM Au-NCs, (c)100 mM H2O2, 3 µM Au-NCs, and 100 µg⋅mL-1 heparin, and (d) 100 mM H2O2 and 100 µg⋅mL-1 heparin. Inset: the corresponding images obtained at a reaction time of 30 min. The experiments were perform in 10 mM phosphate buffer (pH 7.0).


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The steady-state kinetics of the peroxidase-like activity of Au-NCs in the absence and presence of heparin at pH 7 were further investigated (Fig. S3), and the enzyme kinetic parameters were showed in Table S1. Typically, a lower Michaelis-Menten constant (Km) means a higher affinity between the enzyme and the substrate. With H2O2 as the substrate, the Km value of Au-NCs is higher in the presence of heparin, indicating that a higher H2O2 concentration is required to achieve maximal activity. With TMB as the substrate, the Km value of Au-NCs is lower in the presence of heparin, demonstrating the Au-NCs have a higher affinity toward TMB after the adsorption of heparin. The catalytic efficiency of Au-NCs, defined as Kcat/Km, was 15-fold increased in the presence of heparin with TMB as a substrate. However, the catalytic efficiency after the addition of heparin only showed slight increase with H2O2 as a substrate.


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Fig. 2 (A) Comparison of the peroxidase-like activity of Au-NCs in the absence and presence of heparin in different pH values. 200 µM TMB was used as peroxidase substrate. Error bars show the standard deviation of three replicate determinations; (B) The absorbance ratio changes of the catalytic system after and before the addition of heparin in different pH values. A represents the absorbance value in the presence of heparin, while A0 represents the absorbance value in the absence of heparin. Surface charge plays an important role in modulating substrate adsorption. TMB contains two amine groups, thus yielding strong affinity toward a negatively charged NP surface. Previous results indicate the negatively charged Fe3O4 NPs have a strong affinity and catalytic efficiency toward TMB.21 Heparin is known as the most negatively charged biological macromolecule. It can be adsorbed onto the surface of Au-NCs through the interaction between heparin and the BSA template. Previous studies indicate that heparin does not warp around BSA, but rather binds to some site on the protein that bears a positive charge.31,32 The surface charge of Au-NCs will be highly negative after the adsorption of heparin. The highly negatively charged Au-NCs will exhibit higher catalytic activity toward TMB due to the increased affinity by electrostatic attraction.21 To confirm this mechanism, the oxidation of TMB at different ionic strengths was monitored. As shown in Fig. S4, the catalytic activity of Au-NCs was greatly accelerated only at lower ionic concentration (10 mM PBS, 0 M NaCl). The Au-NCs exhibited similar catalytic activity toward TMB oxidation in the absence and presence of heparin at high ionic concentration (10 mM PBS, 1 M NaCl). The high ionic concentration shields the charge attraction and repulsion, resulting in the similar catalytic activity of Au-NCs with and without heparin. This further confirms that electrostatic interaction plays a key role in the catalytic enhancement effect at low ionic concentration condition.


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To further understand the mechanism of the catalysis enhancement, the peroxidase-like activity of Au-NCs with the negatively charged peroxidase substrate ABTS was investigated. ABTS contains two sulfonic acid groups, thus exhibiting stronger affinity toward a positively charged NP surface. As shown in Fig. S5, in the presence of heparin, the absorbance values were only increased slightly below pH 6. The peroxidase-like activity of Au-NCs was not increased at neural pH value. This further suggests the electrostatic attraction of TMB and the negatively charged Au-NCs is the main factor of the catalytic enhancement effect. The zeta potentials of Au-NCs before and after the addition of heparin were performed. As shown in Fig. S6, the apparent zeta potential of Au-NCs decreased from -5.18 mV to -16.20 mV. The considerable decrease in the zeta potential of Au-NCs further demonstrated the adsorption of the highly negatively charged heparin onto the surface of Au-NCs. The aggregation of the NPs might alter their peroxidase-like activity. The dynamic light scattering (DLS) data showed similar hydrodynamic diameters of Au-NCs before and after the addition of heparin (Fig. S7), indicating no aggregates of Au-NCs were formed in the presence of heparin without the addition of TMB and H2O2. To further investigate the catalytic mechanism, the time-dependent DLS and zeta potentials of Au-NCs in the catalysis process were also measured. In the presence of TMB and H2O2, the zeta potentials of Au-NCs increased with the increasing reaction time, indicating the adsorption of positively charged oxidation product of TMB on the surface of AuNCs (Fig. S8). As shown in Fig. S9, the hydrodynamic diameters of Au-NCs increased with the increasing reaction time, indicating the aggregation of Au-NCs during the catalytic process. Interestingly, the aggregation rate of Au-NCs was much slower in the presence of heparin. According to previous report, heparin can inhibit the aggregation of BSA.33 This may be the


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reason that heparin can prevent the Au-NCs from catalytic poisoning, as the aggregation could result in the deactivation of Au-NCs.

Fig. 3 (A) Absorption spectra of the TMB/H2O2/Au-NCs system in the presence of different heparin concentrations (from a to k: 0, 0.5, 1, 2, 2.5, 7.5, 15, 20, 25, 50, 100 µg⋅mL-1); (B) Plots of the absorbance of the system at 652 nm versus the heparin concentrations. Inset: linear plot of heparin, each point is the average of three measurements. The determination of heparin is of crucial significance because the overdose of heparin could induce adverse effects such as thrombocytopenia, hemorrhage, and osteoporosis.34 Based on above interesting phenomena, a sensitive colorimetric method for the detection of heparin was


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developed. Fig. 3 shows the absorbance of the mixture solution is increased with the increasing of the heparin concentrations. The absorbance of the solution is increased linearly in the heparin concentrations from 0.5 µg⋅mL-1 to 25 µg⋅mL-1. The detection limit of heparin at a signal-tonoise ratio of 3 is 0.3 µg⋅mL-1. The sensitivity of the colorimetric method is comparable to previous reports.35-42 The Au-NCs are widely used as fluorescent probes because of their bright emission and low toxicity. The fluorescence responses of Au-NCs toward heparin were also investigated. As shown in Fig. S10, the heparin has almost no effects on the fluorescence of AuNCs. This means heparin could not be detected directly by the fluorescence method. Therefore, the colorimetric method based on the peroxidase-like activity provides an alternative approach to extend the bioanalytical applications of fluorescent Au-NCs. To assess the selectivity of this colorimetric method for heparin determination, the effects of some inorganic anions and biological molecules on the peroxidase-like activity of Au-NCs are studied. As shown in Fig. 4, only heparin resulted a significant absorbance increase of the catalytic system. The results demonstrate these inorganic anions and biological molecules could not interfere the detection of heparin. Heparin has a much higher negative charge density than other species, and it can be easily adsorbed onto the surface of Au-NCs through the interaction with BSA. These unique characters should be the reason of the good selectivity of this method. To demonstrate the validity of the method in biological samples, the heparin concentrations in diluted fetal bovine serum were tested by the standard addition method. As shown in Table S2, the measured recoveries were in the range of 95.3-97.0% with RSD less than 3.42%. The results show this colorimetric has potential applications for the detection of heparin in real biological samples.


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Fig. 4 The absorbance value (A) and corresponding photograph (B) of the catalytic oxidation of TMB-H2O2 by Au-NCs in the presence of various anions and biomolecules. Concentrations: CA (citric acid), CO32-, Ac-, S2-, NO3-, GSH, ATP, Glu (glucose), 10 mM; BSA, CS, HA, Heparin, 100 µg⋅mL-1. Heparinase, a heparin-degrading enzyme, is implicated in various physiological and pathological functions including wound healing, tumor metastasis, and neovascularization.43 As shown in Fig. S11, the effect of heparinase on the peroxidase-like activity of Au-NCs was studied. In the absence of heparin, the peroxidase-like activity of Au-NCs was not influenced even by the addition of 10 µg⋅mL-1 heparinase. Heparinase can hydrolyze heparin into small fragments, and the resulted small fragments of heparin may not be able to bind BSA effectively. The peroxidase-like activity of Au-NCs was further studied by treating heparin with different concentrations of heparinase. As shown in Fig. 5, after treating the heparin with heparinase, the peroxidase-like activity of Au-NCs was weakened, and the absorbance values of the catalytic system were decreased. The results indicate the fragments of heparin can not accelerate the peroxidase-like activity of Au-NCs effectively as heparin itself. The phenomenon allows the


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colorimetric detection of heparinase activity with considerable sensitivity. There is a good linear relationship between the absorbance values and the concentrations of heparinase in the range of 0.1 µg⋅mL-1 to 3 µg⋅mL-1, and the detection limit of heparinase is 0.06 µg⋅mL-1 with a signal-tonoise ratio of 3. The comparison of the analytical performance of this method for heparinase determination with previous reports is shown in Table S3.

Fig. 5 (A) Absorption spectra of the TMB/H2O2/Au-NCs/heparin system in the presence different heparinase concentrations (from a to i: 0, 0.1, 0.2, 0.5, 1, 2, 3, 5, 10 µg⋅mL-1); (B) Plots of the absorbance of the system at 652 nm versus the heparinase concentrations. Inset: linear plot of heparinase. Each point is the average of three measurements.


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The absorbance responses of this system toward some other proteins were also measured. Fig. 6 shows that the addition of 10 µg⋅mL-1 heparinase resulted in 55% decrease of the absorbance. In contrast, the absorbance values showed almost negligible changes in the presence of 100 µg⋅mL-1 BSA, SOD, trypsin, lysozyme, XOD, or EXO I. These results suggest that this method has good selectivity toward the detection of heparinase activity. In order to demonstrate the practical applications of this method, the heparinase concentrations in diluted fetal bovine serum were also measured by standard addition method. As shown in Table S4, the recoveries are ranging from 93.3-96.7% with RSD less than 3.28%. All these data indicate this colorimetric method has great potential for the detection of heparinase activity in biological samples.

Fig. 6 Comparison of the absorbance values of the TMB/H2O2/Au-NCs/heparin system in the presence of different proteins. Concentrations: BSA, SOD, Try (Trypsin), Lys (lysozyme), XOD, ExoI, 100 µg⋅mL-1; heparinase, 10 µg⋅mL-1.

CONCLUSIONS

In summary, we found heparin could greatly boost the peroxidase-like catalytic activity of AuNCs. Based on this finding, the highly sensitive colorimetric detection of heparin and heparinase


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was developed. In the presence of heparin, the catalytic activity of Au-NCs was increased 25fold with TMB as a peroxidase substrate. More importantly, the optimum catalytic activity enhancement of Au-NCs in the presence of heparin was at neural pH, which is beneficial for the biological applications. Finally, the assay based on the peroxidase-like activity of Au-NCs will greatly promote the bioanalytical applications of fluorescent Au-NCs, as some analytes such as heparin could not influence the fluorescence of Au-NCs directly.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS publications website, contains the fluorescence spectra, TEM images, kinetic studies, DLS data, Zeta potential data, and the real sample determinations. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (NSFC, 21605012 and 21705106), Scientific and Technological Research Program of Chongqing Municipal Education Commission (No. KJ1600301), Achievement Transfer Program of


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Institutions of Higher Education in Chongqing (No. KJZH17112), and the Program for TopNotch Young Innovative Talents of Chongqing Normal University (No. 02030307-00043). Dr. Feng also thanks the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (No. TP2016023) and Shanghai Sailing Program (17YF1406400). REFERENCES (1) Wolfenden, R.; Snider, M. J. The depth of chemical time and the power of enzymes as catalysts. Acc. Chem. Res. 2001, 34, 938-945. (2) Nanda, V.; Koder, R. L. Designing artificial enzymes by intuition and computation. Nat Chem. 2010, 2, 15-24. (3) Raynal, M.; Ballester, P.; Vidal-Ferran, A.; Leeuwen, P. Supramolecular catalysis. Part 2: artificial enzyme mimics. Chem. Soc. Rev. 2014, 43, 1734-1787. (4) Dong, Z.; Luo, Q.; Liu, J. Artificial enzymes based on supramolecular scaffolds. Chem. Soc. Rev. 2012, 41, 7890-7908. (5) Wei, H.; Wang, E. Nanomaterials with enzyme-like characteristics (nanozymes): nextgeneration artificial enzymes. Chem. Soc. Rev. 2013, 42, 6060-6093. (6) Wang, X.; Hu, Y.; Wei, H. Nanozymes in bionanotechnology: from sensing to therapeutics and beyond. Inorg. Chem. Front. 2016, 3, 41-60. (7) Lin, Y.; Ren, J.; Qu, X. Catalytically active nanomaterials: a promising candidate for artificial enzymes. Acc. Chem. Res. 2014, 47, 1097-1105. (8) Liu, B.; Liu, J. Surface modification of nanozymes. Nano Res. 2017, 10, 1125-1148.


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(9) Zhou, Y.; Liu, B.; Yang, R.; Liu, J. Filling in the gaps between nanozymes and enzymes: challenges and opportunities. Bioconjugate Chem. 2017, 28, 2903-2909. (10) Gao, L.; Zhuang, J.; Nie, L.; Zhang, J.; Zhang, Y.; Gu, N.; Wang, T.; Feng, J.; Yang, D.; Perrett, S.; Yan, X. Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nat. Nanotechnol 2007, 2, 577-583. (11) Lin, Y.; Ren, J.; Qu, X. Nano-gold as artificial enzymes: hidden talents. Adv. Mater. 2014, 26, 4200-4217. (12) Sun, H.; Zhao, A.; Gao, N.; Li, K.; Ren, J.; Qu, X. Deciphering a nanocarbon-based artificial peroxidase: chemical identification of the catalytically active and substrate-binding sites on graphene quantum dots. Angew. Chem. Int. Ed. 2015, 54, 7176-7180. (13) Weerathunge, P.; Ramanathan, R.; Shukla, R.; Sharma, T.; Bansal, V. Aptamer-controlled reversible inhibition of gold nanozyme activity for pesticide sensing. Anal. Chem. 2014, 86, 11937-11941. (14) Wang, S.; Cazelles, R.; Liao, W.; Vazquez-Gonzalez, M.; Zoabi, A.; Abu-Reziq, R.; Willner, I. Mimicking horseradish peroxidase and NADH peroxidase by heterogeneous Cu2+modified graphene oxide nanoparticles. Nano Lett. 2017, 17, 2043-2048. (15) Zhang, Z.; Zhang, X.; Liu, B.; Liu, J. Molecular imprinting on inorganic nanozymes for hundred-fold enzyme specificity. J. Am. Chem. Soc. 2017, 139, 5412-5419. (16) Tao, Y.; Lin, Y.; Huang, Z.; Ren, J.; Qu, X. Incorporating graphene oxide and gold nanoclusters: a synergistic catalyst with surprisingly high peroxidase-like activity over a broad pH range and its application for cancer cell detection. Adv. Mater. 2013, 25, 2594-2599.


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(17) Liu, B.; Liu, J. Accelerating peroxidase mimicking nanozymes using DNA. Nanoscale 2015, 7, 13831-13835. (18) Fan, K.; Wang, H.; Xi, J.; Liu, Q.; Meng, X.; Duan, D.; Gao, L.; Yan, X. Optimization of Fe3O4 nanozyme activity via single amino acid modification mimicking an enzyme active site. Chem. Commun. 2017, 53, 424-427. (19) Liao, H.; Liu, G.; Liu, Y.; Li, R.; Fu, W.; Hu, L. Aggregation-induced accelerating peroxidase-like activity of gold nanoclusters and their applications for colorimetric Pb2+ detection. Chem. Commun. 2017, 53, 10160-10163. (20) Cheng, H.; Lin, S.; Muhammad, F.; Lin, Y.; Wei, H. Rationally modulate the oxidase-like activity of nanoceria for self-regulated bioassays. ACS sens. 2016, 1, 1336-1343. (21) Yu, F.; Huang, Y.; Cole, A.; Yang, V. The artificial peroxidase activity of magnetic iron oxide nanoparticles and its application to glucose detection. Biomaterials 2009, 30, 4716-4722. (22) Liu, B.; Huang, Z.; Liu, J. Boosting the oxidase mimicking activity of nanoceria by fluoride capping: rivaling protein enzymes and ultrasensitive F- detection. Nanoscale, 2016, 8, 13562-13567. (23) Fang, J.; Zhang, B.; Yao, Q.; Yang, Y.; Xie, J.; Yan, N. Recent advances in the synthesis and catalytic applications of ligand-protected, atomically precise metal nanoclusters. Coord. Chem. Rev. 2016, 322, 1-29. (24) Tao, Y.; Li, M.; Ren, J.; Qu, X. Metal nanoclusters: novel probes for diagnostic and therapeutic applications. Chem. Soc. Rev. 2015, 44, 8636-8663.


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(25) Zhang, L.; Wang, E. Metal nanoclusters: new fluorescent probes for sensors and bioimaging. Nano today 2014, 9, 132-157. (26) Hu, L.; Deng, L.; Alsaiari, S.; Zhang, D.; Khashab, N. "Light-on" sensing of antioxidants using gold nanoclusters. Anal. Chem. 2014, 86, 4989-4994. (27) Shang, L.; Dong, S.; Nienhaus, G. Ultra-small fluorescent metal nanoclusters: Synthesis and biological applications. Nano today 2011, 6, 401-418. (28) Shang, L.; Stockmar, F.; Azadfar, N.; Nienhaus, G. Intracellular thermometry by using fluorescent gold nanoclusters. Angew. Chem. Int. Ed. 2013, 52, 11154-11157. (29) Wang, X.; Wu, Q.; Shan, Z.; Huang, Q. BSA-stabilized Au clusters as peroxidase mimetics for use in xanthine detection. Biosens. Bioelectron. 2011, 26, 3614-3619. (30) Xie, J.; Zheng, Y.; Ying, J. Protein-directed synthesis of highly fluorescent gold nanoclusters. J. Am. Chem. Soc. 2009, 131, 888-889. (31) Hattori, T.; Kimura, K.; Seyrek, E.; Dubin, P. Binding of bovine serum albumin to heparin determined by turbidimetric titration and frontal analysis continuous capillary electrophoresis. Anal. Biochem. 2001, 295, 158-167. (32) Guo, C.; Wang, B.; Wang, L.; Xu, B. Structural basis of single molecular heparin–FX06 interaction revealed by SPM measurements and molecular simulations. Chem. Commun. 2012, 48, 12222-12224. (33) Xu, Y.; Seeman, D.; Yan, Y.; Sun, L.; Post, J.; Dubin, P. Effect of heparin on protein aggregation: inhibition versus promotion. Biomacromolecules 2012, 13, 1642-1651.


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(34) Capila, I.; Linhardt, R. Heparin–protein interactions. Angew. Chem. Int. Ed. 2002, 41, 390-412. (35) Li, J.; Cheng, M.; Li, M. A luminescent and colorimetric probe based on the functionalization of gold nanoparticles by ruthenium(ii) complexes for heparin detection. Analyst 2017, 142, 3733-3739. (36) Chen, Z.; Wang, Z.; Chen, X.; Xu, H,; Liu, J. Chitosan-capped gold nanoparticles for selective and colorimetric sensing of heparin. J. Nanopar. Res. 2013, 15, 1930-1939. (37) You, J.; Liu, Y.; Lu, C.; Tseng, W.; Yu, C. Colorimetric assay of heparin in plasma based on the inhibition of oxidase-like activity of citrate-capped platinum nanoparticles. Biosens. Bioelectron. 2016, 92, 442-448. (38) Qu, F.; Liu, Y.; Lao, H.; Wang, Y.; You, J. Colorimetric detection of heparin with high sensitivity based on the aggregation of gold nanoparticles induced by polymer nanoparticles. New J. Chem. 2017, 41, 10592-10597. (39) Fu, X.; Chen, L.; Li, J.; Lin, M.; You, H.; Wang, W. Label-free colorimetric sensor for ultrasensitive detection of heparin based on color quenching of gold nanorods by graphene oxide. Biosens. Bioelectron. 2012, 34, 227-231. (40) Hemmateenejad, B.; Dorostkar, S.; Shakerizadeh-Shirazi, F.; Shamsipur, M. pHindependent optical sensing of heparin based on ionic liquid-capped gold nanoparticles. Analyst 2013, 138, 4830-4837. (41) Ding, Y.; Shi, L.; Wei, H. A "turn on" fluorescent probe for heparin and its oversulfated chondroitin sulfate contaminant. Chem. Sci. 2015, 6, 6361-6366.


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Page 22 of 23

(42) Cheng, H.; Liu, Y.; Hu, Y.; Ding, Y.; Lin, S.; Cao, W.; Wang, Q.; Wu, J.; Muhammad, F.; Zhao, X.; Zhao, D.; Li, Z.; Xing, H.; Wei, H. Monitoring of heparin activity in live rats using metal−organic framework nanosheets as peroxidase mimics. Anal. Chem. 2017, 89, 11552-11559. (43) Sasisekharan, R.; Moses, M.; Nugent, M.; Cooney, C.; Langer, R. Heparinase inhibits neovascularization. Proc. Natl. Acad. Sci. USA 1994, 91, 1524-1528.


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TOC:

The peroxidase-like catalytic activity of Au-NCs is nearly 25-fold increased after the addition of heparin, and the sensitive colorimetric detection of heparin and heparinase activity is developed.


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Accelerating the Peroxidase-Like Activity of Gold Nanoclusters at

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