Multiplexed Activity of perAuxidase: DNA-Capped AuNPs Act asAdjustable PeroxidaseMustafa Salih Hizir,† Meryem Top,† Mustafa Balcioglu,† Muhit Rana,† Neil M. Robertson,†

Fusheng Shen,† Jia Sheng,†,‡ and Mehmet V. Yigit*,†,‡

†Department of Chemistry, University at Albany, State University of New York, 1400 Washington Avenue, Albany, New York 12222,United States‡The RNA Institute, University at Albany, State University of New York, 1400 Washington Avenue, Albany, New York 12222, UnitedStates

*S Supporting Information

ABSTRACT: In this study, we have investigated the intrinsicperoxidase-like activity of citrate-capped AuNPs (perAuxidase) anddemonstrated that the nanozyme function can be multiplexed andtuned by integrating oligonucleotides on a nanoparticle surface.Systematic studies revealed that by controlling the reactionparameters, the mutiplexing effect can be delayed or advancedand further used for aptasensor applications.

In the past few decades, the investigation on artificialenzymes has been increasing due to multiple advantages

over their biological counterparts. Low-cost, bulk-preparation,and greater stability of artificial enzymes make them attractivefor various applications, particularly for biochemical analysis. Awide variety of inorganic nanomaterials such as metal andmetal-oxide nanoparticles,1−6 transition metal decalcogenide(TMD) nanosheets,7−9 nanocarbon oxides,10−14 and theirvarious hybrid formations15−18 have been reported to displayspecific enzymatic behaviors. Some of these nanomaterials,referred to as nanozymes, are reported to act as mimics ofnuclease, esterase, glucose oxidase, superoxide dismutase,silicatein, catalase, phosphatise, nitrate reductase, and perox-idase.19 In general, nanozymes work in broader pH andtemperature ranges, therefore, for this matter, are advantageousover biological catalysts. For instance, colloidal gold nano-particle (AuNP) is considered as one of the robust nanozymesand a variety of its artificial enzymatic activities have beenreported.19 Particularly, its peroxidase-like activity has attracteda significant attention for biochemical studies.20 Peroxidases areknown to catalyze the oxidation of their substrates in thepresence of peroxide species. AuNPs catalyze the sameoxidation reactions using the same substrates. Among differentfunctional AuNPs, negatively charged citrate-capped AuNP hasbeen investigated predominantly due to its favorable electro-static interaction with the positively charged 3,3′,5,5′-tetramethylbenzidine (TMB) substrate. The intrinsic perox-idase-like activity of gold nanoparticle is highly attractive alone;however, adsorption of single stranded functional oligonucleo-

tides can tune this enzymatic behavior and offer a widespectrum of applications.21

Oligonucleotides are extraordinary biopolymers offeringdiverse functionality to the nanoparticles due to their highlyprogrammable features, target-specific binding or cleavage,structure-switching capability, and unique interactions at thebionanointerfaces. Having these remarkable features, DNAnanotechnology has been integrated into a wide range ofapplications including nanoelectronics, biosensing, environ-mental analysis, gene delivery and manipulation, and etc.22−24

For instance, DNA origami has been employed to engineerprogrammable architectural designs for biological nanodevi-ces25,26 while aptamer or DNAzyme (functional oligonucleo-tides) technology was integrated into biosensor designs forbiological or environmental applications.27,28 Our group haspreviously employed DNA technology to study controllableassembly of gold nanoparticles,29 simultaneous detection ofcirculating miRNAs,30,31 and single-nucleotide polymorphism(SNP) identification.32 As aforementioned, oligonucleotideshold a great potential for various applications due to theversatile manipulation features.For the nanoparticles displaying enzyme-mimicking behav-

iors, DNA binding may significantly enhance nanozyme quality,offer multiplexed enzymatic capacity, and lead to easily

Received: October 16, 2015Accepted: December 11, 2015Published: December 11, 2015

Letter

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© 2015 American Chemical Society 600 DOI: 10.1021/acs.analchem.5b03926Anal. Chem. 2016, 88, 600−605

controllable catalytic activity.33−36 Studies have demonstratedthat the presence of DNA at the interface between nanozymesand their substrates inhibits the enzymatic activity due to thephysical hindrance or electrostatic repulsion.37,38,16,39,40 On theother hand, Liu et al. and Hu et al. reported thatoligonucleotides at the nanointerface enhance the intrinsicenzymatic activity of the nanozymes remarkably by contributingto the enzyme−substrate affinity.41,42 In this study, we haveinvestigated the intrinsic peroxidase-like activity of citrate-capped AuNPs (perAuxidase) and demonstrated that thenanozyme function can be multiplexed and further tuned byintegrating oligonucleotides on the nanoparticle surface.Systematic studies revealed that by controlling the reactionparameters, the mutiplexing effect can be delayed or advancedand further used for aptasensor applications.

■ RESULTS AND DISCUSSION

Here, the peroxidase-like activity of AuNPs (perAuxidase, 15nm in diameter, Figure S9) with and without DNA capping wasvalidated using 3,3′,5,5′-tetramethylbenzidine (TMB) andhydrogen peroxide (H2O2). Before evaluating the nanozymepotential of AuNP, its stability was evaluated in variousconcentrations of reaction buffer (NaOAc) at pH 4.0. Thenanoparticles remained stable up to 50 mM NaOAc at pH 4.0for at least 24h, Figure S1. Later, in order to characterize theperoxidase-like activity of AuNPs and determine the optimumreaction condition, the concentration of each component(AuNPs, H2O2, TMB, and NaOAc) in the reaction was variedsystematically, Scheme S1.To monitor the effect of the AuNPs concentration on the

enzymatic activity, the TMB oxidation reaction was performedwith various concentrations of AuNPs (referred to as

perAuxidase) using H2O2 and TMB in 10 mM NaOAc at pH4.0 (working buffer). As the concentration of AuNPs increasedin the reaction mixture, the observed color change due to TMBoxidation amplified, suggesting that the reaction rate can beincreased with AuNP concentration, Figure 1a. Experimentswith no perAuxidase did not display any color change. Next, theeffect of H2O2 concentration was tested by varying itsconcentration from 0.1 to 30 mM with fixed AuNPconcentration (200 μM perAuxidase). As the H2O2 concen-tration increased, a higher TMB oxidation was observed, Figure1b. Experiments with no H2O2 did not display any colorchange. Before each measurement, the AuNPs were removedwith centrifugation to avoid signal interference from AuNPs.Later, the various concentrations of substrate (TMB) was

used to determine optimum reaction condition to monitor thecolorimetric change. We have observed that TMB concen-trations higher than 0.5 mM was sufficient to display theenzymatic reaction totally, Figure S2a. The dependence on theNaOAc concentration was determined in similar fashion and allconcentrations between 1 and 10 mM displayed same oxidationvalidated visually and spectroscopically, Figure S2b.Then, in order to validate the necessity of each component

for perAuxidase activity of gold nanoparticles, an absence testwas performed. As seen in Figure 2, in the absence of any of thecomponents, the reaction does not display any color change.On the other hand, the oxidation reaction is catalyzed withTMB and H2O2 in NaOAc at pH 4.0 when AuNPs werepresent in the reaction medium, Figure 2. Aforementionedfindings reveal that the absorbance signal of blue color(oxidized TMB) was specific to only the synchronous presenceof all reagents.

Figure 1. Effect of perAuxidase (AuNP) and H2O2 concentration on the catalytic activity of the nanozyme. Increasing concentration of (a)perAuxidase and (b) H2O2 leads to accelerated OD increase at 650 nm and TMB oxidation.

Figure 2. (a) Visual analysis demonstrates that the enzymatic reaction takes place only in the presence of all reagents. (b) Absorbance spectra (inset)and end point readings at 650 nm, at the end of 30 min incubation, shows the necessity of each component for TMB oxidation.

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Next, we studied the tuning of the peroxidase-like activity ofAuNP through its interaction at the biointerface. Because theenzymatic reaction occurs at the surface of the nanozyme, wehypothesized that perAuxidase activity can be manipulated bychanging the surface properties. DNA has been chosen as aninterfacing biopolymer due to its favorable interaction with goldsurface through nucleobases.21 We have observed that the DNAadsorption enhanced the peroxidase activity of the goldnanoparticles significantly after 30 min of reaction. In orderto study this multiplexing effect, 200 μM perAuxidase wasincubated with various concentrations of (5, 10, 20, 30, 40, 50,or 1000 nM) scrambled DNA. Following incubation and TMBoxidation reaction for 30 min, the absorbance readings wererecorded for each sample. Results demonstrate that withincreasing DNA concentrations, the catalytic reaction isamplified which was observed as an OD increase at 650 nm,Figure 3a. Absence test confirmed that the DNA coupled withgold nanoparticles display significantly greater catalytic activitythan gold nanoparticles alone, Figure 3b. On the other hand,the results with the same amount of DNA alone were notstatistically different than the blank samples which suggest thatthe DNA itself does not catalyze the reaction. The experimentswere performed with different pH, temperatures, andincubation times with DNA, Figures S5, S6, and S8. Durabilitytests were performed for perAuxidase and DNA-cappedperAuxidase (perAuxidase-DNA), Figure S7.

Here, ssDNA interacts with citrate-capped AuNPs throughits nucleobases and, in such conformation, negatively chargedDNA backbone is exposed to the environment.21 Wehypothesize that the presence of this multiple negative chargesand the hydrogen bonding moieties provided by DNA basescould attract the positively charged aromatic substrate (TMB)molecule by interacting through intermolecular forces and,therefore, multiplex the enzymatic reaction rate by increasinglocalized substrate concentration around perAuxidase.In order to understand the multiplexing effect in the presence

of DNA, perAuxidase was tested with and without 100 nMscrambled DNA. We have observed that while the peroxidaseactivity was slowed down initially, the enzymatic reaction ismultiplexed in later time points, Figure 4a and inset. The initialboost in the OD at 650 nm with AuNPs was inhibited by thestabilization provided by DNA adsorption in the DNA-cappedAuNPs (perAuxidase-DNA). The multiplexing effect observa-tion was in parallel with the recent report about DNA-cappediron oxide nanoparticles.41 After 10 min, the peak that appearedat 650 nm intensified as the reaction proceeded (Figure 4b andFigure S3) and resulted in an almost 2.5-fold enhancementcompared to the naked perAuxidase at the end of 2 h ofreaction.Following the aforementioned studies showing that DNA

coupled to the nanozyme display a higher TMB oxidation inlater time points, we studied to control the perAuxidase activity

Figure 3. (a) DNA concentration-dependent accelerated TMB oxidation is observed as an absorbance increase at 650 nm. (b) Absorbance end pointdata demonstrate significant enhancement of perAuxidase activity in the presence of both DNA and the perAuxidase.

Figure 4. Absorbance spectra at 650 nm demonstrating (a) slower transition for catalysis in the first 10 min followed by the accelerated enzymatictrend by perAuxidase-DNA (b) the fast formation of oxidized TMB product in first 10 min by perAuxidase only. Inset figures display the first 10 minof the reaction. PerAuxidase-DNA oxidizes 2.5 times more TMB than perAuxidase alone does as indicated by the gap and double head arrow.

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further. Because the reaction kinetics depends on the preciseexperimental parameters, we were able to tune the multiplexingphase to a later time point. As seen in Figure 5, DNAadsorption on perAuxidase enabled us to control the enhance-ment effect back and forward, and change the reaction profileby changing H2O2 concentration. This is an important findingbecause combining the antisense or functional oligonucleotidetechnologies with the artificial enzyme features of goldnanoparticles could be highly useful for oligonucleotide-basedcolorimetric or spectroscopic detection methodologies.Later, we studied the effect of DNA sequence on this

multiplexed catalytic activity. We tested different polynucleo-tide sequences while keeping the total nucleotide concentrationconstant. Polynucleotides with A10, T10, C10, or G10 sequenceswere adsorbed on perAuxidase and tested for catalysis of TMBoxidation reaction. Results demonstrate that polypurine-modified perAuxidases (A10, G10) displayed a remarkableenhancement while poly pyrimidine-modified perAuxidases(T10, C10) were slightly higher than unmodified perAuxidase,Figure 6a. Results demonstrate that the multiplexed effect

observed with surface-adsorbed purine bases is different thanpyrimidine bases, which could be due to the difference in theinteraction between TMB and the surface-adsorbed nucleo-bases.Next, we evaluated the multiplexed catalytic activity in the

presence of different polyA strands with different (A5, A10, A15)lengths while keeping the total nucleotide concentrationconstant. Results suggest that TMB oxidation in all threecases were not statistically different than each other; however,significantly higher than the perAuxidase activity without polyAcapping, Figure 6b. Later, we tested whether RNA has similartendency in this multiplexing effect and performed the studiesusing scrambled RNA and A15 RNA, the RNA counterparts ofscrambled DNA and A15 (DNA), respectively. The results showthat both the RNA and DNA behave similarly and are notstatistically different than each other, Figure 6c. Nearbycatalytic amplification achieved by RNA also implies thepotential of RNA in manipulating the properties of functionalnanomaterials and its possible applications when coupled withnanozymes.

Figure 5. Controlling the multiplexing phase in the perAuxidase reaction profile: (a) higher hydrogen peroxide content (10 mM) results in an earlier(10 min) multiplexing effect while (b) the lower content (5 mM) results in a delayed (30 min) multiplexing effect.

Figure 6. (a) Absorbance (OD at 650 nm) data demonstrate that (a) polypurines enhance the catalysis more efficiently than polypyrimidines and(b) the length of the ssDNA does not influence the multiplexed catalysis. (c) The study with ssRNA molecules demonstrates that they act similar tossDNA in multiplexing the catalysis. (d) The multiplexed peroxidase-like activity can be employed to detect thrombin using aptamer andperAuxidase complex.

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Finally, in order to demonstrate a possible application of thismultiplexing phenomenon, we have used aptamer technologycoupled with AuNPs. A total of 29 nucleobase-long DNAaptamer for thrombin was studied. First, the catalytic activity ofperAuxidase was evaluated with and without thrombin. Theresults show that thrombin itself does not influence theperoxidase-mimicking activity of gold nanoparticles. Later, thenanoparticles were incubated with the aptamer, which increasedthe peroxidase activity (30 min), therefore, the OD at 650 nm.On the other hand, when the aptamer was preincubated withthrombin, inhibiting the adsorption of the aptamer on thenanoparticle surface, this multiplexed effect was diminished,Figure 6d. Findings reveal that the difference in TMB oxidationwith and without DNA adsorption can be employed foraptasensor development.

■ CONCLUSIONTo conclude, our work demonstrates that the intrinsicperoxidase-like activity of artificial gold nanozyme could bemultiplexed or diminished by integrating single stranded DNAor RNA molecules on the nanoparticle surface. This multi-plexing effect can be fine-tuned by altering the nucleotidecomponents of the oligonucleotide sequences or changing thereaction parameters. Furthermore, the mechanism of enhance-ment can be conducted to detect small analytes or biologicalmetabolites using target-specific aptamers. Such a versatileapplication tool holds a great potential for the realms ofenvironmental science, toxicology, and biology.In order to multiplex the inherent enzymatic activity of

certain protein molecules, organic−inorganic hybrid structureshave been employed. Zare and co-workers have coupledprotein structures with copper(II) ions to achieve highercatalytic activity.43 Hou and co-workers studied the immobi-lization of α-amylase using specific nanomaterials to demon-strate the enhanced catalytic capability of the enzyme.44 In arecent study, Ocsoy and co-workers have also achievedenhanced catalytic activity of horseradish peroxidase usingcopper(II) and iron(II) ions.45,46 Our study on the other handreports the multiplexing and adjusting of the intrinsicperoxidase-like activity of gold nanoparticles using DNA andRNA molecules. Though others have shown a greatermultiplexed effect by preparing gold hybrid structures usingdifferent sizes and morphologies,1,16,47,48 multiplexing thecatalytic activity of gold nanoparticles using oligonucleotideswith different nucleobase composition has not been reportedpreviously. In the future, this property could enable us tocharacterize the base composition of DNA or RNA molecules.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.anal-chem.5b03926.

Additional results (Figures S1−S9) and details ofexperimental methods; TEM images, DLS data, andthe durability of the nanoparticles; effects of incubationtime with oligonucleotides, pH, and temperature arereported (PDF).

■ AUTHOR INFORMATIONCorresponding Author*E-mail: myigit@albany.edu. Phone: (1) 518-442-3002.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

We acknowledge the Ministry of National Education, Republicof Turkey, for providing financial support to Mustafa SalihHizir with full scholarship during his doctoral studies. Thiswork was supported by SUNY Albany Start-Up Funds.

■ REFERENCES(1) Gao, L.; Zhuang, J.; Nie, L.; Zhang, J.; Zhang, Y.; Gu, N.; Wang,T.; Feng, J.; Yang, D.; Perrett, S.; Yan, X. Nat. Nanotechnol. 2007, 2,577−583.(2) Wei, H.; Wang, E. Anal. Chem. 2008, 80, 2250−2254.(3) Li, X.; Wen, F.; Creran, B.; Jeong, Y.; Zhang, X.; Rotello, V. M.Small 2012, 8, 3589−3592.(4) Asati, A.; Kaittanis, C.; Santra, S.; Perez, J. M. Anal. Chem. 2011,83, 2547−2553.(5) Artiglia, L.; Agnoli, S.; Paganini, M. C.; Cattelan, M.; Granozzi, G.ACS Appl. Mater. Interfaces 2014, 6, 20130−20136.(6) Vernekar, A. A.; Sinha, D.; Srivastava, S.; Paramasivam, P. U.;D’Silva, P.; Mugesh, G. Nat. Commun. 2014, 5, 5301.(7) Guo, X.; Wang, Y.; Wu, F.; Ni, Y.; Kokot, S. Analyst 2015, 140,1119−1126.(8) Lin, T.; Zhong, L.; Guo, L.; Fu, F.; Chen, G. Nanoscale 2014, 6,11856−11862.(9) Chen, Q.; Chen, J.; Gao, C.; Zhang, M.; Chen, J.; Qiu, H. Analyst2015, 140, 2857−2863.(10) Zhao, R.; Zhao, X.; Gao, X. Chem. - Eur. J. 2015, 21, 960−964.(11) Zheng, X.; Zhu, Q.; Song, H.; Zhao, X.; Yi, T.; Chen, H.; Chen,X. ACS Appl. Mater. Interfaces 2015, 7, 3480−3491.(12) Zhan, L.; Li, C. M.; Wu, W. B.; Huang, C. Z. Chem. Commun.2014, 50, 11526−11528.(13) Yang, Z.; Qian, J.; Yang, X.; Jiang, D.; Du, X.; Wang, K.; Mao,H.; Wang, K. Biosens. Bioelectron. 2015, 65, 39−46.(14) Tao, Y.; Lin, Y.; Ren, J.; Qu, X. Biomaterials 2013, 34, 4810−4817.(15) Gao, Z.; Hou, L.; Xu, M.; Tang, D. Sci. Rep. 2014, 4, 3966.(16) Liu, M.; Zhao, H.; Chen, S.; Yu, H.; Quan, X. ACS Nano 2012,6, 3142−3151.(17) Tao, Y.; Ju, E.; Ren, J.; Qu, X. Adv. Mater. 2015, 27, 1097−1104.(18) Zhang, L. N.; Deng, H. H.; Lin, F. L.; Xu, X. W.; Weng, S. H.;Liu, A. L.; Lin, X. H.; Xia, X. H.; Chen, W. Anal. Chem. 2014, 86,2711−2718.(19) Lin, Y.; Ren, J.; Qu, X. Adv. Mater. 2014, 26, 4200−4217.(20) Wang, S.; Chen, W.; Liu, A. L.; Hong, L.; Deng, H. H.; Lin, X.H. ChemPhysChem 2012, 13, 1199−1204.(21) Liu, J. Phys. Chem. Chem. Phys. 2012, 14, 10485−10496.(22) Doane, T. L.; Alam, R.; Maye, M. M. Nanoscale 2015, 7, 2883−2888.(23) Wei, Q.; Nagi, R.; Sadeghi, K.; Feng, S.; Yan, E.; Ki, S. J.; Caire,R.; Tseng, D.; Ozcan, A. ACS Nano 2014, 8, 1121−1129.(24) Alexander, C. M.; Hamner, K. L.; Maye, M. M.; Dabrowiak, J. C.Bioconjugate Chem. 2014, 25, 1261−1271.(25) Gopinath, A.; Rothemund, P. W. ACS Nano 2014, 8, 12030−12040.(26) Woo, S.; Rothemund, P. W. Nat. Commun. 2014, 5, 4889.(27) Shukoor, M. I.; Altman, M. O.; Han, D.; Bayrac, A. T.; Ocsoy, I.;Zhu, Z.; Tan, W. ACS Appl. Mater. Interfaces 2012, 4, 3007−3011.(28) Wu, P.; Hwang, K.; Lan, T.; Lu, Y. J. Am. Chem. Soc. 2013, 135,5254−5257.(29) Rana, M.; Balcioglu, M.; Yigit, M. BioNanoScience 2014, 4, 195−200.(30) Hizir, M. S.; Balcioglu, M.; Rana, M.; Robertson, N. M.; Yigit,M. V. ACS Appl. Mater. Interfaces 2014, 6, 14772−14778.(31) Balcioglu, M.; Buyukbekar, B. Z.; Yavuz, M. S.; Yigit, M. V. ACSBiomater. Sci. Eng. 2015, 1, 27−36.

Analytical Chemistry Letter

DOI: 10.1021/acs.analchem.5b03926Anal. Chem. 2016, 88, 600−605

604

(32) Robertson, N. M.; Salih Hizir, M.; Balcioglu, M.; Wang, R.;Yavuz, M. S.; Yumak, H.; Ozturk, B.; Sheng, J.; Yigit, M. V. Langmuir2015, 31, 9943−9952.(33) Zhang, D.; Chen, Z.; Omar, H.; Deng, L.; Khashab, N. M. ACSAppl. Mater. Interfaces 2015, 7, 4589−4594.(34) Tao, Y.; Lin, Y.; Ren, J.; Qu, X. Biosens. Bioelectron. 2013, 42,41−46.(35) Deng, H. H.; Weng, S. H.; Huang, S. L.; Zhang, L. N.; Liu, A. L.;Lin, X. H.; Chen, W. Anal. Chim. Acta 2014, 852, 218−222.(36) Wang, Y.; Zhang, X.; Luo, Z.; Huang, X.; Tan, C.; Li, H.; Zheng,B.; Li, B.; Huang, Y.; Yang, J.; Zong, Y.; Ying, Y.; Zhang, H. Nanoscale2014, 6, 12340−12344.(37) Park, K. S.; Kim, M. I.; Cho, D. Y.; Park, H. G. Small 2011, 7,1521−1525.(38) Pautler, R.; Kelly, E. Y.; Huang, P. J.; Cao, J.; Liu, B.; Liu, J. ACSAppl. Mater. Interfaces 2013, 5, 6820−6825.(39) Sharma, T. K.; Ramanathan, R.; Weerathunge, P.;Mohammadtaheri, M.; Daima, H. K.; Shukla, R.; Bansal, V. Chem.Commun. 2014, 50, 15856−15859.(40) Weerathunge, P.; Ramanathan, R.; Shukla, R.; Sharma, T. K.;Bansal, V. Anal. Chem. 2014, 86, 11937−11941.(41) Liu, B.; Liu, J. Nanoscale 2015, 7, 13831−13835.(42) Hu, J.; Ni, P.; Dai, H.; Sun, Y.; Wang, Y.; Jiang, S.; Li, Z. Analyst2015, 140, 3581−3586.(43) Ge, J.; Lei, J.; Zare, R. N. Nat. Nanotechnol. 2012, 7, 428−432.(44) Wang, L. B.; Wang, Y. C.; He, R.; Zhuang, A.; Wang, X.; Zeng,J.; Hou, J. G. J. Am. Chem. Soc. 2013, 135, 1272−1275.(45) Ocsoy, I.; Dogru, E.; Usta, S. Enzyme Microb. Technol. 2015,75−76, 25−29.(46) Somturk, B.; Hancer, M.; Ocsoy, I.; Ozdemir, N. Dalton. Trans.2015, 44, 13845−13852.(47) Wang, X. X.; Wu, Q.; Shan, Z.; Huang, Q. M. Biosens.Bioelectron. 2011, 26, 3614−3619.(48) Wei, H.; Wang, E. Chem. Soc. Rev. 2013, 42, 6060−6093.

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