Nanoscale

PAPER

Cite this: Nanoscale, 2020, 12, 967

Received 29th July 2019,Accepted 19th November 2019

DOI: 10.1039/c9nr06470b

rsc.li/nanoscale

Biomimetic mineralization of nitrile hydrataseinto a mesoporous cobalt-based metal–organicframework for efficient biocatalysis†

Xiaolin Pei, *a,b Yifeng Wu,a Jiapao Wang,a Zhiji Chen,a Wen Liu,a Weike Sub andFangming Liu*a

Nitrile hydratases (NHases) have attracted considerable attention owing to their application in the syn-

thesis of valuable amides under mild conditions. However, the poor stability of NHases is still one of the

main drawbacks for their industrial application. Recently, mesoporous metal–organic frameworks (MOFs)

have been explored as an attractive support material for immobilizing enzymes. Here, we encapsulated a

recombinant cobalt-type NHase from Aurantimonas manganoxydans into the cobalt-based MOF ZIF-67

by a biomimetic mineralization strategy. The nano-catalyst NHase1229@ZIF-67 shows high catalytic

activity for the hydration of 3-cyanopyridine to nicotinamide, and its specific activity reached 29.5

U mg−1. The NHase1229@ZIF-67 nanoparticles show a significant improvement in the thermal stability of

NHase1229. The optimum reaction temperature of NHase1229@ZIF-67 is at 50–55 °C, and it still retained

40% of the maximum activity at 70 °C. However, the free NHase1229 completely lost its catalytic activity at

70 °C. The half-lives of NHase1229@ZIF-67 at 30 and 40 °C were 102.0 h and 26.5 h, respectively.

NHase1229@ZIF-67 nanoparticles exhibit an excellent cycling performance, and their catalytic efficiency did

not significantly decrease in the initial 6 cycles using 0.9 M 3-cyanopyridine as the substrate. In a fed-batch

reaction, NHase1229@ZIF-67 can efficiently hydrate 3-cyanopyridine to nicotinamide, and the space–time

yield was calculated to be 110 g·L−1·h−1. Therefore, the cobalt-type NHase was immobilized in MOF ZIF-67,

which is shown as a potential nanocatalyst for the large-scale industrial preparation of nicotinamide.

Introduction

Enzyme catalysis has been commercialized for the sustainablesynthesis of bulk and fine chemicals due to its numerousenvironmental and economic benefits.1 Nitrile hydratases(NHases, EC 4.2.1.84) catalyse the hydration of various nitriles(R-CN) to the corresponding amides (R-CONH2) under mildconditions, and have attracted considerable academic andcommercial interest because of their application to produceacrylamide, nicotinamide, and 5-cyanovaleramide.2,3

Nicotinamide acts as the physiologically active form of vitaminB3 and has been widely used as an ingredient in pharma-ceutical and cosmetic compositions, as well as food and feed

additives.4 Although NHases have been successfully adapted toproduce valuable amides, the poor stability of NHases is stillone of the main drawbacks for their industrial applicationsbecause of the thermal deactivation and the substratetoxicity.5,6 Many approaches have been developed to overcomethis limitation, including discovering novel thermal enzymes,7

engineering enzymes8,9 and immobilizing enzymes.10,11 Inparticular, the immobilization of enzymes can not onlyimprove their stability and activity, but also reduce the productcosts due to the recyclability of enzymes.12,13 Hitherto, variousmethods have been described for immobilizing enzymes, suchas binding to the solid support, cross-linking and entrappinginto particles.14,15 Pawar and Yadav immobilized NHase usingpoly(vinyl alcohol)/chitosan, and the immobilized NHaseexhibited effective catalytic activity and thermal stability.11,16

NHases were also immobilized as cross-linked enzyme aggre-gates (CLEAs), which also showed enhanced thermal, mechani-cal, storage and operational stabilities.4,10 However, the use ofcross-linker glutaraldehyde in these methods will decrease theactivity of NHases. Therefore, there is a need to develop newmaterials and methods to immobilize NHases with high cata-lytic efficiency.

†Electronic supplementary information (ESI) available. See DOI: 10.1039/c9nr06470b

aCollege of Material, Chemistry and Chemical Engineering, Hangzhou Normal

University, Hangzhou, 310012, PR China. E-mail: pxl@hznu.edu.cn,

fmliu859@sohu.combCollaborative Innovation Center of Yangtze River Delta Region Green

Pharmaceuticals, College of Pharmaceutical Science, Zhejiang University of

Technology, Hangzhou, 310032, PR China

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Metal–organic frameworks (MOFs) have been explored asan attractive support candidate for the immobilization ofenzymes.17,18 MOFs comprise a class of highly ordered porousnanomaterials, which are constructed from organic linkersand inorganic metal nodes.19 MOFs have been used toimmobilize enzymes owing to their tunable porosity, largesurface area and excellent thermal/chemical stability.20,21

Generally, there are four approaches to prepare enzyme@MOFcomposites, including surface adsorption, covalent tethering,encapsulation and pore/channel entrapment.22 Thereinto, theencapsulation of enzymes has emerged as a preferred methodto synthesize enzyme@MOF. Cytochrome c (Cyt c) wasembedded in the zeolitic imidazolate framework ZIF-8 withhigh biological activities, and polyvinylpyrrolidone (PVP) wasrequired to stabilize Cyt c in methanol.23 A biomineralizationprocess was also developed to encapsulate biomacromoleculesin MOFs under physiological conditions by concentrating theframework building blocks, and the resultant biocompositepreserved high bioactivity and high biological, thermal andchemical stabilities.24,25 So far, the enzyme@MOF nano-cata-lysts have been mainly investigated in hydrolysis and redoxreactions.26,27 It must be cautiously considered whether meso-porous MOFs are suitable for other reactions, such as thehydration of nitriles catalysed by NHases.

The NHase gene from Aurantimonas manganoxydans hasbeen cloned and expressed in Escherichia coli.6 The recombi-nant Co-type NHase (NHase1229) consists of two subunits (α-and β-subunit) and non-corrin Co(III) ions. In this study, wedeveloped a biomimetic mineralization of NHase1229 in thecobalt-based MOF ZIF-67 under mild conditions. Co(NO3)2 wasadded into the NHase1229 solution, which was further mixedwith the 2-methylimidazole (mIM) solution to immobilizerecombinant NHase1229 in ZIF-67. The NHase1229@ZIF-67nanoparticles showed excellent catalytic efficiency, thermo-stability and recyclability in the hydration of 3-cyanopyridineto prepare nicotinamide, which might extend the further appli-cation of NHase in industrial scales.

Results and discussionA biomimetic mineralization strategy for immobilizingNHase1229

Recombinant NHase1229 from A. manganoxydans belongs tocobalt-dependent NHases, and it was expressed in the cyto-plasm of E. coli and further purified by nickel affinity columnchromatography (Sodium dodecyl sulfate polyacrylamide gelelectrophoresis, SDS-PAGE, Fig. S1†). A cobalt-based MOFmaterial ZIF-67 [Co(mIM)2] was selected as the support matrixfor immobilizing NHase1229. ZIF-67 is formed by bridgingmIM anions and cobalt cations to result in a regular truncatedrhombic dodecahedral shape with a pore size of approximately0.34 nm, which is too small for bulk enzymes to freely travel.28

Therefore, we encapsulated NHase1229 in MOF ZIF-67 by a bio-mimetic mineralization strategy (Fig. 1). Enzyme molecules canincrease the local concentration of both metal cation Co2+ and

organic ligands mIM, which facilitate the prenucleation andcrystallization of ZIF-67 around the biomacromolecules.24 TheMOF coating could protect the enzyme to enhance its stability.

Synthesis and characterization of ZIF-67

The molar ratio of Co2+/mIM was firstly investigated to preparestable ZIF-67 nanoparticles in aqueous solution at 4 °C.Through comparing the X-ray diffraction (XRD) patterns of theresulting ZIF-67 materials with the standard simulated XRDpattern of ZIF-67 structure data,29 the molar ratio of Co2+/mIMabove 1 : 30 was necessary to synthesize pure-phaseZIF-67 materials in aqueous solution (Fig. 2A). The surfacearea and pore-size of ZIF-67 were investigated using nitrogenadsorption–desorption isotherms. The ZIF-67 crystals exhibit atypical type-I isotherm indicating high porosity (Fig. 2B). Theincreased adsorption volume at low relative pressure indicatesthe existence of micropores in the ZIF-67 material.30 TheBrunauer–Emmett–Teller (BET) surface area was calculated as1085.6 m2 g−1. The Barrett–Joyner–Halenda (BJH) pore-size dis-tribution curve of ZIF-67 (Fig. 2C) is consistent with that inother studies, which further reveals the microporous structureof ZIF-67.31

The morphology of ZIF-67 samples was characterized byscanning electron microscopy (SEM) and transmission elec-tron microscopy (TEM). When the molar ratio of Co2+/mIMwas 1 : 10 and 1 : 20, the resulting materials showed irregularshapes (Fig. S2†). When the molar ratio of Co2+/mIM was 1 : 30and 1 : 40, the ZIF-67 nanoparticles showed a rhombic dodeca-hedral shape as previously reported in ref. 32. The particle sizeof 1 : 30 (Co2+/mIM) was slightly larger than that of 1 : 40 (Co2+/mIM), approximately 100–200 nm (Fig. 2D and E). The TEMimages also reveal the rhombic dodecahedron of ZIF-67 crystals(Fig. 2F). The obtained ZIF-67 nanoparticles were well dispersedin pH 8.0 phosphate buffer solution (PBS) and kept for severalweeks without a settlement. The XRD patterns and SEM imagesare shown in Fig. S3 and S4.† These results suggest that the syn-thesized ZIF-67 crystals show high structural stability the sameas that of the synthesized ZIF-67 in methanol.33

Synthesis and characterization of NHase1229@ZIF-67

The effects of Co(NO3)2, mIM and enzyme amount were inves-tigated on the activities of free NHase1229 andNHase1229@ZIF-67 (Fig. S5†). Therefore, the molar ratio 1 : 30

Fig. 1 One-pot synthetic procedure for encapsulating NHase1229 inMOF ZIF-67.

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of Co2+/mIM was selected to synthesise NHase1229@ZIF-67,and the concentrations of Co(NO3)2 and mIM were 0.013 Mand 0.39 M, respectively. The resulting NHase1229@ZIF-67nanoparticles show high catalytic activity, and their specificactivity was 29.5 U mg−1. The framework structure ofNHase1229@ZIF-67 was identified by XRD analysis. The XRDpattern is identical to that of the bare ZIF-67 and the simu-lated spectrum (Fig. 3A). No extra peaks were generated afterthe encapsulation of NHase1229. However, the intensity ofsome peaks was varied in NHase1229@ZIF-67. These changesmight reflect the encapsulation of NHase1229 in ZIF-67.

N2 absorption-desorption experiments were conducted toinvestigate the pore structure and the BET surface area ofNHase1229@ZIF-67 crystals (Fig. 3B). The isotherms were the

same as those of ZIF-67, which is categorized as type-I hyster-esis loops, indicating the microporous structure inNHase1229@ZIF-67. The BET surface area was calculated as781.2 m2 g−1.

FT-IR spectra of the ZIF-67, NHase1229, andNHase1229@ZIF-67 are shown in Fig. 3C. The absorptionpeaks in the range of 600–1500 cm−1 were attributed to thecharacteristic stretching and bending modes of the imidazolering of mIM, and the peaks at 2960 and 3420 cm−1 corre-sponded to the stretching mode of C–H from the aromatic ringand aliphatic chain of mIM.33 The free NHase1229 exhibitedtwo absorption peaks at 1620 and 3130 cm−1, which wereattributed to CvO stretching vibration and N–H stretchingvibration of the amide group, respectively.34 These peaks werewell presented in NHase1229@ZIF-67, which confirmed thatNHase1229 was immobilized in the MOF ZIF-67.

Thermogravimetric analysis (TGA) curves provide the infor-mation on the thermal stability of ZIF-67 andNHase1229@ZIF-67 (Fig. 4D). The curve of pure ZIF-67 showedan initial weight loss (approximately 15%) related to the removalof guest water molecules and unreacted ligands in the poreswithin the range of 100 to 150 °C.35 The decomposition tempera-ture is located at approximately 460 °C in N2, which ensures thehigh thermal stability of ZIF-67.32 For NHase1229@ZIF-67, the

Fig. 2 Characterization of ZIF-67. (A) XRD patterns, (B) N2 adsorption–desorption isotherms, (C) the pore size distribution, (D) and (E) SEM imagesof ZIF-67 (Co2+/mIM = 1 : 30 and 1 : 40, respectively), and (F) TEM images of ZIF-67 (Co2+/mIM = 1 : 30).

Fig. 3 Characterization of NHase1229@ZIF-67 and ZIF-67. (A) XRD pat-terns, (B) N2 adsorption–desorption isotherms of NHase1229@ZIF-67,(C) FI-IR spectra, and (D) TGA curves. Fig. 4 Morphology of NHase1229@ZIF-67. (A) SEM and (B) TEM images.

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curve showed an initial weight loss (approximately 25%) relatedto the removal of guest molecules including enzymes within therange of 100 to 280 °C. The results indicate that the weight per-centage of NHase1229 is approximately 10% in MOF ZIF-67, andthe total activity recovery is 73%.

The morphology of NHase1229@ZIF-67 was examined bySEM and TEM. SEM images showed that NHase1229@ZIF-67nanoparticles exhibited a truncated rhombic dodecahedralshape the same as that of bare ZIF-67 (Fig. 4A). TEM imagesshowed that the surface of NHase1229@ZIF-67 was not assmooth as the pure ZIF-67 (Fig. 4B), which may be because ofthe aggregations of NHase1229 on the surface of ZIF-67. Panet al. also reported the trapping enzymes on the MOF surfaces,and the orientation of enzymes will affect the catalyticefficiency of enzyme@MOF particles.36 These results deter-mined that enzymes were distributed on or recessed in theMOF surface in the biomimetic mineralization process.

Enzymatic properties of immobilized NHase1229

The enzymatic properties of NHase1229@ZIF-67 were investi-gated using 3-cyanopyridine as the substrate. As shown inFig. 5A, the optimum temperature was 50 °C, and the activityremained above 40% of the maximum activity at 70 °C.However, the free NHase1229 was virtually inactivated at70 °C.6 The optimum pH of NHase1229@ZIF-67 was 8.0,which was different from that of free NHase1229 at pH 6.8(Fig. 5B). This might be due to the degradation of MOFmaterial ZIF-67 in weakly acidic to neutral aqueous solution(Fig. S6†). The immobilized enzyme NHase1229@ZIF-67 wasincubated at various temperatures to examine thermal stabi-lity. The half-lives (τ1/2) of 30 and 40 °C were 102.0 h and26.5 h, respectively (Fig. 5C), which are higher than those offree NHase1229.6 The encapsulation of enzymes in the MOFhas shown great potential to improve the stability of

enzymes.26,37 The ZIF-67 film holds enzymes in confinementto stabilize their tertiary and quaternary structural confor-mation.38 The kinetics of NHase1229@ZIF-67 was calculatedaccording to the classical Michaelis–Menten equation using3-cyanopyridine as the substrate. The maximal reaction rate(Vmax) and apparent Michaelis–Menten constant (Km) were33.9 μmol min−1 mg−1 and 4.3 mM, respectively (Fig. 5D).Compared with free NHase1229, the decreased Km suggeststhat NHase1229@ZIF-67 has a higher substrate affinity toward3-cyanopyridine molecules, which is possibly due to the micro-environments of MOF crystals.23 Therefore, ZIF-67 can efficien-tly protect NHase1229 to enhance its thermal stability; more-over, its porous structure also facilitates the transportation ofthe substrate and product.

Reusability and the fed-batch reaction of NHase1229@ZIF-67

The concentration of 3-cyanopyridine affected the catalyticefficiency of NHase1229@ZIF-67, and the results suggest thatthe optimal concentration of the substrate is 0.9 M (Fig. S7†).The operational stability of NHase1229@ZIF-67 was furtherinvestigated using 3-cyanopyridine as the substrate. In theinitial 6 batch reactions, 0.9 mol L−1 3-cyanopyridine was com-pletely hydrated to nicotinamide in 30 min (Fig. 6A). The con-version of 3-cyanopyridine was gradually decreased to 86%after 10 cycles. In a 20 mL fed-batch reaction, the concen-tration of 3-cyanopyridine and nicotinamide is shown inFig. 6B. In the first two feedings, 3-cyanopyridine was comple-tely converted to nicotinamide in 30 min. The conversion ratedecreased in the next two feeding reactions, which partiallyreflected the inhibition of the product in the reaction mixtureon the enzymatic activity. The space–time yield of nicotina-mide was calculated to be 110 g L−1 h−1. The results indicatethat the nano-catalyst NHase1229@ZIF-67 has technical andeconomic advantages for the biosynthesis of nicotinamide.

Conclusions

NHase1229 was successfully encapsulated into the MOFmaterial ZIF-67 by a biomimetic mineralization strategy. Thesynthesized NHase1229@ZIF-67 nanoparticles show signifi-cant improvement in the thermal stability. NHase1229@ZIF-67

Fig. 5 The enzymatic characteristics of NHase1229@ZIF-67. (A)Temperature profiles, (B) pH profiles, (C) thermal stability profiles, and(D) Michaelis–Menten kinetic curve.

Fig. 6 Production of nicotinamide using NHase1229@ZIF-67. (A)Reusability of NHase1229@ZIF-67 using 3-cyanopyridine as the sub-strate. (B) Time course of a fed-batch production of nicotinamide usingNHase1229@ZIF-67.

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showed an optimum temperature of 50–55 °C, and retainedabove 70% and 40% of the maximum activity at 65 °C and70 °C, respectively. The nano-catalyst showed more excellentthermal stability compared to free NHase1229. The half-livesof NHase1229@ZIF-67 at 30 and 40 °C were 102.0 h and26.5 h, respectively. Moreover, the immobilized NHase exhibi-ted an excellent recycling performance, and there was noreduction of catalytic efficiency in the initial 6 cycles using3-cyanopyridine as the substrate. NHase1229@ZIF-67 showshigh catalytic activity for hydrating 3-cyanopyridine to nicoti-namide, and its specific activity reached 29.5 U mg−1. In a fed-batch reaction, NHase1229@ZIF-67 can efficiently catalyse3-cyanopyridine to nicotinamide. Therefore, the cobalt-basedMOF material ZIF-67 is a suitable host matrix to immobilizeCo-dependent NHases for the large-scale industrial prepa-ration of nicotinamide.

ExperimentalMaterials

Recombinant Escherichia coli harbouring plasmid pENh1229was stored in our laboratory and used to express recombinantNHase1229.6 The chemicals used in this study were purchasedfrom Sinopharm Chemical Reagent (China), Sigma-Aldrich(USA) and Aladdin Chemistry (China) unless otherwise speci-fied. Tryptone and yeast extract were obtained from OXOID(England). Acetonitrile and methanol for HPLC analysis werepurchased from Merck (Germany).

Preparation of ZIF-67

To investigate the effect of the molar ratio of Co2+ to mIM onthe synthesis of ZIF-67, 49.2 mg Co(NO3)2 was dissolved in6.5 mL deionized water (0.026 M), and different amounts ofmIM were dissolved in 6.5 mL deionized water (0.26 M, 0.52M, 0.78 M and 1.04 M, respectively). The two solutions weremixed and stirred at room temperature for 30 min, and theresulting mixture was stored at 4 °C overnight. The purple pre-cipitate was separated by membrane filtration and washedwith deionized water three times. Finally, the resultingpowders were dried under vacuum at −60 °C for 24 h.

Biomimetic mineralization of NHase1229@ZIF-67

1.2 mg recombinant NHase1229 was added into 6.5 mL de-ionized water with 0.026 M Co(NO3)2, and was combined with0.78 M mIM solution and stirred at room temperature for30 min. The mixture was stored at 4 °C overnight. The purple pre-cipitate was separated by membrane filtration and furtherwashed with pre-cooling deionized water three times. The result-ing purple powders were dried under vacuum at −60 °C for 24 h.

Characterization of ZIF-67 and NHase1229@ZIF-67

Nanoparticles ZIF-67 and NHase1229@ZIF-67 were character-ized as described by Yang with minor modifications.39 XRDpatterns of ZIF-67 and NHase1229@ZIF-67 were recordedusing an X-ray diffractometer (D8 Advance, Bruker, Germany)

under the following operating conditions: 40 kV and 40 mAwith Cu Kα radiation at λ = 0.154184 nm and acceptance slotat 0.1 mm. The relative intensity was recorded in the scatteringrange (2θ) of 5–40° at a step of 10° min−1. The morphologiesof the nanoparticles were characterized by Field EmissionScanning Electron Microscopy (FE-SEM, Zeiss Sigma 500, CarlZeiss, Germany) with an in-lens secondary electron detector(InLens) and Transmission Electron Microscopy (TEM,HT7700, Hitachi, Tokyo, Japan). Thermogravimetric analysis(TGA) was performed using a Thermogravimetric Analyzer(TGA Q500, TA Instruments, USA) from room temperature to650 °C at a heating rate of 10 °C min−1 under a N2 atmosphere.Fourier Transform Infrared (FT-IR) spectroscopy measure-ments were performed using an FT-IR spectrometer (NicoletIS5, Thermo Fisher, USA). Nitrogen adsorption–desorptionmeasurements were performed at 77 K on a Surface Area &Pore Size Analyzer (QUADRASORB SI, USA). The surface area iscalculated based on the BET method, and the pore-size distri-bution is based on the BJH method.

Enzymatic characterization of NHase1229@ZIF-67

The activities of NHase1229@ZIF-67 were measured at20–70 °C to determine the optimum reaction temperature. Theoptimum pH of NHase1229@ZIF-67 was determined bymeasuring the activities at different pH values between 6.0and 9.0. The thermal stability was measured by incubatingNHase1229@ZIF-67 in 100 mM phosphate buffer (pH 8.0) at30 and 40 °C, respectively. The samples were withdrawn fordifferent periods to determine the residual activities. Theactivities were measured in 50 mM phosphate buffer (pH 8.0)at 25 °C using 100 mM 3-cyanopyridine as the substrate. Thefirst-order rate constant kinact of the thermal inactivation wasobtained by plotting logarithmic percentages of residualactivity against time. The half-life (t1/2) of the thermal inacti-vation was calculated using the following eqn (1).40

t1=2 ¼ ln 2=kinact ð1ÞThe kinetic analyses were implemented at 30 °C in 100 mM

pH 8.0 phosphate buffer containing 3-cyanopyridine atdifferent concentrations ranging from 0.25 to 15 mM. The Km

and Vmax values were determined using the substrate satur-ation plotting, which followed the Michaelis–Mentenequation.41 All experiments were performed in triplicate.

Reusability assay

A continuous batch reaction was carried out to evaluate the re-usability of NHase1299@ZIF-67 nanoparticles. In a 4 ml reac-tion mixture containing 0.9 M 3-cyanopyridine, 100 mgNHase1299@ZIF-67 was added to start the hydration reactionat 30 °C for 30 min. Then the NHase1229@ZIF-67 was separ-ated by centrifugation (10 000 rpm, 5 min) at 4 °C, and wasfurther added to a fresh substrate solution to start a new cata-lytic reaction. The amounts of nicotinamide and 3-cyanopyri-dine were measured by the HPLC method as previouslydescribed in ref. 6.

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Fed-batch biotransformation in a fixed-bed bioreactor

A fed-batch biocatalytic process was implemented in a 20 mLreaction mixture containing 0.5 g NHase1229@ZIF-67 toincrease the final concentration of nicotinamide. Four batchesof 4.68 g 3-cyanopyridine (0.9 mol L−1 concentration) wereadded to the reaction mixture. The reaction was performed at20 °C at 200 rpm. The samples were periodically withdrawnand analysed by the HPLC method.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This study was financially supported by the Natural ScienceFoundation of Zhejiang Province (grant No. LY15B060011,LY17C050001, and LY18B060009), the China PostdoctoralScience Foundation Funded Project (grant no. 2016M601966),the 2nd Climbing Project for Discipline Construction ofHangzhou Normal University, the National Natural ScienceFoundation of China (grant no. 21576062), and the NationalTraining Program of Innovation and Entrepreneurship forUndergraduates of Hangzhou Normal University.

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972 | Nanoscale, 2020, 12, 967–972 This journal is © The Royal Society of Chemistry 2020

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