Covalent attachment of the enzymes glucose oxidase and catalase to carbon nanotubes enables the tandem catalytic conversion of glucose and H2O2 formed to power autonomous movement of the nanotubes.

Browne, Wesley R.,

Feringa, Ben L.,

Pantarotto, Davide,

English

Covalent attachment of the enzymes glucose oxidase and catalase to carbon nanotubes enables the tandem catalytic conversion of glucose and H2O2 formed to power autonomous movement of the nanotubes.</p

Pantarotto, Davide

Browne, Wesley R.

Feringa, Ben L.

University of Groningen

   University of GroningenAutonomous propulsion of carbon nanotubes powered by a multienzyme ensemblePantarotto, Davide; Browne, Wesley R.; Feringa, Ben L.Published in:Chemical CommunicationsDOI:10.1039/b715310dIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2008Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Pantarotto, D., Browne, W. R., & Feringa, B. L. (2008). Autonomous propulsion of carbon nanotubespowered by a multienzyme ensemble. Chemical Communications, 10(13), 1533-1535.https://doi.org/10.1039/b715310dCopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-11-2019Autonomous propulsion of carbon nanotubes powered by a multienzymeensemblewDavide Pantarotto, Wesley R. Browne and Ben L. Feringa*Received (in Cambridge, UK) 5th October 2007, Accepted 8th November 2007First published as an Advance Article on the web 16th November 2007DOI: 10.1039/b715310dCovalent attachment of the enzymes glucose oxidase and cata-lase to carbon nanotubes enables the tandem catalytic conversionof glucose and H2O2 formed to power autonomous movement ofthe nanotubes.From intracellular transport and cell division to muscle con-traction, nature’s protein-based molecular motors1 serve as aconstant source of inspiration to scientists in developing bothbiohybrid2–5 and wholly synthetic nano-devices6–11 poweredby light12–15 and chemical fuels.16–19 The chemical fuel dihy-drogen peroxide has proven to be exceptionally versatile inpowering microscopic motion;20–25 however, for this fuel to beapplied successfully, eﬃcient conversion of chemical energy ina controlled manner is essential to achieve motion at the levelseen in nature’s use of ATP. Bimetallic20–23,25 and molecular24systems have been employed to drive objects of mm dimensionusing dioxygen bubbles or concentration gradients formed bythe catalytic disproportionation of H2O2. However, withH2O2, its inherent reactivity limits its application in practicaldevices where such controlled energy release is required.Here we describe a biohybrid system comprising enzymefunctionalized carbon nanotubes that show autonomoustranslational movement. Covalent attachment of the enzymesglucose oxidase and catalase to carbon nanotubes enables thetandem catalytic conversion of the ubiquitous natural fuelglucose to kinetic energy. Acting in concert, remarkably, thesetwo enzymes produce gaseous molecular dioxygen to propelnanoobjects—rather than the anticipated mechanism invol-ving dioxygen depletion—and provide exciting opportunitiestowards both autonomous nanoscale transport within biolo-gical microenvironments and in exploring the behaviour ofcomplex multienzyme ensembles.In our approach, in situ generation and rapid consumptionof the H2O2 fuel from the relatively stable and ubiquitousbiomolecule b-D-glucose through enzymatic activation of di-oxygen provides an attractive alternative to the direct use ofH2O2 as fuel. For the basic structure of the biohybrid propul-sion system (Fig. 1) we chose multi-walled carbon nanotubes(MWCNTs), taking advantage of their peculiar aspect ratioand shape and the methods developed recently for theircovalent functionalization.26–33 Furthermore, MWCNTs func-tionalized with biomolecules are attractive targets with poten-tial application as cellular delivery vehicles.30–33 Watersolubilization, by oxidation under strongly acidic conditionsto introduce carboxylic acid groups at the periphery, ofcommercial MWCNTs (diameter 20–80 nm, length 0.5–5 mm)29,34 and puriﬁcation was followed by analysis andstructural characterization using transmission and scanningelectron (TEM, SEM) and atomic force (AFM) microscopictechniques (see ESIw). The enzymes were coupled to thecarboxylic acid functionalized MWCNTs in aqueous solutionusing the coupling reagent 1-ethyl-3-(3-dimethylaminopro-pyl)-carbodiimide (EDC) in a phosphate buﬀer solution(PBS).Three diﬀerent conjugates were prepared (see ESI fordetailsw): 1 GOx functionalized MWCNTs; 2 catalase functio-nalized MWCNTs; 3GOx–catalase functionalized MWCNTs.The individual enzymes were coupled in conjugates 1 and 2 ascontrols and in order to assess their structural and functionalintegrity and stability when attached covalently to MWCNTs.The loading of each enzyme onto the external surface of thenanotubes was calculated both from assaying enzymatic ac-tivity and from the unreacted enzyme remaining after thecoupling reaction (see ESIw). For compound 1 an averageloading of 296 mg of catalase per mg of nanotubes wasdetermined, whereas for 2, 680 mg of GOx per mg of nano-tubes was found. The average enzyme coverage for compound3 was ﬁxed at 50% each of the previous values for theindividual enzymes. For conjugate 3 a decrease in waterFig. 1 A biohybrid propulsion system based on the enzymes glucoseoxidase (GOx) and catalase, immobilized covalently to water-solubleMWCNTs bearing carboxyl functionalities. The conversion of glucoseby GOx produces dihydrogen peroxide, which is decomposed bycatalase to generate dioxygen gas bubbles which propel the MWCNTs.Department of Organic Chemistry, Stratingh Institute for Chemistryand Zernike Institute for Advanced Materials, University ofGroningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.E-mail: b.l.feringa@rug.nl; Fax: +31-503634278; Tel: +31-503634296w Electronic supplementary information (ESI) available: Synthesisand characterization of 1–3, experimental details and video ﬁlesshowing movement of nanotubes in the presence of oxygen. SeeDOI: 10.1039/b715310dThis journal is c The Royal Society of Chemistry 2008 Chem. Commun., 2008, 1533–1535 | 1533COMMUNICATION www.rsc.org/chemcomm | ChemCommsolubility was observed (o1 mg ml1) compared to 1 (stablecolloidal suspension 3.2 mg ml1) and 2 (stable colloidalsuspension 4.4 mg ml1). Aggregates of 3 featuring large linearor spherical bundles ranging from 10–150 mm in length wereobserved by optical microscopy (see ESIw). Therefore moredilute aqueous solutions of 3 (typically 100 mg ml1) were usedfor TEM, SEM and AFM imaging (Figs. 2 and 3).Comparison of SEM images of oxidized MWCNTs(Fig. 2A) and conjugate 3 (Fig. 2B), after sputtering with athin layer of Pt, revealed the coverage of the external surfaceof the nanotubes with the enzymes. Several grains are presenton the surface and analysis of the grain size showed homo-geneous enzyme coverage. AFM analysis established thelength of the MWCNTs prior to and after attachment of bothenzymes (as in 3) as well as the presence of the enzymes on thewalls as can be seen in Fig. 2C,D. Fourier-transform infraredspectroscopic analysis of 1 and 2 veriﬁed that only a slightmodiﬁcation in secondary structure (a-helix and b-sheet) ofthe enzymes is induced upon grafting to the MWCNT surfaceif compared with the free enzyme (see ESIw).Analysis of TEM images of 3 reveals the coverage of theexternal surface of a carbon nanotube by the two enzymes indetail (Fig. 3). In this case negative staining with uranylacetate was necessary to visualize the biomolecules. Fromthe heterogeneous distribution at the end section of the tubeand the dimensions (range 15–30 nm) (Fig. 3, inset), it isreasonable to conclude that several enzyme units are present ineach enzyme aggregate.Optical microscopy enables demonstration of autonomousmovement and the eﬀectiveness of the propulsion system dueto the concerted action of the two enzymes present in 3. Whena sample of 3 was dispersed in an aqueous phosphate buﬀeredsolution at pH 6.8 on a glass cover-slip, with a continuous airﬂow over the liquid ﬁlm (suﬃciently gentle so as to maintain astable oxygen concentration in solution while not disturbingthe thin ﬁlm of liquid), in the presence of glucose (100 mM),within a few seconds gentle bubbling of oxygen was observed.The generation of oxygen bubbles induced a translationalmotion of the small aggregates and bundles of functionalizedMWCNTs 3 in the thin liquid ﬁlm (Fig. 4A, see also video 1,ESIw).A number of mechanisms for generation of kinetic motionusing the catalytic decomposition of H2O2 have been pro-posed, including the generation of oxygen concentration gra-dients and oxygen bubble formation.20–25 In the presentsystem overall the oxidation of glucose by GOx and subse-quent disproportionation of H2O2 by catalase leads to adepletion in the local oxygen concentration and hence move-ment driven by oxygen concentration gradients would beanticipated. Remarkably, the co-immobilization of the GOxand catalase enzymes, in mixed enzyme assemblies, enables theformation of transiently high oxygen concentrations allowingfor oxygen bubble formation and, through this, movement ofthe nanotube objects.Depletion of oxygen in solution (i.e., by halting the con-tinuous ﬂow of air) results eventually in a decrease in themovement of 3 and a decrease in the size of the bubblesFig. 2 SEM and AFM images of nanotubes before and after attach-ment of enzymes. SEM images of: (A) oxidized MWNT (diameter30 nm) and (B) conjugate 3 (diameter 85 nm due to the presence of theenzyme) after Pt treatment. Bar caption 1 mm. AFM analysis of: (C)oxidizedMWNT and (D) conjugate 3 on a ﬂat silicon surface. Averagelengths of 1.1 mm and 0.97 mm were measured for C and D, respec-tively. The edges of the tubes (D) are ill-deﬁned due to the presence ofthe enzymes.Fig. 3 Microscope image of carbon nanotube with enzymes attached.TEM image of conjugate 3. The image was recorded at 100 kV afternegative staining with uranyl acetate. The MWCNT analyzed has anouter diameter of 20 nm. Globular enzymatic particles have variabledimensions (diameter 15–30 nm), suggesting additional aggregationphenomena. The slow drying process of the specimen can inducefurther aggregation of the biomolecules on the nanotubes. The insetshows the distribution of the enzyme on the end part of the tube. Barcaption 200 nm.Fig. 4 Eﬀect of exclusion of oxygen on autonomous movement.Observation by video-microscopy of conjugate 3 after addition of aglucose (100 mM) solution under constant air ﬂow (A). TheMWCNTsaggregates are visible as black irregular shapes. Bubbles generatedfrom the aggregate are visible as spherical objects. The trajectories ofautonomous movement of small aggregates of 3 are shown (see video1, ESI). When air ﬂow is stopped (B), the rate of bubble formation isreduced. In this case larger aggregates are observed. Bar caption50 mm.1534 | Chem. Commun., 2008, 1533–1535 This journal is c The Royal Society of Chemistry 2008formed. GOx converts glucose into gluconolactone with mo-lecular oxygen producing dihydrogen peroxide. Thereforeoxygen is required to sustain the conversion of glucose byGOx. When catalase is present the H2O2 is disproportionatedinto water and dioxygen with the formation of dioxygenbubbles. Bubble formation is preferred rather than dissolutionof the dioxygen produced into the aqueous phase followed byconsumption in subsequent GOx enzymatic cycles. The pre-sence of the continuous ﬂow of air or pure dioxygen over thesample maintains the overall concentration of dioxygen in thesolution and hence the conversion of glucose. In accordancewith this rationale, a similar experiment with 3 under a purenitrogen ﬂow over the ﬁlm led to a complete cessation of thebubble formation and the motion was limited, driven only bythe less eﬃcient production of gluconolactone and water. Itappears that most of the oxygen produced remains dissolvedand/or is consumed again by GOx.Control experiments show that neither 1 nor 2 can engage inthe same autonomous motion observed for 3. The diﬀerence inmotility between 3, containing the enzyme propulsion system,and the corresponding non-functionalized MWCNTs is ex-empliﬁed further in experiments where mixtures of both typesof MWCNTs were examined (videos 1 and 2, see ESIw).Furthermore, aggregates of 3 in thin solvent ﬁlms show thattranslational movement induced by liquid ﬂow or eﬀects dueto temperature dependent Brownian motion is not signiﬁcantwith these nanometre size (200–800 nm) functionalizedMWCNT aggregates. The bundles and aggregates of 3, uponaddition of glucose, were propelled at a speed of approxi-mately 0.2–0.8 cm s1 with the speed decreasing over severalminutes in accordance with the depletion of glucose in solu-tion, thereby reducing enzymatic conversion, and leading to acessation of oxygen bubble formation (Fig. 4A, see also video1, ESIw). However, analysis of the movement of much smallerMWCNT bundles (o100 nm) shows liquid ﬂow and Brow-nian motion contribute signiﬁcantly to the dynamic behaviourof 3 (video 3, see ESIw).Whereas the autonomous movement achieved with 3 ischaotic, the trajectory in the liquid phase appears to bedependent strictly on the shape of the aggregate as well asthe position of the enzymes on the MWCNTs. With thepresent system all our observations point to a propulsionmechanism resulting from local oxygen bubble formation.These results indicate that directionality might be introducedinto these systems by controlling the shape of the aggregatesformed and by topological control of the coverage of thenanotube by the enzymes.33,34 As discussed above, this is aremarkable observation as in solution the non-concerted ac-tion of GOx and catalase would act to deplete the concentra-tion of molecular oxygen in solution. The approach taken heredemonstrates that when held in proximity the kinetic beha-viour of mixed enzyme ensembles can surprise us and achievefunctions which are thermodynamically unexpected. Thesepreliminary observations will not only guide eﬀorts to controldirectionality in these autonomously moving carbon nano-tubes but oﬀer fascinating opportunities in exploring thebehaviour of complex multienzyme ensembles that are other-wise inaccessible ex-vivo.The authors thank the NanoNed consortium and TheNetherlands Organisation for Scientiﬁc Research (NWO-CW) for ﬁnancial support.Notes and references1 Molecular Motors, ed. M. Schliwa, Wiley-VCH, Weinheim, 2003.2 H. Hess, G. D. Bachand and V. Vogel, Chem.–Eur. J., 2004, 10,2110.3 H. Hess and G. D. Bachand, Nanotoday, 2005, 8, 22.4 K. Kinbara and T. Aida, Chem. Rev., 2005, 105, 1377.5 M. G. L. van den Heuvel and C. Dekker, Science, 2007, 317, 333.6 W. R. Browne and B. L. Feringa, Nat. Nanotechnol., 2006, 1, 25.7 V. Balzani, M. Venturi and A. Credi, Molecular Devices andMachines: A Journey in the Nano World, Wiley-VCH, Weinheim,2003.8 R. A. L. Jones, Soft Machines, Nanotechnology and Life, OxfordUniversity Press, Oxford, 2004.9 A. R. Pease, J. O. Jeppesen, J. F. Stoddart, Y. Luo, C. P. Collierand J. R. Heath, Acc. Chem. Res., 2001, 34, 433.10 J.-P. Sauvage, Molecular Machines and Motors, Springer, Berlin,2001.11 E. R. Kay, D. A. Leigh and F. Zerbetto, Angew. Chem., Int. Ed.,2007, 46, 72.12 N. Koumura, R. W. J. Zijlstra, R. A. van Delden, N. Harada andB. L. Feringa, Nature, 1999, 401, 152.13 R. A. van Delden, M. K. J. ter Wiel, M. M. Pollard, J. Vicario, N.Koumura and B. L. Feringa, Nature, 2005, 437, 1337.14 R. Eelkema, M. M. Pollard, J. Vicario, N. Katsonis, B. S. Ramon,C. W. M. Bastiaansen, D. J. Broer and B. L. Feringa,Nature, 2006,440, 163.15 S. Saha and J. F. Stoddart, Chem. Soc. Rev., 2007, 36, 77.16 T. R. Kelly, H. De Silva and R. A. Silva, Nature, 1999, 401, 150.17 D. A. Leigh, J. K. Y. Wong, F. Dehez and F. Zerbetto, Nature,2003, 424, 174.18 S. P. Fletcher, F. Dumur, M. M. Pollard and B. L. Feringa,Science, 2005, 310, 80.19 P. L. Anelli, N. Spencer and J. F. Stoddart, J. Am. Chem. Soc.,1991, 113, 5131.20 R. F. Ismagilov, A. Schwartz, N. Bowden and G. M. Whitesides,Angew. Chem., Int. Ed., 2002, 41, 652.21 T. R. Kline, W. F. Paxton, T. E. Mallouk and A. Sen, Angew.Chem., Int. Ed., 2005, 44, 744.22 J. M. Catchmark, S. Subramanian and A. Sen, Small, 2005, 1,202.23 S. Fornier-Bidoz, A. C. Arsenault, I. Manners and G. A. Ozin,Chem. Commun., 2005, 441.24 J. Vicario, R. Eelkema, W. R. Browne, A. Meetsma, R. M. LaCrois and B. L. Feringa, Chem. Commun., 2005, 3936.25 Y. Wang, R. M. Hernandez, D. J. Bartlett, J. M. Bingham, T. R.Kline, A. Sen and T. E. Mallouk, Langmuir, 2006, 22, 10451.26 J. Chen, M. A. Hamon, H. Hu, Y. S. Chen, A. M. Rao, P. C.Eklund and R. C. Haddon, Science, 1998, 282, 95.27 J. Liu, A. G. Rinzler, H. J. Dai, J. H. Hafner, R. K. Bradley, P. J.Boul, A. Lu, T. Iverson, K. Shelimov, C. B. Huﬀman, F. Rodri-guez-Macias, Y. S. Shon, T. R. Lee, D. T. Colbert and R. E.Smalley, Science, 1998, 280, 1253.28 R. C. Haddon, Acc. Chem. Res., 2002, 35, 997.29 I. D. Rosca, F. Watari, M. Uo and T. Akasaka, Carbon, 2005, 43,3124.30 N. Mano and A. Heller, J. Am. Chem. Soc., 2005, 127, 11574.31 D. Cui, J. Nanosci. Nanotechnol., 2007, 7, 1298.32 K. Kostarelos, L. Lacerda, G. Pastorin, W. Wu, S. Wieckowski, J.Luangsivilay, S. Godefroy, D. Pantarotto, J. P. Briand, S. Muller,M. Prato and A. Bianco, Nat. Nanotechnol., 2007, 2, 108.33 D. Tasis, N. Tagmatarchis, A. Bianco and M. Prato, Chem. Rev.,2006, 106, 1105.34 H. Dumortier, S. Lacotte, G. Pastorin, R. Marega, W. Wu, D.Bonifazi, J.-P. Briand, M. Prato, S. Muller and A. Bianco, NanoLett., 2006, 6, 1522.This journal is c The Royal Society of Chemistry 2008 Chem. Commun., 2008, 1533–1535 | 1535

Autonomous propulsion of carbon nanotubes powered by a multienzyme ensemble

Covalent attachment of the enzymes glucose oxidase and catalase to carbon nanotubes enables the tandem catalytic conversion of glucose and H2O2 formed to power autonomous movement of the nanotubes

NARCIS 

ARTS repository - University of Groningen

   University of GroningenAutonomous propulsion of carbon nanotubes powered by a multienzyme ensemblePantarotto, Davide; Browne, Wesley R.; Feringa, Ben L.Published in:Chemical CommunicationsDOI:10.1039/b715310dIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2008Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Pantarotto, D., Browne, W. R., & Feringa, B. L. (2008). Autonomous propulsion of carbon nanotubespowered by a multienzyme ensemble. Chemical Communications, 10(13), 1533-1535.https://doi.org/10.1039/b715310dCopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 17-07-2023 1 Autonomous Propulsion of Carbon Nanotubes Powered by a Multi-Enzyme Ensemble. Davide Pantarotto, Wesley R. Browne, Ben L. Feringa* Department of Organic Chemistry, Stratingh Institute for Chemistry and Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands *To whom correspondence should be addressed. E-mail: b.l.feringa@rug.nl  Supplementary Information  All the reagents and solvents were obtained from commercial suppliers and used without further purification. Multiwalled carbon nanotubes (CNTs) were purchased from Nanostructured and Amorphous Materials, Inc. (Houston, USA). Catalase from bovine liver and Glucose oxidase from Aspergillus Niger were purchased from Sigma Aldrich and used as received. Characterization of CNTs The acid treatment of commercial CNTs was carried out to dissolve metal species, breakdown amorphous carbon, and introduce reactive groups along the sidewalls of the nanotubes.1 After treatment, the CNTs were characterized by electron microscopy (TEM, SEM) and atomic force microscopy (AFM).  For AFM, a drop of the diluted nanotube suspension in pure water or distilled dichloromethane was deposited onto a clean flat silicon surface. The solvent was allowed Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 2 to evaporate over 10 min. The substrate was analyzed by tapping mode AFM (Digital Instruments Nanoscope III). TEM characterization was carried out on a JEOL 1200EX operating at 100 KV. A drop of diluted nanotube suspension was deposited on a carbon film copper grid. The drop was dry after 5 min. Negative staining was not required. The SEM measurements were performed on a JEOL JSM 7000F field emission SEM operating at 2.0 kV. A nanotube suspension (150 l) was deposited on a glass slit covered with a 120 nm Al top-layer on which a 1 nm Cr layer was sputtered. These samples where allowed to dry at ambient temperatures and used without additional staining. Quantitative analysis of carboxylic sites was performed using the quantitative Kaiser test2 after further functionalisation of the oxidized nanotubes with 1,8-diamino-3,6-dioxaoctane (vide infra). Preparation of water soluble CNTs After gently grinding, commercial CNTs (160.0 mg) were suspended in 150 ml of a mixture of concentrated H2SO4/HNO3 (3/1 v/v) and sonicated for 5 h. The suspension was then cooled to RT and diluted to 500 ml with 2 M aqueous NaOH in an ice bath. The solution was filtered through a PTFE filter (pore size 0.22 m) under vacuum. The black residue collected was washed thoroughly with deionized water until the filtrate was pH 6 and subsequently dried under vacuum at 60 ºC. The product, 92.0 mg, was collected and suspended in 30 ml of Pirahna solution (H2SO4 98% / H2O2 30 vol 4/1 v/v) and stirred r.t. for 1 h. The solution was filtered over a 0.22 m pore size polycarbonate membrane and washed extensively with deionised water. The Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 3 black material on the collected was dried under vacuum at 60 ºC to yield 88.0 mg of product. The material was stored as a solid or as a stable solution (concentration of 1 mg/ml). The IR spectroscopy shows absorptions at 1710 cm-1 (carbonyl stretching) and a broad absorption at 1100 cm-1 (C-O stretching). The loading was calculated using a quantitative Kaiser test2 after functionalization of oxidized carbon nanotubes (typically 20 mg) first with an excess of oxalyl chloride and, after removal of the excess, by treatment with 1,8-diamino-3,6-dioxaoctane in tetrahydrofurane (THF) at 60°C for 2 h. The resulting suspension was filtered and the black solid washed with methanol and dried. To perform the Kaiser test three solutions were prepared in parallel. (I) 10 g of phenol dissolved in 20 mL of ethanol, (II) 2 mL of KCN 1 mM (aqueous solution) dissolved in 98 mL of pyridine, (III) 1.0 g of ninhydrine dissolved in 20 mL of ethanol. Approximately 20 mg of free amino CNTs was weighed accurately. Non-amino CNT was tested also as a control solution and treated in the same way. 75 µl of solution I, 100 µl of solution II and 75 µl of solution III were added to a haemolysis test tube. The test tube is incubated in a heating block at 100°C for 7 min and then removed and added immediately to 4.8 mL of 60% ethanol to make a final volume of 5 mL. The tube was then mixed to render the violet colour homogenous. The absorbance of each sample was measured relative to the blank CNT solution (pale yellow) at 570 nm. The amine loading was calculated from the equation:   [ ] (mg) weight sample t coefficien Extinction10)ml(dilution AbsAbsgmol6×××−= blanksampleµ (Dilution=5mL and Extinction coefficient = 15000 m-1cm-1). Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 4 The values are expressed as micromole of amino groups per gram of material and are an average of at least ten measurements at different dilutions.  Covalent enzyme conjugate 1  Oxidized CNTs (2.0 mg, loading 357±14 mol/g) were dispersed in 500 l of deionised water and activated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) solution (0.2 M in PBS pH 7) for 2 h at room temperature. 500 l of Catalase (62000 U or 1.5 mg) in phosphate buffer solution (PBS pH 7.6) were added. The reaction was carried out within 5 h at r.t. The final suspension was filtered on a 0.22 m pore size nitrocellulose filter and washed with 30 ml of pure water. The black solid obtained (3.4 mg) was suspended in 2.0 ml of pure water and centrifuged twice at 4000 rpm with discharge of the supernatant. The precipitate collected was dissolved in 2.0 ml of water and stored at 4 ºC. A small amount was used to determine the enzymatic activity to calculate the amount of enzyme present (vide infra). The enzymatic activity was conserved for at least 4 months. Covalent enzyme conjugate 2 Oxidized CNTs (2.0 mg, loading 357±14 mol/g) were dispersed in 500 l of deionised water and activated with EDC solution (0.2 M in water) for 2 h at room temperature. 500 l of a solution of -D-Glucose oxygen 1-oxidoreductase (1060 U or 3.6 mg) were added. The reaction was carried out within 5 h at r.t. The final suspension was filtered using a 0.22 m pore size nitrocellulose filter and washed with 120 ml of pure water. The black solid obtained (3.3 mg) was suspended in 4 ml of pure water and centrifuged twice at 2000 rpm separated from the supernatant. The precipitate collected was redissolved in 1.0 Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 5 ml of water and stored at 4 ºC. A small amount was used to determine the enzymatic activity and allowed to calculate the amount of enzyme present (vide infra). The enzymatic activity was conserved for at least 4 months. Covalent enzyme conjugate 3 Oxidized CNTs (5.0 mg, loading 357±14 mol/g) were dispersed in 1 ml of deionised water and activated with EDC solution (0.2 M in water ) for 2 h at room temperature. 1 ml of a solution of Catalase (62000 U) in PBS (pH=7.6) were mixed with 500 l of a solution of -D-Glucose oxygen 1-oxidoreductase (GOx) (1060 U) and added slowly to the activated nanotubes. The reaction was continued for 5 h at r.t. The final suspension was filtered through a 0.22 m pore size nitrocellulose filter and washed with 30 ml of pure water. The black solid obtained (8.2 mg) was suspended in 2 ml of pure water and centrifuged twice at 4000 rpm and separated from the supernatant. The precipitate collected was dissolved in 10 ml of water and stored at 4 ºC. A small amount was used to determine the enzymatic activity to calculate the amount of each enzyme present (vide infra).          Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 6 Catalase enzymatic activity assay  Catalase activity was measured using H2O2 as substrate.3 Catalase dispropotionates H2O2 to form oxygen and water in a pH 7.0 PBS buffer at 25 ºC. For a typical assay the disappearance of H2O2 was monitored by UV spectroscopy at 240 nm.  Table 4. Enzymatic assay activity of conjugate 1. Results from three independent experiments.  Conjugate 1 (mg) Measured timea (min) Enzymatic units b Amount of enzyme c (g) Amount of catalase (g /mg) 1.20 2.18 15797 355 295.83 1.30 2.35 17088 384 295.38 0.80 3.08 10502 251 200.80 aTime expressed in min to decrease absorbance at 240nm from 0.45 to 0.40. bUnits/ml = (3.45*dilution factor)/(time*volume of enzyme in ml)  c From supplier 44500 Units/mg  A solution of 2.9 ml of 0.0036% (w/w) H2O2 in PBS was prepared with a stable absorbance at 240 nm between 0.55 and 0.52. 100l of conjugate 1 solution (weight amount dissolved in 2 ml) was added by inversion (at 25ºC) and the kinetics in the loss of absorbance at 240 nm from 0.45 to 0.40 units was recorded and the number of enzymatic unit was calculated by: Enzymatic Units/ml = (A500/min Gox- A500/min Blank)*3.1*(dF)/(7.5*0.1)  Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 7 Glucose oxidase enzymatic assay GOx activity was detected measuring the production of H2O2 in a 0.1 M acetate buffer at 35 ºC and pH 5.0 upon addition of D(+)-glucose.4 The concentration was monitored with time at 240 nm using a spectrophotometer.  Table 5. Enzymatic assay activity of conjugate 2. Results from three independent experiments. Conjugate 2 (mg) (A500/min Gox- A500/min Blank) dF=100 Enzymatic units a  Amount of enzyme b (mg) Amount of GOx (g /mg) 2.21 0.721 300.4 1.50 678.73 2.32 0.763 315.4 1.57 676.72 4.10 1.349 557.6 2.78 678.04 a Enzymatic Units/ml = (A500/min Gox- A500/min Blank)*3.1*(dF)/(7.5*0.1)  b From supplier 200 Units/mg   A solution of 2.9 ml combining 24 ml of 0.21 mM o-dianisine in 50mM sodium acetate buffer (pH 5.1) and 5 ml of 10% w/v -D-Glucose in deionized water were mixed with 100 l of prepared solution of 60 purpurogallin units/ml of peroxidase type II in cold water, equilibrated at 35ºC and monitored at 500 nm absorbance until stable. Then 100 l of glucose oxidase solution in 50 mM sodium acetate buffer was added and the increasing in absorbance at 500 nm was recorded for approximately 5 min. For the blank test 100 l Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 8 of simple acetate buffer solution was added instead of glucose oxidase solution. The concentration of enzymatic units was calculated by4: Enzymatic Units/ml = (A500/min Gox- A500/min Blank)*3.1*(dilution factor)/(7.5*0.1) where 3.1 is the total volume, 7.5 is the millimolar extinction coefficient of oxidized o-dianisine at 500 nm and 0.1 is the volume of enzyme used in the test.   FT-IR Spectroscopy The secondary structure of native and adsorbed enzymes was determined using FT-IR spectroscopy. All spectra were collected using a Nicolet Magna 550 series II. The spectrometer was equipped with a mercury cadmium telluride detector cooled by liquid nitrogen. The data collection and second-derivative calculations were carried out using the software OMNIC 6.1a provided by Thermo Nicolet Corporation. An average of 170 scans were obtained at a resolution of 2 cm-1 with a gain of 2 and an aperture of 50. Typically, lyophilized compound 1, 2 and 3 (1 mg) were ground gently with IR grade KBr to form a homogeneous grey powder. The grinding was performed under nitrogen atmosphere to prevent absorption of atmospheric water. The spectra of the enzymes, both native and attached to the nanotubes were baseline corrected over the entire spectral window. Secondary structural content of the enzymes was obtained by analysis of their FT-IR spectra in the region from 1600 to 1700 cm-1. To obtain the second-derivative spectrum, a baseline correction was applied to the corrected spectra in the region from 1750 to 2000 cm-1, which is critical to obtain quantitative structural information using the second-derivative analysis method5. The second-derivative spectrum was then corrected Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 9 in the region 1600-1700 cm-1 to obtain a flat baseline. This spectrum was used to obtain the -helix and -sheet contents of the enzymes5,6.  Table 2. Secondary structure of enzymes at 20 ºC from FT-IR amide I band analysis compared with the reference native enzyme.   Assignment of components Native Catalase  Conjugate 1 Native GOx  Conjugate 2 β-sheets (%) (1621 cm-1) 12.3 13.2 18.4 22.5 α-helix (%) (1647 cm-1) 26.0 35.3 28.8 27.6   4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0   Figure 8. FT-IR reflectance spectrum of liophylized catalase Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 10 4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0   Figure 9. FT-IR reflectance spectrum of conjugate 1. 4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0   Figure 10. FT-IR reflectance spectrum of liophylized GOx enzyme.  Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 11 4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0 - 1 Figure 11. FT-IR reflectance spectrum of conjugate 2.  Microscopy characterization AFM images were taken by spreading a drop of the diluted conjugate suspension in pure water onto a clean silicon surface. The solvent was removed by evaporation by a gentle air streamfor 20 min. The substrate was then analyzed by a tapping mode AFM (Digital Instruments Nanoscope III). Pictures were analyzed using WSxM 4.0. TEM characterization was carried out on a JEOL 1200EX operating at 100 KV. A drop of diluted enzyme-nanotube water suspension was deposited on a carbon film copper grid. After 5 min the drop was blow dried and stained for 10 min with a saturated solution of uranyl acetate. The SEM measurements were performed on a JEOL JSM 7000F field emission SEM operating at 2.0 kV. 150 l of enzyme-nanotube water suspension was deposited on a glass slit covered with a 120 nm Al top-layer on which a 1 nm Cr layer was sputtered. These samples where allowed to dry at ambient temperature and used with an additional sputtered staining. Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 12 Optical microscopy analysis: Images and movies of the moving nanotubes were recorded using a Olympus BX 60 microscope, equipped with a Sony 3CCD DXC 950P digital camera, attached to a personal computer with Matrox Inspector 2.1 imaging software. Typically, a 50 l of aqueous enzyme-nanotube conjugate solution was deposited onto a glass coverslip and examined for the presence of aggregates under by optical microscopy. After careful addition of hydrogen peroxide (0.3% v/v) or D-glucose (100 mM), the movement of the carbon nanotube bundles was recorded and analyzed.    Video 1. Observation by optical microscopy of conjugate 3 after addition of a glucose solution (100 mM) under constant air flow. The CNT aggregates are visible as black irregular shape. Round shape oxygen bubbles generated from the aggregate are visible. An amount of non-enzyme-modified CNTs was added also.  Video 2. Observation by video-microscopy of conjugate 3 after addition of a glucose solution 100 mM under constant air flow. The CNT aggregates are visible as irregular black shapes. The round shapes are oxygen bubbles generated by/at the aggregates. An amount of non enzyme modified CNTs was added also.  Video 3 Observation by video-microscopy of a static Brownian diluted solution of conjugate 3 at 25ºC. The very slow movement of fiber-like aggregate is attributed to Brownian motion. Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 13  Video 4. Observation by video-microscopy of conjugate 3 after addition of a glucose solution (100mM) under constant nitrogen flow.  References  1. Liu, J., Rinzler, A. G., Dai, H., Hafner, J. H., Bradley, R. K., Boul, P. J., Lu, A., Iverson, T., Shelimov, K., Huffman, C. B., Rodriguez-Macias, F., Shon, Y. S., Lee, T. R., Colbert, D. T. & Smalley, R. E. Fullerene pipes. Science, 280, 1253 (1998). 2. Kaiser, E., Colescott, R. L., Bossinger, C. D. & Cook, P. I. Color test for detection of free terminal amino groups in solid-phase synthesis of perptides. Anal. Biochem, 34, 595-598 (1970) 3. Beers, R. F. & Sizer, I. W. A Spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195, 133-140 (1952). 4. Bergmeyer, H. U., Gawehn, K. & Grassl, M. Methods of Enzymatic Analysis, Academic Press Inc., New York, Volume I, 457 (1974). 5. Eremin, A. N., Metelitsa, D. I., Shishko, Zh. F., Mikhailova, R. V., Yasenko, M. I. & Lobanok, A. G. Thermal stability of glucose oxidase from Penicillium adametzii.  Appl. Biochem and Microbiol, 37, 579-586 (2001). 6. Karajanagi, S.S., Vertegel, A. A., Kane, R. S. & Dordick, J. S. Structure and Function of Enzymes Adsorbed onto Single-Walled Carbon Nanotubes. Langmuir 20, 11594-11599 (2004). Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007

   University of GroningenAutonomous propulsion of carbon nanotubes powered by a multienzyme ensemblePantarotto, Davide; Browne, Wesley R.; Feringa, Ben L.Published in:Chemical CommunicationsDOI:10.1039/b715310dIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2008Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Pantarotto, D., Browne, W. R., & Feringa, B. L. (2008). Autonomous propulsion of carbon nanotubespowered by a multienzyme ensemble. Chemical Communications, 10(13), 1533-1535.https://doi.org/10.1039/b715310dCopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 26-04-2023 1 Autonomous Propulsion of Carbon Nanotubes Powered by a Multi-Enzyme Ensemble. Davide Pantarotto, Wesley R. Browne, Ben L. Feringa* Department of Organic Chemistry, Stratingh Institute for Chemistry and Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands *To whom correspondence should be addressed. E-mail: b.l.feringa@rug.nl  Supplementary Information  All the reagents and solvents were obtained from commercial suppliers and used without further purification. Multiwalled carbon nanotubes (CNTs) were purchased from Nanostructured and Amorphous Materials, Inc. (Houston, USA). Catalase from bovine liver and Glucose oxidase from Aspergillus Niger were purchased from Sigma Aldrich and used as received. Characterization of CNTs The acid treatment of commercial CNTs was carried out to dissolve metal species, breakdown amorphous carbon, and introduce reactive groups along the sidewalls of the nanotubes.1 After treatment, the CNTs were characterized by electron microscopy (TEM, SEM) and atomic force microscopy (AFM).  For AFM, a drop of the diluted nanotube suspension in pure water or distilled dichloromethane was deposited onto a clean flat silicon surface. The solvent was allowed Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 2 to evaporate over 10 min. The substrate was analyzed by tapping mode AFM (Digital Instruments Nanoscope III). TEM characterization was carried out on a JEOL 1200EX operating at 100 KV. A drop of diluted nanotube suspension was deposited on a carbon film copper grid. The drop was dry after 5 min. Negative staining was not required. The SEM measurements were performed on a JEOL JSM 7000F field emission SEM operating at 2.0 kV. A nanotube suspension (150 l) was deposited on a glass slit covered with a 120 nm Al top-layer on which a 1 nm Cr layer was sputtered. These samples where allowed to dry at ambient temperatures and used without additional staining. Quantitative analysis of carboxylic sites was performed using the quantitative Kaiser test2 after further functionalisation of the oxidized nanotubes with 1,8-diamino-3,6-dioxaoctane (vide infra). Preparation of water soluble CNTs After gently grinding, commercial CNTs (160.0 mg) were suspended in 150 ml of a mixture of concentrated H2SO4/HNO3 (3/1 v/v) and sonicated for 5 h. The suspension was then cooled to RT and diluted to 500 ml with 2 M aqueous NaOH in an ice bath. The solution was filtered through a PTFE filter (pore size 0.22 m) under vacuum. The black residue collected was washed thoroughly with deionized water until the filtrate was pH 6 and subsequently dried under vacuum at 60 ºC. The product, 92.0 mg, was collected and suspended in 30 ml of Pirahna solution (H2SO4 98% / H2O2 30 vol 4/1 v/v) and stirred r.t. for 1 h. The solution was filtered over a 0.22 m pore size polycarbonate membrane and washed extensively with deionised water. The Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 3 black material on the collected was dried under vacuum at 60 ºC to yield 88.0 mg of product. The material was stored as a solid or as a stable solution (concentration of 1 mg/ml). The IR spectroscopy shows absorptions at 1710 cm-1 (carbonyl stretching) and a broad absorption at 1100 cm-1 (C-O stretching). The loading was calculated using a quantitative Kaiser test2 after functionalization of oxidized carbon nanotubes (typically 20 mg) first with an excess of oxalyl chloride and, after removal of the excess, by treatment with 1,8-diamino-3,6-dioxaoctane in tetrahydrofurane (THF) at 60°C for 2 h. The resulting suspension was filtered and the black solid washed with methanol and dried. To perform the Kaiser test three solutions were prepared in parallel. (I) 10 g of phenol dissolved in 20 mL of ethanol, (II) 2 mL of KCN 1 mM (aqueous solution) dissolved in 98 mL of pyridine, (III) 1.0 g of ninhydrine dissolved in 20 mL of ethanol. Approximately 20 mg of free amino CNTs was weighed accurately. Non-amino CNT was tested also as a control solution and treated in the same way. 75 µl of solution I, 100 µl of solution II and 75 µl of solution III were added to a haemolysis test tube. The test tube is incubated in a heating block at 100°C for 7 min and then removed and added immediately to 4.8 mL of 60% ethanol to make a final volume of 5 mL. The tube was then mixed to render the violet colour homogenous. The absorbance of each sample was measured relative to the blank CNT solution (pale yellow) at 570 nm. The amine loading was calculated from the equation:   [ ] (mg) weight sample t coefficien Extinction10)ml(dilution AbsAbsgmol6×××−= blanksampleµ (Dilution=5mL and Extinction coefficient = 15000 m-1cm-1). Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 4 The values are expressed as micromole of amino groups per gram of material and are an average of at least ten measurements at different dilutions.  Covalent enzyme conjugate 1  Oxidized CNTs (2.0 mg, loading 357±14 mol/g) were dispersed in 500 l of deionised water and activated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) solution (0.2 M in PBS pH 7) for 2 h at room temperature. 500 l of Catalase (62000 U or 1.5 mg) in phosphate buffer solution (PBS pH 7.6) were added. The reaction was carried out within 5 h at r.t. The final suspension was filtered on a 0.22 m pore size nitrocellulose filter and washed with 30 ml of pure water. The black solid obtained (3.4 mg) was suspended in 2.0 ml of pure water and centrifuged twice at 4000 rpm with discharge of the supernatant. The precipitate collected was dissolved in 2.0 ml of water and stored at 4 ºC. A small amount was used to determine the enzymatic activity to calculate the amount of enzyme present (vide infra). The enzymatic activity was conserved for at least 4 months. Covalent enzyme conjugate 2 Oxidized CNTs (2.0 mg, loading 357±14 mol/g) were dispersed in 500 l of deionised water and activated with EDC solution (0.2 M in water) for 2 h at room temperature. 500 l of a solution of -D-Glucose oxygen 1-oxidoreductase (1060 U or 3.6 mg) were added. The reaction was carried out within 5 h at r.t. The final suspension was filtered using a 0.22 m pore size nitrocellulose filter and washed with 120 ml of pure water. The black solid obtained (3.3 mg) was suspended in 4 ml of pure water and centrifuged twice at 2000 rpm separated from the supernatant. The precipitate collected was redissolved in 1.0 Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 5 ml of water and stored at 4 ºC. A small amount was used to determine the enzymatic activity and allowed to calculate the amount of enzyme present (vide infra). The enzymatic activity was conserved for at least 4 months. Covalent enzyme conjugate 3 Oxidized CNTs (5.0 mg, loading 357±14 mol/g) were dispersed in 1 ml of deionised water and activated with EDC solution (0.2 M in water ) for 2 h at room temperature. 1 ml of a solution of Catalase (62000 U) in PBS (pH=7.6) were mixed with 500 l of a solution of -D-Glucose oxygen 1-oxidoreductase (GOx) (1060 U) and added slowly to the activated nanotubes. The reaction was continued for 5 h at r.t. The final suspension was filtered through a 0.22 m pore size nitrocellulose filter and washed with 30 ml of pure water. The black solid obtained (8.2 mg) was suspended in 2 ml of pure water and centrifuged twice at 4000 rpm and separated from the supernatant. The precipitate collected was dissolved in 10 ml of water and stored at 4 ºC. A small amount was used to determine the enzymatic activity to calculate the amount of each enzyme present (vide infra).          Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 6 Catalase enzymatic activity assay  Catalase activity was measured using H2O2 as substrate.3 Catalase dispropotionates H2O2 to form oxygen and water in a pH 7.0 PBS buffer at 25 ºC. For a typical assay the disappearance of H2O2 was monitored by UV spectroscopy at 240 nm.  Table 4. Enzymatic assay activity of conjugate 1. Results from three independent experiments.  Conjugate 1 (mg) Measured timea (min) Enzymatic units b Amount of enzyme c (g) Amount of catalase (g /mg) 1.20 2.18 15797 355 295.83 1.30 2.35 17088 384 295.38 0.80 3.08 10502 251 200.80 aTime expressed in min to decrease absorbance at 240nm from 0.45 to 0.40. bUnits/ml = (3.45*dilution factor)/(time*volume of enzyme in ml)  c From supplier 44500 Units/mg  A solution of 2.9 ml of 0.0036% (w/w) H2O2 in PBS was prepared with a stable absorbance at 240 nm between 0.55 and 0.52. 100l of conjugate 1 solution (weight amount dissolved in 2 ml) was added by inversion (at 25ºC) and the kinetics in the loss of absorbance at 240 nm from 0.45 to 0.40 units was recorded and the number of enzymatic unit was calculated by: Enzymatic Units/ml = (A500/min Gox- A500/min Blank)*3.1*(dF)/(7.5*0.1)  Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 7 Glucose oxidase enzymatic assay GOx activity was detected measuring the production of H2O2 in a 0.1 M acetate buffer at 35 ºC and pH 5.0 upon addition of D(+)-glucose.4 The concentration was monitored with time at 240 nm using a spectrophotometer.  Table 5. Enzymatic assay activity of conjugate 2. Results from three independent experiments. Conjugate 2 (mg) (A500/min Gox- A500/min Blank) dF=100 Enzymatic units a  Amount of enzyme b (mg) Amount of GOx (g /mg) 2.21 0.721 300.4 1.50 678.73 2.32 0.763 315.4 1.57 676.72 4.10 1.349 557.6 2.78 678.04 a Enzymatic Units/ml = (A500/min Gox- A500/min Blank)*3.1*(dF)/(7.5*0.1)  b From supplier 200 Units/mg   A solution of 2.9 ml combining 24 ml of 0.21 mM o-dianisine in 50mM sodium acetate buffer (pH 5.1) and 5 ml of 10% w/v -D-Glucose in deionized water were mixed with 100 l of prepared solution of 60 purpurogallin units/ml of peroxidase type II in cold water, equilibrated at 35ºC and monitored at 500 nm absorbance until stable. Then 100 l of glucose oxidase solution in 50 mM sodium acetate buffer was added and the increasing in absorbance at 500 nm was recorded for approximately 5 min. For the blank test 100 l Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 8 of simple acetate buffer solution was added instead of glucose oxidase solution. The concentration of enzymatic units was calculated by4: Enzymatic Units/ml = (A500/min Gox- A500/min Blank)*3.1*(dilution factor)/(7.5*0.1) where 3.1 is the total volume, 7.5 is the millimolar extinction coefficient of oxidized o-dianisine at 500 nm and 0.1 is the volume of enzyme used in the test.   FT-IR Spectroscopy The secondary structure of native and adsorbed enzymes was determined using FT-IR spectroscopy. All spectra were collected using a Nicolet Magna 550 series II. The spectrometer was equipped with a mercury cadmium telluride detector cooled by liquid nitrogen. The data collection and second-derivative calculations were carried out using the software OMNIC 6.1a provided by Thermo Nicolet Corporation. An average of 170 scans were obtained at a resolution of 2 cm-1 with a gain of 2 and an aperture of 50. Typically, lyophilized compound 1, 2 and 3 (1 mg) were ground gently with IR grade KBr to form a homogeneous grey powder. The grinding was performed under nitrogen atmosphere to prevent absorption of atmospheric water. The spectra of the enzymes, both native and attached to the nanotubes were baseline corrected over the entire spectral window. Secondary structural content of the enzymes was obtained by analysis of their FT-IR spectra in the region from 1600 to 1700 cm-1. To obtain the second-derivative spectrum, a baseline correction was applied to the corrected spectra in the region from 1750 to 2000 cm-1, which is critical to obtain quantitative structural information using the second-derivative analysis method5. The second-derivative spectrum was then corrected Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 9 in the region 1600-1700 cm-1 to obtain a flat baseline. This spectrum was used to obtain the -helix and -sheet contents of the enzymes5,6.  Table 2. Secondary structure of enzymes at 20 ºC from FT-IR amide I band analysis compared with the reference native enzyme.   Assignment of components Native Catalase  Conjugate 1 Native GOx  Conjugate 2 β-sheets (%) (1621 cm-1) 12.3 13.2 18.4 22.5 α-helix (%) (1647 cm-1) 26.0 35.3 28.8 27.6   4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0   Figure 8. FT-IR reflectance spectrum of liophylized catalase Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 10 4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0   Figure 9. FT-IR reflectance spectrum of conjugate 1. 4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0   Figure 10. FT-IR reflectance spectrum of liophylized GOx enzyme.  Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 11 4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0 - 1 Figure 11. FT-IR reflectance spectrum of conjugate 2.  Microscopy characterization AFM images were taken by spreading a drop of the diluted conjugate suspension in pure water onto a clean silicon surface. The solvent was removed by evaporation by a gentle air streamfor 20 min. The substrate was then analyzed by a tapping mode AFM (Digital Instruments Nanoscope III). Pictures were analyzed using WSxM 4.0. TEM characterization was carried out on a JEOL 1200EX operating at 100 KV. A drop of diluted enzyme-nanotube water suspension was deposited on a carbon film copper grid. After 5 min the drop was blow dried and stained for 10 min with a saturated solution of uranyl acetate. The SEM measurements were performed on a JEOL JSM 7000F field emission SEM operating at 2.0 kV. 150 l of enzyme-nanotube water suspension was deposited on a glass slit covered with a 120 nm Al top-layer on which a 1 nm Cr layer was sputtered. These samples where allowed to dry at ambient temperature and used with an additional sputtered staining. Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 12 Optical microscopy analysis: Images and movies of the moving nanotubes were recorded using a Olympus BX 60 microscope, equipped with a Sony 3CCD DXC 950P digital camera, attached to a personal computer with Matrox Inspector 2.1 imaging software. Typically, a 50 l of aqueous enzyme-nanotube conjugate solution was deposited onto a glass coverslip and examined for the presence of aggregates under by optical microscopy. After careful addition of hydrogen peroxide (0.3% v/v) or D-glucose (100 mM), the movement of the carbon nanotube bundles was recorded and analyzed.    Video 1. Observation by optical microscopy of conjugate 3 after addition of a glucose solution (100 mM) under constant air flow. The CNT aggregates are visible as black irregular shape. Round shape oxygen bubbles generated from the aggregate are visible. An amount of non-enzyme-modified CNTs was added also.  Video 2. Observation by video-microscopy of conjugate 3 after addition of a glucose solution 100 mM under constant air flow. The CNT aggregates are visible as irregular black shapes. The round shapes are oxygen bubbles generated by/at the aggregates. An amount of non enzyme modified CNTs was added also.  Video 3 Observation by video-microscopy of a static Brownian diluted solution of conjugate 3 at 25ºC. The very slow movement of fiber-like aggregate is attributed to Brownian motion. Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 13  Video 4. Observation by video-microscopy of conjugate 3 after addition of a glucose solution (100mM) under constant nitrogen flow.  References  1. Liu, J., Rinzler, A. G., Dai, H., Hafner, J. H., Bradley, R. K., Boul, P. J., Lu, A., Iverson, T., Shelimov, K., Huffman, C. B., Rodriguez-Macias, F., Shon, Y. S., Lee, T. R., Colbert, D. T. & Smalley, R. E. Fullerene pipes. Science, 280, 1253 (1998). 2. Kaiser, E., Colescott, R. L., Bossinger, C. D. & Cook, P. I. Color test for detection of free terminal amino groups in solid-phase synthesis of perptides. Anal. Biochem, 34, 595-598 (1970) 3. Beers, R. F. & Sizer, I. W. A Spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195, 133-140 (1952). 4. Bergmeyer, H. U., Gawehn, K. & Grassl, M. Methods of Enzymatic Analysis, Academic Press Inc., New York, Volume I, 457 (1974). 5. Eremin, A. N., Metelitsa, D. I., Shishko, Zh. F., Mikhailova, R. V., Yasenko, M. I. & Lobanok, A. G. Thermal stability of glucose oxidase from Penicillium adametzii.  Appl. Biochem and Microbiol, 37, 579-586 (2001). 6. Karajanagi, S.S., Vertegel, A. A., Kane, R. S. & Dordick, J. S. Structure and Function of Enzymes Adsorbed onto Single-Walled Carbon Nanotubes. Langmuir 20, 11594-11599 (2004). Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007

University of GroningenAutonomous propulsion of carbon nanotubes powered by a multienzyme ensemblePantarotto, Davide; Browne, Wesley R.; Feringa, Ben L.Published in:Chemical CommunicationsDOI:10.1039/b715310dIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2008Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Pantarotto, D., Browne, W. R., & Feringa, B. L. (2008). Autonomous propulsion of carbon nanotubespowered by a multienzyme ensemble. Chemical Communications, 10(13), 1533-1535.https://doi.org/10.1039/b715310dCopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 30-12-2025 1 Autonomous Propulsion of Carbon Nanotubes Powered by a Multi-Enzyme Ensemble. Davide Pantarotto, Wesley R. Browne, Ben L. Feringa* Department of Organic Chemistry, Stratingh Institute for Chemistry and Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands *To whom correspondence should be addressed. E-mail: b.l.feringa@rug.nl  Supplementary Information  All the reagents and solvents were obtained from commercial suppliers and used without further purification. Multiwalled carbon nanotubes (CNTs) were purchased from Nanostructured and Amorphous Materials, Inc. (Houston, USA). Catalase from bovine liver and Glucose oxidase from Aspergillus Niger were purchased from Sigma Aldrich and used as received. Characterization of CNTs The acid treatment of commercial CNTs was carried out to dissolve metal species, breakdown amorphous carbon, and introduce reactive groups along the sidewalls of the nanotubes.1 After treatment, the CNTs were characterized by electron microscopy (TEM, SEM) and atomic force microscopy (AFM).  For AFM, a drop of the diluted nanotube suspension in pure water or distilled dichloromethane was deposited onto a clean flat silicon surface. The solvent was allowed Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 2 to evaporate over 10 min. The substrate was analyzed by tapping mode AFM (Digital Instruments Nanoscope III). TEM characterization was carried out on a JEOL 1200EX operating at 100 KV. A drop of diluted nanotube suspension was deposited on a carbon film copper grid. The drop was dry after 5 min. Negative staining was not required. The SEM measurements were performed on a JEOL JSM 7000F field emission SEM operating at 2.0 kV. A nanotube suspension (150 l) was deposited on a glass slit covered with a 120 nm Al top-layer on which a 1 nm Cr layer was sputtered. These samples where allowed to dry at ambient temperatures and used without additional staining. Quantitative analysis of carboxylic sites was performed using the quantitative Kaiser test2 after further functionalisation of the oxidized nanotubes with 1,8-diamino-3,6-dioxaoctane (vide infra). Preparation of water soluble CNTs After gently grinding, commercial CNTs (160.0 mg) were suspended in 150 ml of a mixture of concentrated H2SO4/HNO3 (3/1 v/v) and sonicated for 5 h. The suspension was then cooled to RT and diluted to 500 ml with 2 M aqueous NaOH in an ice bath. The solution was filtered through a PTFE filter (pore size 0.22 m) under vacuum. The black residue collected was washed thoroughly with deionized water until the filtrate was pH 6 and subsequently dried under vacuum at 60 ºC. The product, 92.0 mg, was collected and suspended in 30 ml of Pirahna solution (H2SO4 98% / H2O2 30 vol 4/1 v/v) and stirred r.t. for 1 h. The solution was filtered over a 0.22 m pore size polycarbonate membrane and washed extensively with deionised water. The Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 3 black material on the collected was dried under vacuum at 60 ºC to yield 88.0 mg of product. The material was stored as a solid or as a stable solution (concentration of 1 mg/ml). The IR spectroscopy shows absorptions at 1710 cm-1 (carbonyl stretching) and a broad absorption at 1100 cm-1 (C-O stretching). The loading was calculated using a quantitative Kaiser test2 after functionalization of oxidized carbon nanotubes (typically 20 mg) first with an excess of oxalyl chloride and, after removal of the excess, by treatment with 1,8-diamino-3,6-dioxaoctane in tetrahydrofurane (THF) at 60°C for 2 h. The resulting suspension was filtered and the black solid washed with methanol and dried. To perform the Kaiser test three solutions were prepared in parallel. (I) 10 g of phenol dissolved in 20 mL of ethanol, (II) 2 mL of KCN 1 mM (aqueous solution) dissolved in 98 mL of pyridine, (III) 1.0 g of ninhydrine dissolved in 20 mL of ethanol. Approximately 20 mg of free amino CNTs was weighed accurately. Non-amino CNT was tested also as a control solution and treated in the same way. 75 µl of solution I, 100 µl of solution II and 75 µl of solution III were added to a haemolysis test tube. The test tube is incubated in a heating block at 100°C for 7 min and then removed and added immediately to 4.8 mL of 60% ethanol to make a final volume of 5 mL. The tube was then mixed to render the violet colour homogenous. The absorbance of each sample was measured relative to the blank CNT solution (pale yellow) at 570 nm. The amine loading was calculated from the equation:   [ ] (mg) weight sample t coefficien Extinction10)ml(dilution AbsAbsgmol6×××−= blanksampleµ (Dilution=5mL and Extinction coefficient = 15000 m-1cm-1). Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 4 The values are expressed as micromole of amino groups per gram of material and are an average of at least ten measurements at different dilutions.  Covalent enzyme conjugate 1  Oxidized CNTs (2.0 mg, loading 357±14 mol/g) were dispersed in 500 l of deionised water and activated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) solution (0.2 M in PBS pH 7) for 2 h at room temperature. 500 l of Catalase (62000 U or 1.5 mg) in phosphate buffer solution (PBS pH 7.6) were added. The reaction was carried out within 5 h at r.t. The final suspension was filtered on a 0.22 m pore size nitrocellulose filter and washed with 30 ml of pure water. The black solid obtained (3.4 mg) was suspended in 2.0 ml of pure water and centrifuged twice at 4000 rpm with discharge of the supernatant. The precipitate collected was dissolved in 2.0 ml of water and stored at 4 ºC. A small amount was used to determine the enzymatic activity to calculate the amount of enzyme present (vide infra). The enzymatic activity was conserved for at least 4 months. Covalent enzyme conjugate 2 Oxidized CNTs (2.0 mg, loading 357±14 mol/g) were dispersed in 500 l of deionised water and activated with EDC solution (0.2 M in water) for 2 h at room temperature. 500 l of a solution of -D-Glucose oxygen 1-oxidoreductase (1060 U or 3.6 mg) were added. The reaction was carried out within 5 h at r.t. The final suspension was filtered using a 0.22 m pore size nitrocellulose filter and washed with 120 ml of pure water. The black solid obtained (3.3 mg) was suspended in 4 ml of pure water and centrifuged twice at 2000 rpm separated from the supernatant. The precipitate collected was redissolved in 1.0 Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 5 ml of water and stored at 4 ºC. A small amount was used to determine the enzymatic activity and allowed to calculate the amount of enzyme present (vide infra). The enzymatic activity was conserved for at least 4 months. Covalent enzyme conjugate 3 Oxidized CNTs (5.0 mg, loading 357±14 mol/g) were dispersed in 1 ml of deionised water and activated with EDC solution (0.2 M in water ) for 2 h at room temperature. 1 ml of a solution of Catalase (62000 U) in PBS (pH=7.6) were mixed with 500 l of a solution of -D-Glucose oxygen 1-oxidoreductase (GOx) (1060 U) and added slowly to the activated nanotubes. The reaction was continued for 5 h at r.t. The final suspension was filtered through a 0.22 m pore size nitrocellulose filter and washed with 30 ml of pure water. The black solid obtained (8.2 mg) was suspended in 2 ml of pure water and centrifuged twice at 4000 rpm and separated from the supernatant. The precipitate collected was dissolved in 10 ml of water and stored at 4 ºC. A small amount was used to determine the enzymatic activity to calculate the amount of each enzyme present (vide infra).          Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 6 Catalase enzymatic activity assay  Catalase activity was measured using H2O2 as substrate.3 Catalase dispropotionates H2O2 to form oxygen and water in a pH 7.0 PBS buffer at 25 ºC. For a typical assay the disappearance of H2O2 was monitored by UV spectroscopy at 240 nm.  Table 4. Enzymatic assay activity of conjugate 1. Results from three independent experiments.  Conjugate 1 (mg) Measured timea (min) Enzymatic units b Amount of enzyme c (g) Amount of catalase (g /mg) 1.20 2.18 15797 355 295.83 1.30 2.35 17088 384 295.38 0.80 3.08 10502 251 200.80 aTime expressed in min to decrease absorbance at 240nm from 0.45 to 0.40. bUnits/ml = (3.45*dilution factor)/(time*volume of enzyme in ml)  c From supplier 44500 Units/mg  A solution of 2.9 ml of 0.0036% (w/w) H2O2 in PBS was prepared with a stable absorbance at 240 nm between 0.55 and 0.52. 100l of conjugate 1 solution (weight amount dissolved in 2 ml) was added by inversion (at 25ºC) and the kinetics in the loss of absorbance at 240 nm from 0.45 to 0.40 units was recorded and the number of enzymatic unit was calculated by: Enzymatic Units/ml = (A500/min Gox- A500/min Blank)*3.1*(dF)/(7.5*0.1)  Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 7 Glucose oxidase enzymatic assay GOx activity was detected measuring the production of H2O2 in a 0.1 M acetate buffer at 35 ºC and pH 5.0 upon addition of D(+)-glucose.4 The concentration was monitored with time at 240 nm using a spectrophotometer.  Table 5. Enzymatic assay activity of conjugate 2. Results from three independent experiments. Conjugate 2 (mg) (A500/min Gox- A500/min Blank) dF=100 Enzymatic units a  Amount of enzyme b (mg) Amount of GOx (g /mg) 2.21 0.721 300.4 1.50 678.73 2.32 0.763 315.4 1.57 676.72 4.10 1.349 557.6 2.78 678.04 a Enzymatic Units/ml = (A500/min Gox- A500/min Blank)*3.1*(dF)/(7.5*0.1)  b From supplier 200 Units/mg   A solution of 2.9 ml combining 24 ml of 0.21 mM o-dianisine in 50mM sodium acetate buffer (pH 5.1) and 5 ml of 10% w/v -D-Glucose in deionized water were mixed with 100 l of prepared solution of 60 purpurogallin units/ml of peroxidase type II in cold water, equilibrated at 35ºC and monitored at 500 nm absorbance until stable. Then 100 l of glucose oxidase solution in 50 mM sodium acetate buffer was added and the increasing in absorbance at 500 nm was recorded for approximately 5 min. For the blank test 100 l Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 8 of simple acetate buffer solution was added instead of glucose oxidase solution. The concentration of enzymatic units was calculated by4: Enzymatic Units/ml = (A500/min Gox- A500/min Blank)*3.1*(dilution factor)/(7.5*0.1) where 3.1 is the total volume, 7.5 is the millimolar extinction coefficient of oxidized o-dianisine at 500 nm and 0.1 is the volume of enzyme used in the test.   FT-IR Spectroscopy The secondary structure of native and adsorbed enzymes was determined using FT-IR spectroscopy. All spectra were collected using a Nicolet Magna 550 series II. The spectrometer was equipped with a mercury cadmium telluride detector cooled by liquid nitrogen. The data collection and second-derivative calculations were carried out using the software OMNIC 6.1a provided by Thermo Nicolet Corporation. An average of 170 scans were obtained at a resolution of 2 cm-1 with a gain of 2 and an aperture of 50. Typically, lyophilized compound 1, 2 and 3 (1 mg) were ground gently with IR grade KBr to form a homogeneous grey powder. The grinding was performed under nitrogen atmosphere to prevent absorption of atmospheric water. The spectra of the enzymes, both native and attached to the nanotubes were baseline corrected over the entire spectral window. Secondary structural content of the enzymes was obtained by analysis of their FT-IR spectra in the region from 1600 to 1700 cm-1. To obtain the second-derivative spectrum, a baseline correction was applied to the corrected spectra in the region from 1750 to 2000 cm-1, which is critical to obtain quantitative structural information using the second-derivative analysis method5. The second-derivative spectrum was then corrected Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 9 in the region 1600-1700 cm-1 to obtain a flat baseline. This spectrum was used to obtain the -helix and -sheet contents of the enzymes5,6.  Table 2. Secondary structure of enzymes at 20 ºC from FT-IR amide I band analysis compared with the reference native enzyme.   Assignment of components Native Catalase  Conjugate 1 Native GOx  Conjugate 2 β-sheets (%) (1621 cm-1) 12.3 13.2 18.4 22.5 α-helix (%) (1647 cm-1) 26.0 35.3 28.8 27.6   4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0   Figure 8. FT-IR reflectance spectrum of liophylized catalase Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 10 4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0   Figure 9. FT-IR reflectance spectrum of conjugate 1. 4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0   Figure 10. FT-IR reflectance spectrum of liophylized GOx enzyme.  Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 11 4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0 - 1 Figure 11. FT-IR reflectance spectrum of conjugate 2.  Microscopy characterization AFM images were taken by spreading a drop of the diluted conjugate suspension in pure water onto a clean silicon surface. The solvent was removed by evaporation by a gentle air streamfor 20 min. The substrate was then analyzed by a tapping mode AFM (Digital Instruments Nanoscope III). Pictures were analyzed using WSxM 4.0. TEM characterization was carried out on a JEOL 1200EX operating at 100 KV. A drop of diluted enzyme-nanotube water suspension was deposited on a carbon film copper grid. After 5 min the drop was blow dried and stained for 10 min with a saturated solution of uranyl acetate. The SEM measurements were performed on a JEOL JSM 7000F field emission SEM operating at 2.0 kV. 150 l of enzyme-nanotube water suspension was deposited on a glass slit covered with a 120 nm Al top-layer on which a 1 nm Cr layer was sputtered. These samples where allowed to dry at ambient temperature and used with an additional sputtered staining. Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 12 Optical microscopy analysis: Images and movies of the moving nanotubes were recorded using a Olympus BX 60 microscope, equipped with a Sony 3CCD DXC 950P digital camera, attached to a personal computer with Matrox Inspector 2.1 imaging software. Typically, a 50 l of aqueous enzyme-nanotube conjugate solution was deposited onto a glass coverslip and examined for the presence of aggregates under by optical microscopy. After careful addition of hydrogen peroxide (0.3% v/v) or D-glucose (100 mM), the movement of the carbon nanotube bundles was recorded and analyzed.    Video 1. Observation by optical microscopy of conjugate 3 after addition of a glucose solution (100 mM) under constant air flow. The CNT aggregates are visible as black irregular shape. Round shape oxygen bubbles generated from the aggregate are visible. An amount of non-enzyme-modified CNTs was added also.  Video 2. Observation by video-microscopy of conjugate 3 after addition of a glucose solution 100 mM under constant air flow. The CNT aggregates are visible as irregular black shapes. The round shapes are oxygen bubbles generated by/at the aggregates. An amount of non enzyme modified CNTs was added also.  Video 3 Observation by video-microscopy of a static Brownian diluted solution of conjugate 3 at 25ºC. The very slow movement of fiber-like aggregate is attributed to Brownian motion. Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 13  Video 4. Observation by video-microscopy of conjugate 3 after addition of a glucose solution (100mM) under constant nitrogen flow.  References  1. Liu, J., Rinzler, A. G., Dai, H., Hafner, J. H., Bradley, R. K., Boul, P. J., Lu, A., Iverson, T., Shelimov, K., Huffman, C. B., Rodriguez-Macias, F., Shon, Y. S., Lee, T. R., Colbert, D. T. & Smalley, R. E. Fullerene pipes. Science, 280, 1253 (1998). 2. Kaiser, E., Colescott, R. L., Bossinger, C. D. & Cook, P. I. Color test for detection of free terminal amino groups in solid-phase synthesis of perptides. Anal. Biochem, 34, 595-598 (1970) 3. Beers, R. F. & Sizer, I. W. A Spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195, 133-140 (1952). 4. Bergmeyer, H. U., Gawehn, K. & Grassl, M. Methods of Enzymatic Analysis, Academic Press Inc., New York, Volume I, 457 (1974). 5. Eremin, A. N., Metelitsa, D. I., Shishko, Zh. F., Mikhailova, R. V., Yasenko, M. I. & Lobanok, A. G. Thermal stability of glucose oxidase from Penicillium adametzii.  Appl. Biochem and Microbiol, 37, 579-586 (2001). 6. Karajanagi, S.S., Vertegel, A. A., Kane, R. S. & Dordick, J. S. Structure and Function of Enzymes Adsorbed onto Single-Walled Carbon Nanotubes. Langmuir 20, 11594-11599 (2004). Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007

   University of GroningenAutonomous propulsion of carbon nanotubes powered by a multienzyme ensemblePantarotto, Davide; Browne, Wesley R.; Feringa, Ben L.Published in:Chemical CommunicationsDOI:10.1039/b715310dIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2008Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Pantarotto, D., Browne, W. R., & Feringa, B. L. (2008). Autonomous propulsion of carbon nanotubespowered by a multienzyme ensemble. Chemical Communications, 10(13), 1533-1535.https://doi.org/10.1039/b715310dCopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 30-10-2023 1 Autonomous Propulsion of Carbon Nanotubes Powered by a Multi-Enzyme Ensemble. Davide Pantarotto, Wesley R. Browne, Ben L. Feringa* Department of Organic Chemistry, Stratingh Institute for Chemistry and Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands *To whom correspondence should be addressed. E-mail: b.l.feringa@rug.nl  Supplementary Information  All the reagents and solvents were obtained from commercial suppliers and used without further purification. Multiwalled carbon nanotubes (CNTs) were purchased from Nanostructured and Amorphous Materials, Inc. (Houston, USA). Catalase from bovine liver and Glucose oxidase from Aspergillus Niger were purchased from Sigma Aldrich and used as received. Characterization of CNTs The acid treatment of commercial CNTs was carried out to dissolve metal species, breakdown amorphous carbon, and introduce reactive groups along the sidewalls of the nanotubes.1 After treatment, the CNTs were characterized by electron microscopy (TEM, SEM) and atomic force microscopy (AFM).  For AFM, a drop of the diluted nanotube suspension in pure water or distilled dichloromethane was deposited onto a clean flat silicon surface. The solvent was allowed Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 2 to evaporate over 10 min. The substrate was analyzed by tapping mode AFM (Digital Instruments Nanoscope III). TEM characterization was carried out on a JEOL 1200EX operating at 100 KV. A drop of diluted nanotube suspension was deposited on a carbon film copper grid. The drop was dry after 5 min. Negative staining was not required. The SEM measurements were performed on a JEOL JSM 7000F field emission SEM operating at 2.0 kV. A nanotube suspension (150 l) was deposited on a glass slit covered with a 120 nm Al top-layer on which a 1 nm Cr layer was sputtered. These samples where allowed to dry at ambient temperatures and used without additional staining. Quantitative analysis of carboxylic sites was performed using the quantitative Kaiser test2 after further functionalisation of the oxidized nanotubes with 1,8-diamino-3,6-dioxaoctane (vide infra). Preparation of water soluble CNTs After gently grinding, commercial CNTs (160.0 mg) were suspended in 150 ml of a mixture of concentrated H2SO4/HNO3 (3/1 v/v) and sonicated for 5 h. The suspension was then cooled to RT and diluted to 500 ml with 2 M aqueous NaOH in an ice bath. The solution was filtered through a PTFE filter (pore size 0.22 m) under vacuum. The black residue collected was washed thoroughly with deionized water until the filtrate was pH 6 and subsequently dried under vacuum at 60 ºC. The product, 92.0 mg, was collected and suspended in 30 ml of Pirahna solution (H2SO4 98% / H2O2 30 vol 4/1 v/v) and stirred r.t. for 1 h. The solution was filtered over a 0.22 m pore size polycarbonate membrane and washed extensively with deionised water. The Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 3 black material on the collected was dried under vacuum at 60 ºC to yield 88.0 mg of product. The material was stored as a solid or as a stable solution (concentration of 1 mg/ml). The IR spectroscopy shows absorptions at 1710 cm-1 (carbonyl stretching) and a broad absorption at 1100 cm-1 (C-O stretching). The loading was calculated using a quantitative Kaiser test2 after functionalization of oxidized carbon nanotubes (typically 20 mg) first with an excess of oxalyl chloride and, after removal of the excess, by treatment with 1,8-diamino-3,6-dioxaoctane in tetrahydrofurane (THF) at 60°C for 2 h. The resulting suspension was filtered and the black solid washed with methanol and dried. To perform the Kaiser test three solutions were prepared in parallel. (I) 10 g of phenol dissolved in 20 mL of ethanol, (II) 2 mL of KCN 1 mM (aqueous solution) dissolved in 98 mL of pyridine, (III) 1.0 g of ninhydrine dissolved in 20 mL of ethanol. Approximately 20 mg of free amino CNTs was weighed accurately. Non-amino CNT was tested also as a control solution and treated in the same way. 75 µl of solution I, 100 µl of solution II and 75 µl of solution III were added to a haemolysis test tube. The test tube is incubated in a heating block at 100°C for 7 min and then removed and added immediately to 4.8 mL of 60% ethanol to make a final volume of 5 mL. The tube was then mixed to render the violet colour homogenous. The absorbance of each sample was measured relative to the blank CNT solution (pale yellow) at 570 nm. The amine loading was calculated from the equation:   [ ] (mg) weight sample t coefficien Extinction10)ml(dilution AbsAbsgmol6×××−= blanksampleµ (Dilution=5mL and Extinction coefficient = 15000 m-1cm-1). Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 4 The values are expressed as micromole of amino groups per gram of material and are an average of at least ten measurements at different dilutions.  Covalent enzyme conjugate 1  Oxidized CNTs (2.0 mg, loading 357±14 mol/g) were dispersed in 500 l of deionised water and activated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) solution (0.2 M in PBS pH 7) for 2 h at room temperature. 500 l of Catalase (62000 U or 1.5 mg) in phosphate buffer solution (PBS pH 7.6) were added. The reaction was carried out within 5 h at r.t. The final suspension was filtered on a 0.22 m pore size nitrocellulose filter and washed with 30 ml of pure water. The black solid obtained (3.4 mg) was suspended in 2.0 ml of pure water and centrifuged twice at 4000 rpm with discharge of the supernatant. The precipitate collected was dissolved in 2.0 ml of water and stored at 4 ºC. A small amount was used to determine the enzymatic activity to calculate the amount of enzyme present (vide infra). The enzymatic activity was conserved for at least 4 months. Covalent enzyme conjugate 2 Oxidized CNTs (2.0 mg, loading 357±14 mol/g) were dispersed in 500 l of deionised water and activated with EDC solution (0.2 M in water) for 2 h at room temperature. 500 l of a solution of -D-Glucose oxygen 1-oxidoreductase (1060 U or 3.6 mg) were added. The reaction was carried out within 5 h at r.t. The final suspension was filtered using a 0.22 m pore size nitrocellulose filter and washed with 120 ml of pure water. The black solid obtained (3.3 mg) was suspended in 4 ml of pure water and centrifuged twice at 2000 rpm separated from the supernatant. The precipitate collected was redissolved in 1.0 Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 5 ml of water and stored at 4 ºC. A small amount was used to determine the enzymatic activity and allowed to calculate the amount of enzyme present (vide infra). The enzymatic activity was conserved for at least 4 months. Covalent enzyme conjugate 3 Oxidized CNTs (5.0 mg, loading 357±14 mol/g) were dispersed in 1 ml of deionised water and activated with EDC solution (0.2 M in water ) for 2 h at room temperature. 1 ml of a solution of Catalase (62000 U) in PBS (pH=7.6) were mixed with 500 l of a solution of -D-Glucose oxygen 1-oxidoreductase (GOx) (1060 U) and added slowly to the activated nanotubes. The reaction was continued for 5 h at r.t. The final suspension was filtered through a 0.22 m pore size nitrocellulose filter and washed with 30 ml of pure water. The black solid obtained (8.2 mg) was suspended in 2 ml of pure water and centrifuged twice at 4000 rpm and separated from the supernatant. The precipitate collected was dissolved in 10 ml of water and stored at 4 ºC. A small amount was used to determine the enzymatic activity to calculate the amount of each enzyme present (vide infra).          Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 6 Catalase enzymatic activity assay  Catalase activity was measured using H2O2 as substrate.3 Catalase dispropotionates H2O2 to form oxygen and water in a pH 7.0 PBS buffer at 25 ºC. For a typical assay the disappearance of H2O2 was monitored by UV spectroscopy at 240 nm.  Table 4. Enzymatic assay activity of conjugate 1. Results from three independent experiments.  Conjugate 1 (mg) Measured timea (min) Enzymatic units b Amount of enzyme c (g) Amount of catalase (g /mg) 1.20 2.18 15797 355 295.83 1.30 2.35 17088 384 295.38 0.80 3.08 10502 251 200.80 aTime expressed in min to decrease absorbance at 240nm from 0.45 to 0.40. bUnits/ml = (3.45*dilution factor)/(time*volume of enzyme in ml)  c From supplier 44500 Units/mg  A solution of 2.9 ml of 0.0036% (w/w) H2O2 in PBS was prepared with a stable absorbance at 240 nm between 0.55 and 0.52. 100l of conjugate 1 solution (weight amount dissolved in 2 ml) was added by inversion (at 25ºC) and the kinetics in the loss of absorbance at 240 nm from 0.45 to 0.40 units was recorded and the number of enzymatic unit was calculated by: Enzymatic Units/ml = (A500/min Gox- A500/min Blank)*3.1*(dF)/(7.5*0.1)  Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 7 Glucose oxidase enzymatic assay GOx activity was detected measuring the production of H2O2 in a 0.1 M acetate buffer at 35 ºC and pH 5.0 upon addition of D(+)-glucose.4 The concentration was monitored with time at 240 nm using a spectrophotometer.  Table 5. Enzymatic assay activity of conjugate 2. Results from three independent experiments. Conjugate 2 (mg) (A500/min Gox- A500/min Blank) dF=100 Enzymatic units a  Amount of enzyme b (mg) Amount of GOx (g /mg) 2.21 0.721 300.4 1.50 678.73 2.32 0.763 315.4 1.57 676.72 4.10 1.349 557.6 2.78 678.04 a Enzymatic Units/ml = (A500/min Gox- A500/min Blank)*3.1*(dF)/(7.5*0.1)  b From supplier 200 Units/mg   A solution of 2.9 ml combining 24 ml of 0.21 mM o-dianisine in 50mM sodium acetate buffer (pH 5.1) and 5 ml of 10% w/v -D-Glucose in deionized water were mixed with 100 l of prepared solution of 60 purpurogallin units/ml of peroxidase type II in cold water, equilibrated at 35ºC and monitored at 500 nm absorbance until stable. Then 100 l of glucose oxidase solution in 50 mM sodium acetate buffer was added and the increasing in absorbance at 500 nm was recorded for approximately 5 min. For the blank test 100 l Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 8 of simple acetate buffer solution was added instead of glucose oxidase solution. The concentration of enzymatic units was calculated by4: Enzymatic Units/ml = (A500/min Gox- A500/min Blank)*3.1*(dilution factor)/(7.5*0.1) where 3.1 is the total volume, 7.5 is the millimolar extinction coefficient of oxidized o-dianisine at 500 nm and 0.1 is the volume of enzyme used in the test.   FT-IR Spectroscopy The secondary structure of native and adsorbed enzymes was determined using FT-IR spectroscopy. All spectra were collected using a Nicolet Magna 550 series II. The spectrometer was equipped with a mercury cadmium telluride detector cooled by liquid nitrogen. The data collection and second-derivative calculations were carried out using the software OMNIC 6.1a provided by Thermo Nicolet Corporation. An average of 170 scans were obtained at a resolution of 2 cm-1 with a gain of 2 and an aperture of 50. Typically, lyophilized compound 1, 2 and 3 (1 mg) were ground gently with IR grade KBr to form a homogeneous grey powder. The grinding was performed under nitrogen atmosphere to prevent absorption of atmospheric water. The spectra of the enzymes, both native and attached to the nanotubes were baseline corrected over the entire spectral window. Secondary structural content of the enzymes was obtained by analysis of their FT-IR spectra in the region from 1600 to 1700 cm-1. To obtain the second-derivative spectrum, a baseline correction was applied to the corrected spectra in the region from 1750 to 2000 cm-1, which is critical to obtain quantitative structural information using the second-derivative analysis method5. The second-derivative spectrum was then corrected Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 9 in the region 1600-1700 cm-1 to obtain a flat baseline. This spectrum was used to obtain the -helix and -sheet contents of the enzymes5,6.  Table 2. Secondary structure of enzymes at 20 ºC from FT-IR amide I band analysis compared with the reference native enzyme.   Assignment of components Native Catalase  Conjugate 1 Native GOx  Conjugate 2 β-sheets (%) (1621 cm-1) 12.3 13.2 18.4 22.5 α-helix (%) (1647 cm-1) 26.0 35.3 28.8 27.6   4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0   Figure 8. FT-IR reflectance spectrum of liophylized catalase Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 10 4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0   Figure 9. FT-IR reflectance spectrum of conjugate 1. 4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0   Figure 10. FT-IR reflectance spectrum of liophylized GOx enzyme.  Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 11 4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0 - 1 Figure 11. FT-IR reflectance spectrum of conjugate 2.  Microscopy characterization AFM images were taken by spreading a drop of the diluted conjugate suspension in pure water onto a clean silicon surface. The solvent was removed by evaporation by a gentle air streamfor 20 min. The substrate was then analyzed by a tapping mode AFM (Digital Instruments Nanoscope III). Pictures were analyzed using WSxM 4.0. TEM characterization was carried out on a JEOL 1200EX operating at 100 KV. A drop of diluted enzyme-nanotube water suspension was deposited on a carbon film copper grid. After 5 min the drop was blow dried and stained for 10 min with a saturated solution of uranyl acetate. The SEM measurements were performed on a JEOL JSM 7000F field emission SEM operating at 2.0 kV. 150 l of enzyme-nanotube water suspension was deposited on a glass slit covered with a 120 nm Al top-layer on which a 1 nm Cr layer was sputtered. These samples where allowed to dry at ambient temperature and used with an additional sputtered staining. Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 12 Optical microscopy analysis: Images and movies of the moving nanotubes were recorded using a Olympus BX 60 microscope, equipped with a Sony 3CCD DXC 950P digital camera, attached to a personal computer with Matrox Inspector 2.1 imaging software. Typically, a 50 l of aqueous enzyme-nanotube conjugate solution was deposited onto a glass coverslip and examined for the presence of aggregates under by optical microscopy. After careful addition of hydrogen peroxide (0.3% v/v) or D-glucose (100 mM), the movement of the carbon nanotube bundles was recorded and analyzed.    Video 1. Observation by optical microscopy of conjugate 3 after addition of a glucose solution (100 mM) under constant air flow. The CNT aggregates are visible as black irregular shape. Round shape oxygen bubbles generated from the aggregate are visible. An amount of non-enzyme-modified CNTs was added also.  Video 2. Observation by video-microscopy of conjugate 3 after addition of a glucose solution 100 mM under constant air flow. The CNT aggregates are visible as irregular black shapes. The round shapes are oxygen bubbles generated by/at the aggregates. An amount of non enzyme modified CNTs was added also.  Video 3 Observation by video-microscopy of a static Brownian diluted solution of conjugate 3 at 25ºC. The very slow movement of fiber-like aggregate is attributed to Brownian motion. Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007 13  Video 4. Observation by video-microscopy of conjugate 3 after addition of a glucose solution (100mM) under constant nitrogen flow.  References  1. Liu, J., Rinzler, A. G., Dai, H., Hafner, J. H., Bradley, R. K., Boul, P. J., Lu, A., Iverson, T., Shelimov, K., Huffman, C. B., Rodriguez-Macias, F., Shon, Y. S., Lee, T. R., Colbert, D. T. & Smalley, R. E. Fullerene pipes. Science, 280, 1253 (1998). 2. Kaiser, E., Colescott, R. L., Bossinger, C. D. & Cook, P. I. Color test for detection of free terminal amino groups in solid-phase synthesis of perptides. Anal. Biochem, 34, 595-598 (1970) 3. Beers, R. F. & Sizer, I. W. A Spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195, 133-140 (1952). 4. Bergmeyer, H. U., Gawehn, K. & Grassl, M. Methods of Enzymatic Analysis, Academic Press Inc., New York, Volume I, 457 (1974). 5. Eremin, A. N., Metelitsa, D. I., Shishko, Zh. F., Mikhailova, R. V., Yasenko, M. I. & Lobanok, A. G. Thermal stability of glucose oxidase from Penicillium adametzii.  Appl. Biochem and Microbiol, 37, 579-586 (2001). 6. Karajanagi, S.S., Vertegel, A. A., Kane, R. S. & Dordick, J. S. Structure and Function of Enzymes Adsorbed onto Single-Walled Carbon Nanotubes. Langmuir 20, 11594-11599 (2004). Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2007

University of Groningen Digital Archive

  1 
Autonomous Propulsion of Carbon Nanotubes Powered by a Multi-
Enzyme Ensemble. 
Davide Pantarotto, Wesley R. Browne, Ben L. Feringa* 
Department of Organic Chemistry, Stratingh Institute for Chemistry and Zernike Institute 
for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, 
The Netherlands 
*To whom correspondence should be addressed. E-mail: b.l.feringa@rug.nl 
 
Supplementary Information 
 
All the reagents and solvents were obtained from commercial suppliers and used without 
further  purification.  Multiwalled  carbon  nanotubes  (CNTs)  were  purchased  from 
Nanostructured and Amorphous Materials, Inc. (Houston, USA). Catalase from bovine 
liver and Glucose oxidase from Aspergillus Niger were purchased from Sigma Aldrich 
and used as received. 
Characterization of CNTs 
The  acid  treatment  of  commercial  CNTs  was  carried  out  to  dissolve  metal  species, 
breakdown amorphous carbon, and introduce reactive groups along the sidewalls of the 
nanotubes.
1 After treatment, the CNTs were characterized by electron microscopy (TEM, 
SEM) and atomic force microscopy (AFM). 
 
For  AFM,  a  drop  of  the  diluted  nanotube  suspension  in  pure  water  or  distilled 
dichloromethane was deposited onto a clean flat silicon surface. The solvent was allowed 
Supplementary Material (ESI) for Chemical Communications
This journal is (c) The Royal Society of Chemistry 2007  2 
to evaporate over 10 min. The substrate was analyzed by tapping mode AFM (Digital 
Instruments Nanoscope III). 
TEM characterization was carried out on a JEOL 1200EX operating at 100 KV. A drop 
of diluted nanotube suspension was deposited on a carbon film copper grid. The drop was 
dry after 5 min. Negative staining was not required. 
The SEM measurements were performed on a JEOL JSM 7000F field emission SEM 
operating at 2.0 kV. A nanotube suspension (150 ￿l) was deposited on a glass slit covered 
with a 120 nm Al top-layer on which a 1 nm Cr layer was sputtered. These samples 
where allowed to dry at ambient temperatures and used without additional staining. 
Quantitative analysis of carboxylic sites was performed using the quantitative Kaiser test
2 
after  further  functionalisation  of  the  oxidized  nanotubes  with  1,8-diamino-3,6-
dioxaoctane (vide infra). 
Preparation of water soluble CNTs 
After  gently grinding,  commercial CNTs (160.0 mg) were suspended in 150 ml of  a 
mixture of concentrated H2SO4/HNO3 (3/1 v/v) and sonicated for 5 h. The suspension 
was then cooled to RT and diluted to 500 ml with 2 M aqueous NaOH in an ice bath. The 
solution was filtered through a PTFE filter (pore size 0.22 ￿m) under vacuum. The black 
residue collected was washed thoroughly with deionized water until the filtrate was pH 6 
and subsequently dried under vacuum at 60 ºC. 
The product, 92.0 mg, was collected and suspended in 30 ml of Pirahna solution (H2SO4 
98% / H2O2 30 vol 4/1 v/v) and stirred r.t. for 1 h. The solution was filtered over a 0.22 
￿m pore size polycarbonate membrane and washed extensively with deionised water. The 
Supplementary Material (ESI) for Chemical Communications
This journal is (c) The Royal Society of Chemistry 2007  3 
black material on the collected was dried under vacuum at 60 ºC to yield 88.0 mg of 
product. 
The material was stored as a solid or as a stable solution (concentration of 1 mg/ml). 
The IR spectroscopy shows absorptions at 1710 cm
-1 (carbonyl stretching) and a broad 
absorption at 1100 cm
-1 (C-O stretching). 
The loading was calculated using a quantitative Kaiser test
2 after functionalization of 
oxidized carbon nanotubes (typically 20 mg) first with an excess of oxalyl chloride and, 
after  removal  of  the  excess,  by  treatment  with  1,8-diamino-3,6-dioxaoctane  in 
tetrahydrofurane (THF) at 60°C for 2 h. The resulting suspension was filtered and the 
black solid washed with methanol and dried. To perform the Kaiser test three solutions 
were prepared in parallel. (I) 10 g of phenol dissolved in 20 mL of ethanol, (II) 2 mL of 
KCN 1 mM (aqueous solution) dissolved in 98 mL of pyridine, (III) 1.0 g of ninhydrine 
dissolved in 20 mL of ethanol. Approximately 20 mg of free amino CNTs was weighed 
accurately. Non-amino CNT was tested also as a control solution and treated in the same 
way. 75 ml of solution I, 100 ml of solution II and 75 ml of solution III were added to a 
haemolysis test tube. The test tube is incubated in a heating block at 100°C for 7 min and 
then removed and added immediately to 4.8 mL of 60% ethanol to make a final volume 
of  5  mL.  The  tube  was  then  mixed  to  render  the  violet  colour  homogenous.  The 
absorbance  of  each  sample  was  measured  relative  to  the  blank  CNT  solution  (pale 
yellow) at 570 nm. The amine loading was calculated from the equation:  
 
[ ]
  (mg)  weight  sample   t  coefficien   Extinction
10 ) ml ( dilution  Abs Abs
g
mol
6
´
´ ´ -
=
blank sample m
 
(Dilution=5mL and Extinction coefficient = 15000 m
-1cm
-1). 
Supplementary Material (ESI) for Chemical Communications
This journal is (c) The Royal Society of Chemistry 2007  4 
The values are expressed as micromole of amino groups per gram of material and are an 
average of at least ten measurements at different dilutions. 
 
Covalent enzyme conjugate 1  
Oxidized CNTs (2.0 mg, loading 357±14 ￿mol/g) were dispersed in 500 ￿l of deionised 
water  and  activated  with  1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide  (EDC) 
solution (0.2 M in PBS pH 7) for 2 h at room temperature. 500 ￿l of Catalase (62000 U 
or 1.5 mg) in phosphate buffer solution (PBS pH 7.6) were added. The reaction was 
carried out within 5 h at r.t. The final suspension was filtered on a 0.22 ￿m pore size 
nitrocellulose filter and washed with 30 ml of pure water. The black solid obtained (3.4 
mg) was suspended in 2.0 ml of pure water and centrifuged twice at 4000 rpm with 
discharge of the supernatant. The precipitate collected was dissolved in 2.0 ml of water 
and stored at 4 ºC. A small amount was used to determine the enzymatic activity to 
calculate  the  amount  of  enzyme  present  (vide  infra).  The  enzymatic  activity  was 
conserved for at least 4 months. 
Covalent enzyme conjugate 2 
Oxidized CNTs (2.0 mg, loading 357±14 ￿mol/g) were dispersed in 500 ￿l of deionised 
water and activated with EDC solution (0.2 M in water) for 2 h at room temperature. 500 
￿l of a solution of ￿-D-Glucose oxygen 1-oxidoreductase (1060 U or 3.6 mg) were added. 
The reaction was carried out within 5 h at r.t. The final suspension was filtered using a 
0.22 ￿m pore size nitrocellulose filter and washed with 120 ml of pure water. The black 
solid obtained (3.3 mg) was suspended in 4 ml of pure water and centrifuged twice at 
2000 rpm separated from the supernatant. The precipitate collected was redissolved in 1.0 
Supplementary Material (ESI) for Chemical Communications
This journal is (c) The Royal Society of Chemistry 2007  5 
ml of water and stored at 4 ºC. A small amount was used to determine the enzymatic 
activity  and  allowed  to  calculate  the  amount  of  enzyme  present  (vide  infra).  The 
enzymatic activity was conserved for at least 4 months. 
Covalent enzyme conjugate 3 
Oxidized CNTs (5.0 mg, loading 357±14 ￿mol/g) were dispersed in 1 ml of deionised 
water and activated with EDC solution (0.2 M in water ) for 2 h at room temperature. 1 
ml of a solution of Catalase (62000 U) in PBS (pH=7.6) were mixed with 500 ￿l of a 
solution of ￿-D-Glucose oxygen 1-oxidoreductase (GOx) (1060 U) and added slowly to 
the activated nanotubes. The reaction was continued for 5 h at r.t. The final suspension 
was filtered through a 0.22 ￿m pore size nitrocellulose filter and washed with 30 ml of 
pure water. The black solid obtained (8.2 mg) was suspended in 2 ml of pure water and 
centrifuged  twice  at  4000  rpm  and  separated  from  the  supernatant.  The  precipitate 
collected was dissolved in 10 ml of water and stored at 4 ºC. A small amount was used to 
determine the enzymatic activity to calculate the amount of each enzyme present (vide 
infra).  
 
 
 
 
 
 
 
 
Supplementary Material (ESI) for Chemical Communications
This journal is (c) The Royal Society of Chemistry 2007  6 
Catalase enzymatic activity assay  
Catalase activity was measured using H2O2 as substrate.
3 Catalase dispropotionates H2O2 
to form oxygen and water in a pH 7.0 PBS buffer at 25 ºC.  For a typical assay the 
disappearance of H2O2 was monitored by UV spectroscopy at 240 nm. 
 
Table  4.  Enzymatic  assay  activity  of  conjugate  1.  Results  from  three  independent 
experiments. 
 
Conjugate 1 
(mg) 
Measured time
a 
(min) 
Enzymatic 
units 
b 
Amount of 
enzyme 
c (￿g) 
Amount of 
catalase (￿g /mg) 
1.20  2.18  15797  355  295.83 
1.30  2.35  17088  384  295.38 
0.80  3.08  10502  251  200.80 
aTime expressed in min to decrease absorbance at 240nm from 0.45 to 0.40. 
bUnits/ml = (3.45*dilution factor)/(time*volume of enzyme in ml)  
c From supplier 44500 Units/mg 
 
A  solution  of  2.9  ml  of  0.0036%  (w/w)  H2O2  in  PBS  was  prepared  with  a  stable 
absorbance at 240 nm between 0.55 and 0.52. 100￿l of conjugate 1 solution (weight 
amount dissolved in 2 ml) was added by inversion (at 25ºC) and the kinetics in the loss of 
absorbance at 240 nm from 0.45 to 0.40 units was recorded and the number of enzymatic 
unit was calculated by: 
Enzymatic Units/ml = (￿A500/min Gox- ￿A500/min Blank)*3.1*(dF)/(7.5*0.1)  
Supplementary Material (ESI) for Chemical Communications
This journal is (c) The Royal Society of Chemistry 2007  7 
Glucose oxidase enzymatic assay 
GOx activity was detected measuring the production of H2O2 in a 0.1 M acetate buffer at 
35 ºC and pH 5.0 upon addition of D(+)-glucose.
4 The concentration was monitored with 
time at 240 nm using a spectrophotometer. 
 
Table 5. Enzymatic assay activity of conjugate 2. Results from three independent 
experiments. 
Conjugate 2 
(mg) 
(￿A500/min 
Gox- ￿A500/min 
Blank) dF=100 
Enzymatic units
 a   Amount of 
enzyme
 b (mg) 
Amount of 
GOx (￿g /mg) 
2.21  0.721  300.4  1.50  678.73 
2.32  0.763  315.4  1.57  676.72 
4.10  1.349  557.6  2.78  678.04 
a Enzymatic Units/ml = (￿A500/min Gox- ￿A500/min Blank)*3.1*(dF)/(7.5*0.1)  
b From supplier 200 Units/mg  
 
A solution of 2.9 ml combining 24 ml of 0.21 mM o-dianisine in 50mM sodium acetate 
buffer (pH 5.1) and 5 ml of 10% w/v ￿-D-Glucose in deionized water were mixed with 
100 ￿l of prepared solution of 60 purpurogallin units/ml of peroxidase type II in cold 
water, equilibrated at 35ºC and monitored at 500 nm absorbance until stable. Then 100 ￿l 
of glucose oxidase solution in 50 mM sodium acetate buffer was added and the increasing 
in absorbance at 500 nm was recorded for approximately 5 min. For the blank test 100 ￿l 
Supplementary Material (ESI) for Chemical Communications
This journal is (c) The Royal Society of Chemistry 2007  8 
of simple acetate buffer solution was added instead of  glucose oxidase solution. The 
concentration of enzymatic units was calculated by
4: 
Enzymatic Units/ml = (￿A500/min Gox- ￿A500/min Blank)*3.1*(dilution factor)/(7.5*0.1) 
where 3.1 is the total volume, 7.5 is the millimolar extinction coefficient of oxidized o-
dianisine at 500 nm and 0.1 is the volume of enzyme used in the test.  
 
FT-IR Spectroscopy 
The secondary structure of native and adsorbed enzymes was determined using FT-IR 
spectroscopy.  All  spectra  were  collected  using  a  Nicolet  Magna  550  series  II.  The 
spectrometer was equipped with a mercury cadmium telluride detector cooled by liquid 
nitrogen. The data collection and second-derivative calculations were carried out using 
the software OMNIC 6.1a provided by Thermo Nicolet Corporation. An average of 170 
scans were obtained at a resolution of 2 cm
-1 with a gain of 2 and an aperture of 50. 
Typically, lyophilized compound 1, 2 and 3 (1 mg) were ground gently with IR grade 
KBr to form a homogeneous grey powder. The grinding was performed under nitrogen 
atmosphere to prevent absorption of atmospheric water. The spectra of the enzymes, both 
native and  attached  to  the nanotubes were  baseline  corrected  over  the  entire  spectral 
window. Secondary structural content of the enzymes was obtained by analysis of their 
FT-IR spectra in the region from 1600 to 1700 cm
-1. To obtain the second-derivative 
spectrum, a baseline correction was applied to the corrected spectra in the region from 
1750 to 2000 cm
-1, which is critical to obtain quantitative structural information using the 
second-derivative analysis method
5. The second-derivative spectrum was then corrected 
Supplementary Material (ESI) for Chemical Communications
This journal is (c) The Royal Society of Chemistry 2007  9 
in the region 1600-1700 cm
-1 to obtain a flat baseline. This spectrum was used to obtain 
the ￿-helix and ￿-sheet contents of the enzymes
5,6. 
 
Table 2. Secondary structure of enzymes at 20 ºC from FT-IR amide I band analysis 
compared with the reference native enzyme.
  
 
Assignment of components  Native Catalase   Conjugate 1  Native GOx   Conjugate 2 
b-sheets (%) (1621 cm
-1)  12.3  13.2  18.4  22.5 
a-helix (%) (1647 cm
-1)  26.0  35.3  28.8  27.6 
 
 
4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0  
 
Figure 8. FT-IR reflectance spectrum of liophylized catalase 
Supplementary Material (ESI) for Chemical Communications
This journal is (c) The Royal Society of Chemistry 2007  10 
4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0
 
 
 
Figure 9. FT-IR reflectance spectrum of conjugate 1. 
4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0
 
 
 
Figure 10. FT-IR reflectance spectrum of liophylized GOx enzyme. 
 
Supplementary Material (ESI) for Chemical Communications
This journal is (c) The Royal Society of Chemistry 2007  11 
4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0
 
- 1  
Figure 11. FT-IR reflectance spectrum of conjugate 2. 
 
Microscopy characterization 
AFM images were taken by spreading a drop of the diluted conjugate suspension in pure 
water onto a clean silicon surface. The solvent was removed by evaporation by a gentle 
air streamfor 20 min. The substrate was then analyzed by a tapping mode AFM (Digital 
Instruments Nanoscope III). Pictures were analyzed using WSxM 4.0. 
TEM characterization was carried out on a JEOL 1200EX operating at 100 KV. A drop 
of diluted enzyme-nanotube water suspension was deposited on a carbon film copper 
grid. After 5 min the drop was blow dried and stained for 10 min with a saturated solution 
of uranyl acetate. 
The SEM measurements were performed on a JEOL JSM 7000F field emission SEM 
operating at 2.0 kV. 150 ￿l of enzyme-nanotube water suspension was deposited on a 
glass slit covered with a 120 nm Al top-layer on which a 1 nm Cr layer was sputtered. 
These samples where allowed to dry at ambient temperature and used with an additional 
sputtered staining. 
Supplementary Material (ESI) for Chemical Communications
This journal is (c) The Royal Society of Chemistry 2007  12 
Optical microscopy analysis: Images and movies of the moving nanotubes were recorded 
using a Olympus BX 60 microscope, equipped with a Sony 3CCD DXC 950P digital 
camera, attached to a personal computer with Matrox Inspector 2.1 imaging software. 
Typically, a 50 ￿l of aqueous enzyme-nanotube conjugate solution was deposited onto a 
glass coverslip and examined for the presence of aggregates under by optical microscopy. 
After  careful  addition  of  hydrogen  peroxide (0.3%  v/v)  or  D-glucose  (100 mM),  the 
movement of the carbon nanotube bundles was recorded and analyzed. 
 
 
 
Video 1. Observation by optical microscopy of conjugate 3 after addition of a glucose 
solution (100 mM) under constant air flow. The CNT aggregates are visible as black 
irregular shape. Round shape oxygen bubbles generated from the aggregate are visible. 
An amount of non-enzyme-modified CNTs was added also. 
 
Video 2. Observation by video-microscopy of conjugate 3 after addition of a glucose 
solution 100 mM under constant air flow. The CNT aggregates are visible as irregular 
black shapes. The round shapes are oxygen bubbles generated by/at the aggregates. An 
amount of non enzyme modified CNTs was added also. 
 
Video 3 Observation by video-microscopy of a static Brownian diluted solution of 
conjugate 3 at 25ºC. The very slow movement of fiber-like aggregate is attributed to 
Brownian motion. 
Supplementary Material (ESI) for Chemical Communications
This journal is (c) The Royal Society of Chemistry 2007  13 
 
Video 4. Observation by video-microscopy of conjugate 3 after addition of a glucose 
solution (100mM) under constant nitrogen flow. 
 
References  
1.  Liu, J., Rinzler, A. G., Dai, H., Hafner, J. H., Bradley, R. K., Boul, P. J., Lu, A., 
Iverson, T., Shelimov, K., Huffman, C. B., Rodriguez-Macias, F., Shon, Y. S., 
Lee, T. R., Colbert, D. T. & Smalley, R. E. Fullerene pipes. Science, 280, 1253 
(1998). 
2.  Kaiser, E., Colescott, R. L., Bossinger, C. D. & Cook, P. I. Color test for detection 
of free terminal amino groups in solid-phase synthesis of perptides. Anal. 
Biochem, 34, 595-598 (1970) 
3.  Beers, R. F. & Sizer, I. W. A Spectrophotometric method for measuring the 
breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195, 133-140 (1952). 
4.  Bergmeyer, H. U., Gawehn, K. & Grassl, M. Methods of Enzymatic Analysis, 
Academic Press Inc., New York, Volume I, 457 (1974). 
5.  Eremin, A. N., Metelitsa, D. I., Shishko, Zh. F., Mikhailova, R. V., Yasenko, M. 
I. & Lobanok, A. G. Thermal stability of glucose oxidase from Penicillium 
adametzii.  Appl. Biochem and Microbiol, 37, 579-586 (2001). 
6.  Karajanagi, S.S., Vertegel, A. A., Kane, R. S. & Dordick, J. S. Structure and 
Function of Enzymes Adsorbed onto Single-Walled Carbon Nanotubes. Langmuir 
20, 11594-11599 (2004). 
Supplementary Material (ESI) for Chemical Communications
This journal is (c) The Royal Society of Chemistry 2007

A Spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase.

Color test for detection of free terminal amino groups in solid-phase synthesis of perptides.

Fullerene pipes.

Methods of Enzymatic Analysis,

Structure and Function of Enzymes Adsorbed onto Single-Walled Carbon Nanotubes.

Thermal stability of glucose oxidase from Penicillium adametzii.

University of Groningen Research Database

  
 University of Groningen
Autonomous propulsion of carbon nanotubes powered by a multienzyme ensemble
Pantarotto, Davide; Browne, Wesley; Feringa, B.L.
Published in:
Chemical Communications
DOI:
10.1039/b715310d
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
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Publication date:
2008
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Pantarotto, D., Browne, W. R., & Feringa, B. L. (2008). Autonomous propulsion of carbon nanotubes
powered by a multienzyme ensemble. Chemical Communications, 10(13), 1533-1535. DOI:
10.1039/b715310d
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Download date: 10-02-2018
 1 
Autonomous Propulsion of Carbon Nanotubes Powered by a Multi-
Enzyme Ensemble. 
Davide Pantarotto, Wesley R. Browne, Ben L. Feringa* 
Department of Organic Chemistry, Stratingh Institute for Chemistry and Zernike Institute 
for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, 
The Netherlands 
*To whom correspondence should be addressed. E-mail: b.l.feringa@rug.nl 
 
Supplementary Information 
 
All the reagents and solvents were obtained from commercial suppliers and used without 
further purification. Multiwalled carbon nanotubes (CNTs) were purchased from 
Nanostructured and Amorphous Materials, Inc. (Houston, USA). Catalase from bovine 
liver and Glucose oxidase from Aspergillus Niger were purchased from Sigma Aldrich 
and used as received. 
Characterization of CNTs 
The acid treatment of commercial CNTs was carried out to dissolve metal species, 
breakdown amorphous carbon, and introduce reactive groups along the sidewalls of the 
nanotubes.1 After treatment, the CNTs were characterized by electron microscopy (TEM, 
SEM) and atomic force microscopy (AFM). 
 
For AFM, a drop of the diluted nanotube suspension in pure water or distilled 
dichloromethane was deposited onto a clean flat silicon surface. The solvent was allowed 
Supplementary Material (ESI) for Chemical Communications
This journal is (c) The Royal Society of Chemistry 2007
 2 
to evaporate over 10 min. The substrate was analyzed by tapping mode AFM (Digital 
Instruments Nanoscope III). 
TEM characterization was carried out on a JEOL 1200EX operating at 100 KV. A drop 
of diluted nanotube suspension was deposited on a carbon film copper grid. The drop was 
dry after 5 min. Negative staining was not required. 
The SEM measurements were performed on a JEOL JSM 7000F field emission SEM 
operating at 2.0 kV. A nanotube suspension (150 l) was deposited on a glass slit covered 
with a 120 nm Al top-layer on which a 1 nm Cr layer was sputtered. These samples 
where allowed to dry at ambient temperatures and used without additional staining. 
Quantitative analysis of carboxylic sites was performed using the quantitative Kaiser test2 
after further functionalisation of the oxidized nanotubes with 1,8-diamino-3,6-
dioxaoctane (vide infra). 
Preparation of water soluble CNTs 
After gently grinding, commercial CNTs (160.0 mg) were suspended in 150 ml of a 
mixture of concentrated H2SO4/HNO3 (3/1 v/v) and sonicated for 5 h. The suspension 
was then cooled to RT and diluted to 500 ml with 2 M aqueous NaOH in an ice bath. The 
solution was filtered through a PTFE filter (pore size 0.22 m) under vacuum. The black 
residue collected was washed thoroughly with deionized water until the filtrate was pH 6 
and subsequently dried under vacuum at 60 ºC. 
The product, 92.0 mg, was collected and suspended in 30 ml of Pirahna solution (H2SO4 
98% / H2O2 30 vol 4/1 v/v) and stirred r.t. for 1 h. The solution was filtered over a 0.22 
m pore size polycarbonate membrane and washed extensively with deionised water. The 
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 3 
black material on the collected was dried under vacuum at 60 ºC to yield 88.0 mg of 
product. 
The material was stored as a solid or as a stable solution (concentration of 1 mg/ml). 
The IR spectroscopy shows absorptions at 1710 cm-1 (carbonyl stretching) and a broad 
absorption at 1100 cm-1 (C-O stretching). 
The loading was calculated using a quantitative Kaiser test2 after functionalization of 
oxidized carbon nanotubes (typically 20 mg) first with an excess of oxalyl chloride and, 
after removal of the excess, by treatment with 1,8-diamino-3,6-dioxaoctane in 
tetrahydrofurane (THF) at 60°C for 2 h. The resulting suspension was filtered and the 
black solid washed with methanol and dried. To perform the Kaiser test three solutions 
were prepared in parallel. (I) 10 g of phenol dissolved in 20 mL of ethanol, (II) 2 mL of 
KCN 1 mM (aqueous solution) dissolved in 98 mL of pyridine, (III) 1.0 g of ninhydrine 
dissolved in 20 mL of ethanol. Approximately 20 mg of free amino CNTs was weighed 
accurately. Non-amino CNT was tested also as a control solution and treated in the same 
way. 75 µl of solution I, 100 µl of solution II and 75 µl of solution III were added to a 
haemolysis test tube. The test tube is incubated in a heating block at 100°C for 7 min and 
then removed and added immediately to 4.8 mL of 60% ethanol to make a final volume 
of 5 mL. The tube was then mixed to render the violet colour homogenous. The 
absorbance of each sample was measured relative to the blank CNT solution (pale 
yellow) at 570 nm. The amine loading was calculated from the equation:  
 
[ ]
 (mg) weight sample t coefficien Extinction
10)ml(dilution AbsAbs
g
mol
6
×
××−
=
blanksampleµ
 
(Dilution=5mL and Extinction coefficient = 15000 m-1cm-1). 
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 4 
The values are expressed as micromole of amino groups per gram of material and are an 
average of at least ten measurements at different dilutions. 
 
Covalent enzyme conjugate 1  
Oxidized CNTs (2.0 mg, loading 357±14 mol/g) were dispersed in 500 l of deionised 
water and activated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) 
solution (0.2 M in PBS pH 7) for 2 h at room temperature. 500 l of Catalase (62000 U 
or 1.5 mg) in phosphate buffer solution (PBS pH 7.6) were added. The reaction was 
carried out within 5 h at r.t. The final suspension was filtered on a 0.22 m pore size 
nitrocellulose filter and washed with 30 ml of pure water. The black solid obtained (3.4 
mg) was suspended in 2.0 ml of pure water and centrifuged twice at 4000 rpm with 
discharge of the supernatant. The precipitate collected was dissolved in 2.0 ml of water 
and stored at 4 ºC. A small amount was used to determine the enzymatic activity to 
calculate the amount of enzyme present (vide infra). The enzymatic activity was 
conserved for at least 4 months. 
Covalent enzyme conjugate 2 
Oxidized CNTs (2.0 mg, loading 357±14 mol/g) were dispersed in 500 l of deionised 
water and activated with EDC solution (0.2 M in water) for 2 h at room temperature. 500 
l of a solution of -D-Glucose oxygen 1-oxidoreductase (1060 U or 3.6 mg) were added. 
The reaction was carried out within 5 h at r.t. The final suspension was filtered using a 
0.22 m pore size nitrocellulose filter and washed with 120 ml of pure water. The black 
solid obtained (3.3 mg) was suspended in 4 ml of pure water and centrifuged twice at 
2000 rpm separated from the supernatant. The precipitate collected was redissolved in 1.0 
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 5 
ml of water and stored at 4 ºC. A small amount was used to determine the enzymatic 
activity and allowed to calculate the amount of enzyme present (vide infra). The 
enzymatic activity was conserved for at least 4 months. 
Covalent enzyme conjugate 3 
Oxidized CNTs (5.0 mg, loading 357±14 mol/g) were dispersed in 1 ml of deionised 
water and activated with EDC solution (0.2 M in water ) for 2 h at room temperature. 1 
ml of a solution of Catalase (62000 U) in PBS (pH=7.6) were mixed with 500 l of a 
solution of -D-Glucose oxygen 1-oxidoreductase (GOx) (1060 U) and added slowly to 
the activated nanotubes. The reaction was continued for 5 h at r.t. The final suspension 
was filtered through a 0.22 m pore size nitrocellulose filter and washed with 30 ml of 
pure water. The black solid obtained (8.2 mg) was suspended in 2 ml of pure water and 
centrifuged twice at 4000 rpm and separated from the supernatant. The precipitate 
collected was dissolved in 10 ml of water and stored at 4 ºC. A small amount was used to 
determine the enzymatic activity to calculate the amount of each enzyme present (vide 
infra).  
 
 
 
 
 
 
 
 
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Catalase enzymatic activity assay  
Catalase activity was measured using H2O2 as substrate.3 Catalase dispropotionates H2O2 
to form oxygen and water in a pH 7.0 PBS buffer at 25 ºC. For a typical assay the 
disappearance of H2O2 was monitored by UV spectroscopy at 240 nm. 
 
Table 4. Enzymatic assay activity of conjugate 1. Results from three independent 
experiments. 
 
Conjugate 1 
(mg) 
Measured timea 
(min) 
Enzymatic 
units b 
Amount of 
enzyme c (g) 
Amount of 
catalase (g /mg) 
1.20 2.18 15797 355 295.83 
1.30 2.35 17088 384 295.38 
0.80 3.08 10502 251 200.80 
aTime expressed in min to decrease absorbance at 240nm from 0.45 to 0.40. 
bUnits/ml = (3.45*dilution factor)/(time*volume of enzyme in ml)  
c From supplier 44500 Units/mg 
 
A solution of 2.9 ml of 0.0036% (w/w) H2O2 in PBS was prepared with a stable 
absorbance at 240 nm between 0.55 and 0.52. 100l of conjugate 1 solution (weight 
amount dissolved in 2 ml) was added by inversion (at 25ºC) and the kinetics in the loss of 
absorbance at 240 nm from 0.45 to 0.40 units was recorded and the number of enzymatic 
unit was calculated by: 
Enzymatic Units/ml = (A500/min Gox- A500/min Blank)*3.1*(dF)/(7.5*0.1)  
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Glucose oxidase enzymatic assay 
GOx activity was detected measuring the production of H2O2 in a 0.1 M acetate buffer at 
35 ºC and pH 5.0 upon addition of D(+)-glucose.4 The concentration was monitored with 
time at 240 nm using a spectrophotometer. 
 
Table 5. Enzymatic assay activity of conjugate 2. Results from three independent 
experiments. 
Conjugate 2 
(mg) 
(A500/min 
Gox- A500/min 
Blank) dF=100 
Enzymatic units a  Amount of 
enzyme b (mg) 
Amount of 
GOx (g /mg) 
2.21 0.721 300.4 1.50 678.73 
2.32 0.763 315.4 1.57 676.72 
4.10 1.349 557.6 2.78 678.04 
a
 Enzymatic Units/ml = (A500/min Gox- A500/min Blank)*3.1*(dF)/(7.5*0.1)  
b From supplier 200 Units/mg  
 
A solution of 2.9 ml combining 24 ml of 0.21 mM o-dianisine in 50mM sodium acetate 
buffer (pH 5.1) and 5 ml of 10% w/v -D-Glucose in deionized water were mixed with 
100 l of prepared solution of 60 purpurogallin units/ml of peroxidase type II in cold 
water, equilibrated at 35ºC and monitored at 500 nm absorbance until stable. Then 100 l 
of glucose oxidase solution in 50 mM sodium acetate buffer was added and the increasing 
in absorbance at 500 nm was recorded for approximately 5 min. For the blank test 100 l 
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of simple acetate buffer solution was added instead of glucose oxidase solution. The 
concentration of enzymatic units was calculated by4: 
Enzymatic Units/ml = (A500/min Gox- A500/min Blank)*3.1*(dilution factor)/(7.5*0.1) 
where 3.1 is the total volume, 7.5 is the millimolar extinction coefficient of oxidized o-
dianisine at 500 nm and 0.1 is the volume of enzyme used in the test.  
 
FT-IR Spectroscopy 
The secondary structure of native and adsorbed enzymes was determined using FT-IR 
spectroscopy. All spectra were collected using a Nicolet Magna 550 series II. The 
spectrometer was equipped with a mercury cadmium telluride detector cooled by liquid 
nitrogen. The data collection and second-derivative calculations were carried out using 
the software OMNIC 6.1a provided by Thermo Nicolet Corporation. An average of 170 
scans were obtained at a resolution of 2 cm-1 with a gain of 2 and an aperture of 50. 
Typically, lyophilized compound 1, 2 and 3 (1 mg) were ground gently with IR grade 
KBr to form a homogeneous grey powder. The grinding was performed under nitrogen 
atmosphere to prevent absorption of atmospheric water. The spectra of the enzymes, both 
native and attached to the nanotubes were baseline corrected over the entire spectral 
window. Secondary structural content of the enzymes was obtained by analysis of their 
FT-IR spectra in the region from 1600 to 1700 cm-1. To obtain the second-derivative 
spectrum, a baseline correction was applied to the corrected spectra in the region from 
1750 to 2000 cm-1, which is critical to obtain quantitative structural information using the 
second-derivative analysis method5. The second-derivative spectrum was then corrected 
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 9 
in the region 1600-1700 cm-1 to obtain a flat baseline. This spectrum was used to obtain 
the -helix and -sheet contents of the enzymes5,6. 
 
Table 2. Secondary structure of enzymes at 20 ºC from FT-IR amide I band analysis 
compared with the reference native enzyme.  
 
Assignment of components Native Catalase  Conjugate 1 Native GOx  Conjugate 2 
β-sheets (%) (1621 cm-1) 12.3 13.2 18.4 22.5 
α-helix (%) (1647 cm-1) 26.0 35.3 28.8 27.6 
 
 
4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0
 
 
Figure 8. FT-IR reflectance spectrum of liophylized catalase 
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4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0
 
 
 
Figure 9. FT-IR reflectance spectrum of conjugate 1. 
4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0
 
 
 
Figure 10. FT-IR reflectance spectrum of liophylized GOx enzyme. 
 
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4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0
 
- 1
 
Figure 11. FT-IR reflectance spectrum of conjugate 2. 
 
Microscopy characterization 
AFM images were taken by spreading a drop of the diluted conjugate suspension in pure 
water onto a clean silicon surface. The solvent was removed by evaporation by a gentle 
air streamfor 20 min. The substrate was then analyzed by a tapping mode AFM (Digital 
Instruments Nanoscope III). Pictures were analyzed using WSxM 4.0. 
TEM characterization was carried out on a JEOL 1200EX operating at 100 KV. A drop 
of diluted enzyme-nanotube water suspension was deposited on a carbon film copper 
grid. After 5 min the drop was blow dried and stained for 10 min with a saturated solution 
of uranyl acetate. 
The SEM measurements were performed on a JEOL JSM 7000F field emission SEM 
operating at 2.0 kV. 150 l of enzyme-nanotube water suspension was deposited on a 
glass slit covered with a 120 nm Al top-layer on which a 1 nm Cr layer was sputtered. 
These samples where allowed to dry at ambient temperature and used with an additional 
sputtered staining. 
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Optical microscopy analysis: Images and movies of the moving nanotubes were recorded 
using a Olympus BX 60 microscope, equipped with a Sony 3CCD DXC 950P digital 
camera, attached to a personal computer with Matrox Inspector 2.1 imaging software. 
Typically, a 50 l of aqueous enzyme-nanotube conjugate solution was deposited onto a 
glass coverslip and examined for the presence of aggregates under by optical microscopy. 
After careful addition of hydrogen peroxide (0.3% v/v) or D-glucose (100 mM), the 
movement of the carbon nanotube bundles was recorded and analyzed. 
 
 
 
Video 1. Observation by optical microscopy of conjugate 3 after addition of a glucose 
solution (100 mM) under constant air flow. The CNT aggregates are visible as black 
irregular shape. Round shape oxygen bubbles generated from the aggregate are visible. 
An amount of non-enzyme-modified CNTs was added also. 
 
Video 2. Observation by video-microscopy of conjugate 3 after addition of a glucose 
solution 100 mM under constant air flow. The CNT aggregates are visible as irregular 
black shapes. The round shapes are oxygen bubbles generated by/at the aggregates. An 
amount of non enzyme modified CNTs was added also. 
 
Video 3 Observation by video-microscopy of a static Brownian diluted solution of 
conjugate 3 at 25ºC. The very slow movement of fiber-like aggregate is attributed to 
Brownian motion. 
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 13 
 
Video 4. Observation by video-microscopy of conjugate 3 after addition of a glucose 
solution (100mM) under constant nitrogen flow. 
 
References  
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Iverson, T., Shelimov, K., Huffman, C. B., Rodriguez-Macias, F., Shon, Y. S., 
Lee, T. R., Colbert, D. T. & Smalley, R. E. Fullerene pipes. Science, 280, 1253 
(1998). 
2. Kaiser, E., Colescott, R. L., Bossinger, C. D. & Cook, P. I. Color test for detection 
of free terminal amino groups in solid-phase synthesis of perptides. Anal. 
Biochem, 34, 595-598 (1970) 
3. Beers, R. F. & Sizer, I. W. A Spectrophotometric method for measuring the 
breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195, 133-140 (1952). 
4. Bergmeyer, H. U., Gawehn, K. & Grassl, M. Methods of Enzymatic Analysis, 
Academic Press Inc., New York, Volume I, 457 (1974). 
5. Eremin, A. N., Metelitsa, D. I., Shishko, Zh. F., Mikhailova, R. V., Yasenko, M. 
I. & Lobanok, A. G. Thermal stability of glucose oxidase from Penicillium 
adametzii.  Appl. Biochem and Microbiol, 37, 579-586 (2001). 
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Function of Enzymes Adsorbed onto Single-Walled Carbon Nanotubes. Langmuir 
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Autonomous propulsion of carbon nanotubes powered by a multienzyme ensemble

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