Chapter 8

Enzyme-Nanotube-Based Composites Used forChemical and Biological Decontamination

Cerasela Zoica Dinu,1,2,* Indrakant V. Borkar,1 Shyam Sundhar Bale,1Guangyu Zhu,1 Karl Sanford,3 Gregg Whited,3 Ravi S. Kane,1

and Jonathan S. Dordick1

1Department of Chemical and Biological Engineering and RensselaerNanotechnology Center, Rensselaer Polytechnic Institute, Troy, NY 12180

2Department of Chemical Engineering, West Virginia University,Morgantown, WV 26506

3Genencor International, Palo Alto, CA 94304*cerasela-zoica.dinu@mail.wvu.edu

“Smart” coatings capable of both detecting and activelyeliminating hazardous agents are being investigated as newways for combating chemical and biological contamination.Specifically, we took advantage of the unique surface propertiesof carbon nanotubes (e.g. high surface area, controllablemorphology and size etc.) to immobilize a cocktail of enzymesand subsequently we incorporated the enzyme-carbon nanotubeconjugates into latex-based paint. Operational properties of theenzymes were optimized to yield high loading and activity onthe nanotube support, and long-term operational stability in thecomposite. This “green” namely enzyme-based technology,eliminates the risks associated with chemical decontaminationthat uses corrosive agents and generates substantial amountsof residual waste. Moreover, the “green”-based compositesdeveloped herein are cost-effective and decontaminate oncontact without imposing logistical burden to the personnel.

Introduction

There is a critical need to develop and deploy safe and effective meansto decontaminate chemical and biological warfare agents before they are

© 2010 American Chemical Society

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In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

used against military, civilian, agricultural, or other targets. To this end, wedeveloped enzyme-based composites in the forms of paints capable to effectivelyrecognize and neutralize the warfare agents. These composites are mechanicallyrobust, safe, user and environmentally friendly, and stable under long-termoperation and storage. Specifically, we took advantage of the unique surfaceproperties of carbon nanotubes (high surface area, controllable morphology andsize, increase in the number of active sites on their surface by user-directedfunctionalization) to immobilize a cocktail of enzymes (including perhydrolaseS54V and chloroperoxidase). We then incorporated the resulting nanostructuredenzyme-carbon nanotube conjugates into latex-based paint to generate activecomposites to be tested for decontamination against spore of the biological agentB.cereus, asimulant of B. anthracis and 2-chloroethyl ethyl sulfide (CEES), amustard gas analog (Figure 1). Carbon nanotubes enabled high enzyme loading,extended stability without enzyme leaching, and structural reinforcement ofthe paints. The use of biocatalysts inherently makes this technology safe,environmentally benign, operational at mild conditions while requiring lowenergy input. Importantly, the availability of suites of enzymes with desiredactivities endows flexibility and multi-functionality to the proposed technology.

Experimental

Enzyme Coupling to MWNTs

Perhydrolase S54V (AcT) solution was provided by Genencor InternationalInc. (Palo Alto, CA). Chloroperoxidase (CPO) was purchased from Sigma, St.Louis, MO. AcT and CPO were covalently attached to acid functionalized multiwalled carbon nanotubes (MWNTs) via a three-step process (2). First, carboxylicacid groups were created on MWNT (purity > 95%, outer diameter 15 ± 5 nm,length 5-20 µm, NanoLab, Inc., Newton, MA) by acid treatment. Typically, aratio of 3:1 of sulfuric to nitric acid, v/v, total of 60 ml (H2SO4, 95-98%, HNO3,68%-70%, Fisher Scientific, Hampton, NH) were added to 100 mg MWNTs andthe suspension was sonicated at room temperature for 6 h in a VWR ultrasoniccleaner (model 50T, 45 W). The functionalized MWNTs were then diluted in200 ml Milli-Q water and filtered through a 0.2 µm filter membrane (Isoporetype GTTP, polycarbonate, Millipore, Billerica, MA). The nanotubes on thefilter membrane were further redispersed by sonication in Milli-Q water; thefiltration was repeated several times to remove any residual acids and solubilizedimpurities. The functionalized MWNTs were subsequently used in the secondreaction step, for spacer binding. Specifically, 2 mg functionalized MWNTs weredispersed in 2 ml of 2-(N-morpholino)ethanesulfonic acid sodium salt (MES)buffer (50 mM, pH 4.7) containing 160 mM 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC, Acros Organics, Morris Plains, NJ) and 80mM N-hydroxysuccinimide (NHS, Pierce, Rockford, IL) by brief sonication.After 15 min shaking at 200 rpm and room temperature, the EDC/NHS activatedMWNTs were filtered, washed thoroughly with MES and redispersed in 10 mlof amino-dPEG12-acid (1 mg/ml, Quanta Biodesign, Powell, OH) solution inpotassium phosphate buffer (PB, 50 mM, pH 7.1). The mixture was further

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In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

Figure 1. Enzymes are immobilized onto multi-walled carbon nanotubes usingcovalent binding and a spacer. The spacer assures the enhanced flexibilityand greater substrate accessibility of the immobilized enzyme (1). The

resulting enzyme-nanotube conjugates are incorporated into paint and lead toactive biocomposites capable to generate agents for chemical and biological

decontamination.

incubated for 3 h at room temperature with shaking at 200 rpm. Subsequently,the PEG-functionalized MWNTs were filtered, washed to remove any residualamino-dPEG12-acid and used for the third reaction step, namely enzyme couplingreaction (again via the EDC/NHS as previously described). For the AcT,PEG-functionalized MWNTs were redispersed in 10 ml PB, 50 mM, pH 7.1containing 4 mg AcT while for CPO, the PEG-functionalized MWNTs wereredispersed in 10 ml citrate buffer (5 mM, pH 3.2) containing 4 mg CPO. Bothenzyme-nanotube mixtures were incubated for at room temperature for 3 h withshaking at 200 rpm. The resulting conjugates were subsequently filtered andwashed extensively with the corresponding buffers, while the supernatants werecollected and used for the bicinchoninic acid (BCA, Pierce, Rockford, IL) proteinassay in order to evaluate enzyme loading.

Preparation of Composites Containing Enzyme-MWNTs

Enzyme-nanotube latex-based composites were prepared by adding watersuspension of enzyme-nanotube conjugates (different concentrations) intolatex-based paint (typically 0.2 ml) in a glass vial (2.5 cm diameter). The twocomponents were mixed thoroughly using a pipette tip and the mixture wasair-dried under the hood for about 48 h.

Enzyme Activity Test

The activity of AcT was determined by measuring the concentration ofperacidic acid (PAA) generated by the conjugates or composites (3). In a typicalreaction, 10.6 µl hydrogen peroxide solution (H2O2, stock solution 30%, Sigma,St. Louis, MO, final concentration 100 mM) was added to a mixture of 0.8 mlpropylene glycol diacetate (PGD, Sigma, St. Louis, MO, final concentration 100

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In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

mM in PB, 50 mM, pH 7.1) and 0.2 ml AcT solution (2.0 µg/ml final concentrationfor free AcT or equivalent concentration of AcT for AcT-nanotube conjugates).The mixture was incubated for 20 min at 200 rpm and room temperature. PAAassay was conducted by diluting 25 µl of reaction solution 400-times in Milli-Qwater and subsequently mixing 100 µl of the diluted solution with 0.9 ml reagentassay (where the reagent was prepared by mixing 5 ml potassium citrate buffer,125 mM, pH 5.0 with 50 µl 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonicacid solution of 100 mM in Milli-Q water, Fisher Scientific, Hampton, NH and10 µl potassium iodide, 25 mM solution in Milli-Q water). The mixture wasthen incubated at room temperature for another 3 min and the absorbance at420 nm was measured on a UV-Vis spectrophotometer. PAA concentration wascalculated as:

where 0.242 is the calibrated constant correlation between concentration of PAAand absorbance at 420 nm and 400 is the dilution factor.

The activity of CPO was measured by following the conversion ofmonochlorodimedon to dichlorodimedon in the presence of H2O2 and potassiumchloride, at 25°C, pH = 2.75, and 278 nm. Specifically, a reaction mixturecontaining 98 mM citric acid, 98 mM potassium phosphate, 0.096 mMmonochlorodimedon (in potassium chloride buffer, 20 mM), 19 mM potassiumchloride, 0.006% (v/v) H2O2 and equivalent of 0.01 - 0.05 unit of CPO (all finalconcentrations) were monitored at 278 nm on the UV-Vis spectrophotometer forapproximately 5 minutes (all reagents were purchased from Sigma, St. Louis,MO).

Alternatively, for preliminary tests on chemical decontamination, 10 g/m2 of2-chloroethyl ethyl sulfide (CEES, Sigma, St. Louis, MO) were incubated withthe paint composites containg different concentrations of CPO-based conjugatesand CEES degradation was monitored using gas chromatography.

Spore Growth Conditions

Bacillus Cereus (B. cereus) 14737was purchased fromATCC (Manassas, VA)and routinely cultured in nutrient broth (3 g/L beef extract, 5 g/L peptone) (Difco,Detroit, MI, USA) prepared in Milli-Q water for 48 h. The samples were nextcentrifuged at 3000 rpm for 3min and sporulationwas induced by resuspending thecells in GYCmedia at 30°C and 180 rpm for 48 h (4). All reagents were purchasedfrom Sigma, unless otherwise mentioned.To end the sporulation, the solution wascentrifuged and the sediment redispersed in Milli-Q water; the procedure wasrepeated 5 times. Subsequently, the washed sediment was redispersed in lysosomesolution (2 mg/ml, Sigma, St. Louis, MO) and incubated at room temperature and200 rpm for another 3 h. The spores were recovered by centrifugation at 3000 rpmfor 3 min, washed and stored at 4°C in Milli-Q water. Spore purity was confirmedusing Schaeffer-Fulton staining method (5). Spore concentration was estimatedusing standard plate count technique. The sporicidal efficiency of composite filmswas determined by using a diluted suspension containing 106 CFU/ml spore.

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In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

Results

Operational properties of the enzymes were optimized to yield high loadingand activity and long-term stability in the composites. The loading of AcT-basedconjugates was around 0.06 mg AcT/mg MWNTs, while the activity of theseconjugates was about 24% of the activity of the free AcT. The capability ofgenerating sufficiently high amount of PAA was investigated in reaction withB.cereus spores. For instance, following 20 min incubation of the AcT-basedpaint composite (0.16 % conjugate concentration in the paint) with B. cereus sporesolution, the supernatant was capable of killing > 99% of spores initially chargedat 106 CFU/ml. Furthermore, preliminary results on chemical decontaminationof the composites containing 0.16 mg CPO/mg MWNTs (activity conjugatesof about 30% when compared with free CPO activity) showed more than 98%degradation of 10 g/m2 blister agent CEES (where 10 g/m2 is considered astandard in chemical decontamination).

Conclusion

Water-soluble enzyme-nanotube conjugates were prepared by covalentattachment of enzymes onto carbon nanotube supports. Uniformly incorporationof the conjugates into latex-based paint led to composites. These compositescontained “green” namely enzyme-based technology, were user friendly andgenerated reactive species capable of decontaminating chemical (CEES) andbiological agents (spores).

Acknowledgments

This work was supported by a contract (W911SR-05-0038) from the U.S.Army under a subcontract fromGenencor International, and a contract fromDTRA(HDTRA1-08-1-0022).

References

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2. Jiang, K.; Schadler, L. S.; Siegel, R. W.; Zhang, X.; Zhang, H.; Terrones, M.J. Mater. Chem. 2004, 14, 37–39.

3. Pinkernell, U.; Luke, H.-J.; Karst, U. 1997, 122, 567–571.4. Kwon, S. J.; Lee, M. Y.; Ku, B.; Sherman, D. H.; Dordick, J. S. ACS Chem.

Biol. 2007, 2, 419–25.5. O’Mahony, T.; Rekhif, N.; Cavadini, C.; Fitzgerald, G. F. J. Appl. Microbiol.

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In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.