* Corresponding author. Tel.: #81-11-706-2256; fax: #81-11-706-2256.E-mail address: nishin@ees.hokudai.ac.jp (N. Nishi).

Biomaterials 22 (2001) 3121}3126

UV-irradiation-induced DNA immobilization and functionalutilization of DNA on nonwoven cellulose fabric

Masanori Yamada���, Kozue Kato�, Kazuna Shindo�, Motoyoshi Nomizu�, MasahiroHaruki�, Nobuo Sakairi�, Kousaku Ohkawa�, Hiroyuki Yamamoto�, Norio Nishi��*

�Laboratory of Bio-Material Chemistry, Division of Bioscience, Graduate School of Environmental Earth Science,Hokkaido University, Kita-ku, Sapporo 060-0810, Japan

�Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567, Japan

Received 24 January 2000; accepted 24 January 2000

Abstract

Immobilization of double-stranded DNA onto nonwoven cellulose fabric by UV irradiation and utilization of DNA-immobilizedcloth were examined. The immobilized DNA was found to be stable in water, with the maximum amount of fabric-immobilized DNAbeing approximately 20 mg/g of nonwoven fabric. The DNA-immobilized cloth could e!ectively accumulate endocrine disruptors andharmful DNA intercalating pollutants, such as dibenzo-p-dioxin, dibenzofuran, biphenyl, benzo[a]pyrene and ethidium bromide.Additionally, DNA-immobilized cloth was found to bind metal ions, such as Ag�, Cu��, and Zn��. The maximum amounts of boundAg�, Cu��, and Zn�� onto DNA-immobilized cloth (1 g) were approximately 5, 2, and 1 mg, respectively. DNA-immobilized clothcontaining Ag� showed antibacterial activity against Escherichia coli and Staphylococcus aureus. DNA-immobilized cloth withoutmetal ion and with Cu�� or Zn�� did not show antibacterial activity. These results suggest that immobilized DNA imparts usefulfunctionality to cloth. DNA-immobilized cloth prepared by UV irradiation has potential to serve as a useful biomaterial for medical,engineering, and environmental application. � 2001 Elsevier Science Ltd. All rights reserved.

Keywords: Immobilized DNA; Intercalation; UV irradiation; Antibacterial activity; Endocrine disruptor; Functional biomaterial

1. Introduction

DNA, one of the most important materials for lifeprocesses, can be regarded as a naturally occurring andhighly speci"c functional biopolymer. Although "lmsand "bers can be prepared from DNA, they are watersoluble and have low mechanical strength. Therefore,utilization of DNA as a functional material is limited.However, there are several reports of DNA-columns[1}4], DNA-sensors [5}7], and DNA-"lms [8,9] whichutilize DNA as a functional material. Recently, we pre-pared DNA-"lms and gels conjugated with alginic acidor collagen and showed their advantages [10}13].DNA-alginic acid "lms were found to adsorb DNA-binding intercalating reagents, such as ethidium bromide[10]. DNA-alginic acid "lms also adsorbed benzo[a]-

pyrene [10], an endocrine disruptor and DNA-bindingintercalating molecule [14,15]. The adsorption mecha-nism suggested involves intercalation into double-stranded DNA through the planar structure of the aro-matic rings. Endocrine disruptors having planar struc-tures, such as dioxin and polychlorobiphenyl (PCB)derivatives, may also intercalate into the double-stranded DNA.

Beukers et al. showed that thymine}thymine dimerformation was induced when DNA was subjected to UVirradiation [16}18]. This convenient method has beenapplied for the immobilization of DNA onto cellulosepowder [1]. Using the DNA-immobilized cellulose pow-der, a DNA polymerase was successfully isolated fromMicrococcus luteus [1]. This UV irradiation method isuseful for immobilization of DNA onto a cellulose sub-strate by stable binding(s).

On the other hand, Ag� has high antibacterial activitywith low toxicity for humans. Silver compounds, such assilver sulpfdiazine, have been used as chemotherapeuticreagents for the treatment of burns [19,20]. Ag� has also

0142-9612/01/$ - see front matter � 2001 Elsevier Science Ltd. All rights reserved.PII: S 0 1 4 2 - 9 6 1 2 ( 0 1 ) 0 0 0 6 1 - 8

Fig. 1. Structure of endocrine disruptor derivatives.

been found to form reversible complexes with DNA orsynthetic polynucleotides [21]. Previously, we reportedthat DNA-alginate "lm could carry Ag� and that Ag�-containing "lm had an antibacterial activity againstEscherichia coli (E. coli) and Staphylococcus aureus(S. aureus) [13].

In the present study, we report the immobilization ofDNA onto nonwoven cellulose fabric by UV irradiation.These nonwoven fabrics are made only from cellulose"bers and have characteristics of both cloth and paper.We also report the utilization of such DNA-immobilizedcloth as functional material for the accumulation andremoval of endocrine disruptors and harmful DNA inter-calating pollutants. Additionally, we demonstrate thepreparation of Ag�-containing DNA-immobilized clothand its antibacterial activity.

2. Materials and methods

2.1. Materials

Double-stranded DNA from salmon milt (Na salt,molecular weight; 5�10�&) was obtained from YukiFine Chemical Co. Ltd., Tokyo, Japan. DNA intercalat-ing materials, including dibenzo-p-dioxin, dibenzofuran,biphenyl, benzo[a]pyrene, and ethidium bromide, werepurchased from Wako Pure Chemical Industries Ltd.,Osaka, Japan and Tokyo Kasei Industries Ltd., Tokyo,Japan. Silver nitrate, copper sulfate, and zinc sulfate werepurchased from Wako Pure Chemical Industries Ltd.Escherichia coli and Staphylococcus aureus were giftsfrom Tamatsukuri Co. Ltd., Japan. Nonwoven cellulosefabric was a gift from Otsuka Pharmaceutical Co. Ltd.,Japan.

2.2. Immobilization of DNA onto nonwoven cellulose fabricby UV irradiation

Nonwoven cellulose fabric (30 mg, approximately2�2 cm�) was immersed in 50 ml of 0.1 M sodium hy-droxide solution followed by 50 ml of 0.01 M hydrochloricacid solution for 15 min each, and then rinsed with distil-led water (100 ml�5 times). The nonwoven fabric wasdried at room temperature. Aqueous DNA solution(330 �l, 10 mg/ml DNA in H

�O) was applied onto the

nonwoven fabric and dried at room temperature, thenirradiated with UV light (R-52G, Ultraviolet Inc., USA)at 254 nm for 2 h. The intensity of UV irradiation was5600 �W/cm� at the sample position. The UV treatednonwoven fabric was rinsed with distilled water(50 ml�7 times) to remove nonimmobilized DNA andthe DNA-immobilized cloth was stored in water. Theamount of immobilized DNA applied onto the non-woven fabric was determined by the following procedure:DNA-immobilized cloth was hydrolyzed by 1 M hydro-

chloric acid solution at 1003C for 1 h. The amount ofDNA in the solution was quantitated by absorption at260 nm using a UV}VIS Spectrophotometer U-2000A(Hitachi Co. Ltd., Tokyo, Japan).

2.3. Accumulation and removal of endocrine disruptorsby DNA-immobilized cellulose cloth

Dibenzo-p-dioxin, dibenzofuran, biphenyl and benzo-[a]pyrene were used as model endocrine disruptors totest their accumulation into the DNA-immobilized cloth.Their chemical structures are shown in Fig. 1. Dibenzo-p-dioxin and dibenzofuran are derivatives of dioxin, andbiphenyl is a derivative of polychlorobiphenyl (PCB).These reagents show very low solubility in water. Weused saturated aqueous solutions of these reagents. Ben-zo[a]pyrene was dissolved in a mixed solvent(water : acetone : methanol"7 : 2 : 1) since it has almostno solubility in water. Aqueous ethidium bromide solu-tion (10 �M), a well-studied double-stranded DNA inter-calating reagent [22], was also used as a control. Theaccumulation of these reagents was examined by thefollowing procedures: DNA-immobilized cloth (30 mg)was put into the respective saturated aqueous solutions(7 ml) and incubated for 24 h at room temperature.DNA-immobilized cloth was put out from their aqueoussolutions. The accumulation of these reagents was con-"rmed by the absorption spectra of their aqueous solu-tions in the absence or presence of DNA-immobilizedcloth.

2.4. Preparation of DNA-immobilized cellulose clothcontaining metal ions

DNA-immobilized cloth (30 mg) containing metal ionwas prepared by immersing in various concentrations ofaqueous silver nitrate, copper sulfate and zinc sulfatesolutions (10 ml) for 1 h at room temperature. Then thecloths were rinsed with distilled water (50 ml�5 times) toremove the nonimmobilized metal ion, and the cloth was

3122 M. Yamada et al. / Biomaterials 22 (2001) 3121}3126

Fig. 2. Stability of DNA-immobilized cellulose cloth at various pHconditions. DNA-immobilized cloth was incubated in bu!er solution atroom temperature, and the amount of eluted DNA from DNA-immobi-lized cloth was measured at various time intervals. �, 10 mM HClsolution (pH 2); �, 10 mM NaH

�PO

�bu!er (pH 5); �, 10 mM

NaH�PO

�}Na

�HPO

�bu!er (pH 7); �, Na

�HPO

�bu!er (pH 9); �,

10 mM NaOH solution (pH 12). Each value represents the mean ofthree separation determinations $5%. Duplicate experiments gavesimilar results.

dried for 12 h at room temperature. The amount of metalion bound to the DNA-immobilized cloth was examinedby an atomic absorption spectrophotometer. Metal ioncontaining DNA-immobilized cloths (30 mg) were hy-drolyzed by 1 M hydrochloric acid solution (10 ml) at1003C for 60 min, and the amount of metal ions wasmeasured by an atomic absorption spectrophotometerSPCA-626D (Shimazu Co. Ltd., Kyoto, Japan).

2.5. Antibacterial tests

Antibacterial activity of DNA-immobilized cloth con-taining metal ions was evaluated by a bacterial culturemethod. Two types of bacteria, E. coli and S. aureus, wereused. Each of these bacteria was transferred to 10 ml ofmedium (nutrient broth, beef extract; 0.5 g, bacto-pep-tone; 0.1 g, NaCl; 0.1 g per 100 ml) solidi"ed with agar(1.5%) on a sterile culture plate (H10 cm). The solidi"edmedium was covered with appropriate sterile metal-con-taining DNA-immobilized cloth (approximately 2�2 cm�), and then incubated at 283C for 48 h. After incuba-tion, antibacterial activity of each cloth was judged bymeasuring the area of clear zones, where bacteria did notgrow.

3. Results and discussion

3.1. DNA-immobilized cellulose cloth

Double-stranded DNA solution was cast onto non-woven cellulose fabric and dried at room temperature,then treated with UV irradiation. The amount of DNAimmobilized onto the nonwoven fabric was found toincrease depending on time length of UV irradiation andreached a constant value in approximately 15 min (datanot shown). The maximum amount of DNA immobilizedonto the nonwoven fabric (1 g) was approximately 20 mg.The resulting DNA-immobilized cloth was stable inaqueous solution and insoluble even when it was soakedin water for long periods (100 days). These results sugges-ted the DNA was immobilized by stable binding as wellas previously described [1]. The stability of the DNA-immobilized cloth was examined in various aqueoussolutions (pH 2}12), then the amount of eluted DNAfrom the DNA-immobilized cloth was determined(Fig. 2). The DNA-immobilized cloth was found to bestable under neutral and basic conditions (pH 5}12).However, DNA was eluted into the solution when it wasincubated under acidic conditions (Fig. 2). This resultsuggested that DNA phosphodiester bonds were hy-drolyzed under the acidic conditions. These results alsoindicated that DNA-immobilized cloth should be amen-able for use as a biomaterial under neutral and basicconditions.

Next, we determined the e!ects of salt concentrationon the elution of DNA from the DNA-immobilized cloth(Fig. 3). When sodium chloride or magnesium chloridewas added to the solution, the amount of eluted DNAfrom the DNA-immobilized cloth decreased. Less than3% of the DNA that was initially immobilized onto thecloth was eluted upon incubation with 10 or 100 mM

aqueous salt solution for 48 h. Metal ions, especiallymagnesium ion, bind to phosphate groups of DNA byelectrostatic interactions [23,24]. These results indicatethat immobilized DNA on the nonwoven fabric is stabil-ized by electrostatic interaction with metal ions.

3.2. Accumulation and removal of endocrine disruptorsby DNA-immobilized cellulose cloth

We examined the interaction of the DNA-immobilizedcloth with endocrine disruptors, such as dibenzo-p-diox-in, dibenzofuran, biphenyl, and benzo[a]pyrene. TheDNA-immobilized cloth was incubated in the saturatedaqueous solutions of endocrine disruptors, and then theamount of the resulting compound in solution was mea-sured by UV spectrophotometry. Fig. 4A shows the ab-sorption spectra of aqueous dibenzofuran solutions inthe absence or presence of DNA-immobilized cloth.When DNA-immobilized cloth was added to saturatedaqueous dibenzofuran solution, absorption peaks at 250and 280 nm decreased. Figs. 4B, C, and D show absorp-tion spectra of aqueous dibenzo-p-dioxin, biphenyl, andbenzo[a]pyrene solutions in the absence or presence ofthe DNA-immobilized cloth, respectively. The absorp-tion peaks of these reagents also decreased following theaddition of DNA-immobilized cloth. An aqueous

M. Yamada et al. / Biomaterials 22 (2001) 3121}3126 3123

Fig. 3. E!ects of salts on stability of DNA-immobilized cellulose cloth.DNA-immobilized cloth was incubated in the presence of (a) sodiumchloride aqueous solution; (b) magnesium chloride and analyzed theamount of eluted DNA from DNA-immobilized cloth. Concentrationsof the salts were �, 0 mM; �, 1 mM; �, 10 mM; �, 100 mM. Each valuerepresents the mean of three separation determinations $5%. Duplic-ate experiments gave similar results.

Fig. 4. Absorption spectra of aqueous solutions of endocrine disruptorderivatives in the absence (a) and presence (b) of DNA-immobilizedcellulose cloth. (A) Aqueous dibenzofuran solution; (B) aqueousdibenzo-p-dioxin solution; (C) aqueous biphenyl solution; (D) ben-zo[a]pyrene solutions (water : acetone : methanol"7 : 2 : 1). Duplicateexperiments (n"3}5) gave similar results.

ethidium bromide solution, widely used as a double-stranded DNA intercalating reagent [22], also gavesimilar results. The initially white DNA-immobilizednonwoven cellulose fabric turned red following intercala-tion of ethidium bromide into the double-stranded DNA.These results suggest that the DNA-immobilized clothcan capture intercalating reagents similar to that ofa double-stranded DNA described previously [22].

When single-stranded DNA-immobilized cloth was in-cubated in benzo[a]pyrene solution, a decrease in theabsorbtion was not induced (data not shown). Further-more, diethylstylbestrol and bisphenol A, which are en-docrine disruptors and do not interact with DNA [25],did not show decrease in absorbtion even in the presenceof double-stranded DNA-immobilized cloth. These re-sults suggest that endocrine disruptors shown in Fig. 1bind to the double-stranded DNA-immobilized cloth byintercalation or in some similar manner. It is likely thatendocrine disruptors with planar structures (Fig. 1)intercalate into double-stranded DNA-immobilizedcloth. DNA-immobilized cloth may be useful in environ-mental science for the selective accumulation andremoval of DNA intercalating materials, such as dioxin

and PCB, designated as endocrine disruptors. Since theintercalation of these endocrine disruptors into DNA isspeci"c, the double-stranded DNA-immobilized clothwould be useful for the removal of highly minuteamounts of dioxin included in milk, for example, bynot removing the other nutrients such as proteins andvitamins.

3.3. Accumulation of metal ions into DNA-immobilizedcellulose cloth and associated antibacterial activity

Next, we measured the accumulation of metal ions intothe DNA-immobilized cloth. DNA immobilized clothwas incubated in metal ions containing aqueous solutionfor 1 h. Then, the DNA-immobilized cloth was

3124 M. Yamada et al. / Biomaterials 22 (2001) 3121}3126

Fig. 5. Antibacterial activity of DNA-immobilized cellulose cloth con-taining (a) and not containing silver ion (b) for E. coli. Duplicateexperiments (n"3) gave similar results.

hydrolyzed with 1 M hydrochloric acid solution and theamount of accumulated metal was analyzed by atomicabsorption spectrophotometry. The maximum amountsof Ag�, Cu�� and Zn�� bound to the DNA-immobilizedcloth (1 g) were approximately 5, 2, and 1 mg, respective-ly. Positively charged metal ions were shown to bind tophosphate groups by electrostatic interaction [23,24].Among them, only Ag� is monovalent. In addition, Ag�

was described to bind to adenine}thymine base pairs[21]. These could be the reasons why larger amounts ofAg� bind to DNA-immobilized cloth as compared withother metal ions.

Next, we examined the antibacterial activities ofDNA-immobilized cloth containing metal ions usingE. coli and S. aureus. Culture plates, in which E. coli wascultured in the presence of DNA-immobilized cloth withor without Ag� are shown in Fig. 5. A clear zone, whereE. coli did not grow, was observed around theDNA-immobilized cloth containing Ag� (Fig. 5a), whileDNA-immobilized cloth without Ag� did not show anti-bacterial activity (Fig. 5b). When we tested the anti-bacterial activity using S. aureus, Ag�-containingDNA-immobilized cloth also showed antibacterial activ-ity (data not shown). When Cu�� and Zn�� were asso-ciated to DNA-immobilized cloth, no antibacterialactivity was observed (data not shown). These resultsclearly suggest that antibacterial activity was caused byAg� bound to DNA-immobilized cloth. DNA-immobi-lized cloth containing Ag� could potentially be used asan antibacterial material.

4. Conclusions

We prepared DNA-immobilized nonwoven cellulosefabric using UV irradiation. This DNA-immobilizedcloth was stable in various pH and salt conditions. ThisUV irradiation method may be useful for the immobiliz-ation of DNA onto not only the cellulose substrates, suchas cellulose "bers and cellulose "lters, but also the inter-nal organs and inorganic materials.

The DNA-immobilized cloth e!ectively accumulatedendocrine disruptors and DNA-binding intercalating re-agents. Additionally, DNA-immobilized cloth was foundto carry metal ions, such as Ag�, Cu��, and Zn��. Theseresults suggest that the DNA-immobilized cloth can beused not only for the accumulation of harmful chemicalcompounds but also for the isolation of DNA-bindingprotein and the biochemical analysis of the immunolo-gical interaction.

Furthermore, DNA-immobilized cloth containingAg� showed antibacterial activity. Therefore, DNA-im-mobilized cellulose cloth prepared with the antibacterialactivity may have potential utility as a novel biomaterialfor medical, engineering, and environmental puri"cation.

Acknowledgements

This work was supported by the Grants-in-Aid forScienti"c Research from the Ministry of Education,Science, Sports, and Culture of Japan (Nos. 11694114,11450359 and 10555327) and also by Hokkaido Founda-tion for the Promotion of Scienti"c and Industrial Tech-nology (Hokscitec). We also thank Dr. Terrence Burke(National Cancer Institute, National Institutes of Health,Bethesda, MD, USA) for critical reading of the manu-script.

References

[1] Litman RM. A deoxyribonucleic acid polymerase from micrococ-cus luteus (micrococcus lysodeikticus) isolated on deoxyribonuc-leic acid-cellulose. J Biol Chem 1968;243:6222}33.

[2] Cavalieri LF, Carroll E. A DNA-acrylamide gel column for ana-lyzing proteins that bind to DNA, I. DNA polymerase. Proc NatAcad Sci USA 1970;67:807}12.

[3] Rickwood D. An improved method for the insolubilization ofDNA for a$nity chromatography. Biochim Biophys Acta 1972;269:47}50.

[4] Mizutani T. Adsorption chromatography of nucleic acids onsilicone-coated porous glass. J Biochem 1983;94:163}9.

[5] Yamaguchi S, Shimomura T, Tatsuma T, Oyama N. Adsorption,immobilization, and hybridization of DNA studied by the use ofquartz crystal oscillators. Anal Chem 1993;65:1925}7.

[6] Skuridin SG, Yevdokimov YM, E"mov VS, Hall JM, TurnerAPF. A new approach for creating double-stranded DNA biosen-sors. Biosensors Bioelectron 1996;11:903}11.

[7] Wang J, Rivas G, Cai X, Palecek E, Nielsen P, Shiraishi H,Dontha N, Luo D, Parrado C, Chicharro M, Farias PAM, ValeraFS, Grant DH, Ozsoz M, Flair MN. DNA electrochemical bio-sensors for environmental monitoring a review. Anal Chim Acta1997;347:1}8.

[8] Tanaka K, Okahata Y. A DNA}lipid complex in organic mediaand formation of an aligned cast "lm. J Am Chem Soc 1996;118:10679}83.

[9] Ijiro K, Okahata Y. A DNA}lipid complex soluble in organicsolvents J Chem Soc Chem Commun 1992:1339}41.

[10] Iwata K, Sawadaishi T, Nishimura S, Tokura S, Nishi N. Utiliz-ation of DNA as functional materials: preparation of "lters con-taining DNA insolubilized with alginic acid gel. Int J BiolMacromol 1996;18:149}50.

M. Yamada et al. / Biomaterials 22 (2001) 3121}3126 3125

[11] Kitamura H, Matsuura E, Nagata A, Sakairi S, Tokura S, NishiN. DNA}alginate complex recognized by autoantibodies againstDNA. Int J Biol Macromol 1997;20:75}7.

[12] Kitamura H, Iwamoto C, Sakairi N, Tokura S, Nishi N. Markede!ect of DNA on collagen "brillogenesis in vitro. Int J BiolMacromol 1997;20:241}4.

[13] Kitamura H, Kondo Y, Sakairi N, Nishi N. Preparation andcharacterization of antibacterial alginate "lm containing DNA asa carrier of silver ion. Nucl Acids Symp Ser 1997;37:273}4.

[14] Nagata C, Fujita H, Imamura A. Semi-empirical self-consistent"eld molecular orbital calculation of transition energy and oscil-lator strength for the stacked complexes of pyrene and 3,4-ben-zopyrene with DNA bases. Bull Chem Soc Jpn 1967;40:2564}8.

[15] Geacintov NE, Prusik T, Khosro"an JM. Properties of ben-zopyrene}DNA complexes investigated by #uorescence and trip-let #ash photolysis techniques. J Am Chem Soc 1976;98:6444}52.

[16] Beukers R, Ylstra J, Berends W. The e!ect of ultraviolet light onsome components of the nucleic acids. II. In rapidly frozen solu-tions. Rec Trav Chim 1958;77:729}32.

[17] Beukers R, Berends W. Isolation and identi"cation of the irradia-tion product of thymine. Biochim Biophys Acta 1960;41:550}1.

[18] Glisin VR, Doty P. The cross-linking of DNA by ultravioletradiation. Biochim Biophys Acta 1967;142:314}22.

[19] Ho!man S. Silver sulfadiazine: an antibacterial agent for topicaluse in burns. A review of the literature. Scand J Plast ReconstrSurg 1984;18:119}26.

[20] Boeckx W, Blondeel PN, Vandersteen K, Wolf-Peeters CD,Schmitz A. E!ect of cerium nitrate}silver sulphadiazine on deepdermal burns: a histological hypothesis. Burns 1992;18:456}62.

[21] Luk KFS, Maki AH, Hoover RJ. Studies of heavy metal bindingwith polynucleotides using optical detection of magnetic reson-ance. Silver(I) binding. J Am Chem Soc 1975;97:1241}2.

[22] Fuller W, Waring MJ. A molecular model for the interaction ofethidium bromide with deoxyribonucleic acid. Ber BunsengesPhys Chem 1964;68:805}8.

[23] Zimmer C, Luck G, Triebel H. Conformation and reactivity ofDNA. IV. Base binding ability of transition metal ions to nativeDNA and e!ect on helix conformation with special reference toDNA}Zn(II) complex. Biopolymers 1974;13:425}53.

[24] Eichhorn GL, Shin YA. Interaction of metal ions with poly-nucleotides and related compounds. XII. The relative e!ect ofvarious metal ions on DNA helicity. J Am Chem Soc 1968;90:7323}8.

[25] Tso POP, Lu P. Interaction of nucleic acids, I. Physical binding ofthymine, adenine, steroids, and aromatic hydrocarbons to nucleicacids. Proc Nat Acad Sci USA 1964;51:17}24.

3126 M. Yamada et al. / Biomaterials 22 (2001) 3121}3126