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Analytica Chimica Acta 647 (2009) 249–254

Contents lists available at ScienceDirect

Analytica Chimica Acta

journa l homepage: www.e lsev ier .com/ locate /aca

elective accumulation of harmful compounds by the DNA-inorganicybrid-immobilized glass bead

asanori Yamada ∗, Akari Hamaiepartment of Chemistry, Faculty of Science, Okayama University of Science, Ridaicho 1-1, Okayama 700-0005, Japan

r t i c l e i n f o

rticle history:eceived 8 February 2009eceived in revised form 5 June 2009ccepted 10 June 2009vailable online 17 June 2009

a b s t r a c t

Previously, we reported the DNA-inorganic hybrid material including double-stranded DNA by mix-ing the aqueous DNA solution and silane coupling reagents. Here, we immobilized the DNA-inorganichybrid material onto the glass bead and prepared the DNA-immobilized glass bead column. The DNA-immobilized glass beads were stable in water and the amount of eluted DNA from the DNA-glass beads

eywords:NAunctional materialntercalation

did not change for more than 1 week. Additionally, this DNA-immobilized column selectively accumu-lated the harmful compounds with the planar structure, such as dioxin- and polychlorinated biphenyl(PCB)-derivatives, and these accumulation percentages were 50–70%. Furthermore, the DNA-immobilizedglass bead was recycled nine times by the application of ethanol solution and the accumulative ratio wasmaintained at more than 60% and did not appear to be decreasing. Therefore, these DNA-columns might

electar st

NA-immobilized columnrganic–inorganic hybrid material

have a potential for the scompounds with the plan

. Introduction

Harmful chemicals in food, water, and solid have become one oforldwide problems [1–3]. Especially, polychlorinated dibenzo-p-ioxin, dibenzofuran, and biphenyl have been emerged as potentialarcinogenic compounds or endocrine disruptors. They exert phys-ological effects at very low concentrations [1]. However, theseompounds, which have been existed at the low concentration andhe wide area, are difficult to selectively accumulate. Therefore, anfficient system for removing and enriching harmful compoundsrom polluted water is definitely required.

The conversion of natural products to a useful material is impor-ant for environmental safety and product cost through a greenhemistry approach. Especially, the utilization of double-strandedNA, one of the most important materials of genetic process in a

ife thing, has been attracted [4,5]. Although DNA is readily puri-ed from salmon milts or shellfish gonads, large amount of theseNA-enriched materials have been discarded as waste by indus-

ry. Furthermore, DNA has a highly specific function, such as thentercalation, the groove binding, and the complementary inter-ction between nucleic acid bases [6,7]. Therefore, the conversion

f this discarded DNA to a functional material would be ben-ficial to utilize the unique properties of DNA. As a result, thetilization of DNA to the electrical [8,9], optical [10–13], medical14,15], and device [16,17] materials has been reported. Recently, we

∗ Corresponding author. Tel.: +81 86 256 9550; fax: +81 86 256 9757.E-mail address: myamada@chem.ous.ac.jp (M. Yamada).

003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2009.06.024

ive removal and separation of DNA-intercalating molecules and harmfulructure from experimental or industrial drainages.

© 2009 Elsevier B.V. All rights reserved.

prepared the water-insoluble and nuclease-resistant DNA-matrixby the UV irradiation [18] and proposed the UV-irradiated DNA-immobilized glass bead column as an environmental material, suchas the removal of harmful compounds [19] and heavy metal ions[20]. However, since these UV-irradiated DNA matrices did not havea mechanical strength, the UV-irradiated DNA-column could notuse for a long time. Additionally, the preparation of UV-irradiatedDNA-column necessitated a high power UV lamp of 254 nm.

Organic–inorganic hybrid materials with both the properties ofthe flexibility and the mechanical strength have been attractedas the novel functional materials, such as for electrical and opti-cal devices, biocompatible, and sensors materials [21–25]. Mostrecently, we also prepared a flexible DNA-inorganic hybrid filmwith the mechanical strength by mixing double-stranded DNAand silane coupling reagents [26]. These hybrid films were water-insoluble and resistant to hydrolysis by a nuclease. Furthermore,DNA-inorganic hybrid film could selectively accumulate harmfulcompounds with a planar structure, such as dibenzo-p-dioxin,dibenzofuran, and biphenyl, by the intercalation into double-stranded DNA [26]. However, the accumulative ratio of the harmfulcompound by the DNA-inorganic hybrid film was approximately40% and was not so high. Therefore, the improvements of DNA-inorganic hybrid material for the environmental application werenecessary.

In the present study, we immobilized the DNA-inorganic hybridmaterial onto the glass bead and prepared the DNA-immobilizedcolumn. The DNA-immobilized glass beads (DNA-glass beads) werestable in water and the amount of eluted DNA from the DNA-glass beads did not change for more than 1 week. Additionally, this

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NA-inorganic hybrid material-immobilized glass bead columnDNA-column) selectively accumulated the harmful compoundsith the planar structure, such as dioxin- and polychlorinated

iphenyl (PCB)-derivatives. Furthermore, this DNA-immobilizedolumn could recycle by the addition of organic solvent, such asthanol.

. Experimental

.1. Materials

Double-stranded DNA (sodium salt from salmon milt, molecu-ar weight; >5 × 106) was obtained from Yuki Fine Chemical Co. Ltd.,okyo, Japan, and used without further purification. Silane couplingeagents bis(trimethoxysilylpropyl)amine (SiNSi) was purchasedrom Gelest Inc., Morrisville, PA. Glass beads were purchased fromhe As one Corp., Osaka, Japan. The particle size of glass bead was.500–0.710 mm. Ethidium bromide, dibenzo-p-dioxin, dibenzofu-an, biphenyl, bisphenol A, and diethylstilbestrol were obtainedrom Wako Pure Chemical Industries Ltd., Osaka, Japan, Tokyo Kaseindustries Ltd., Tokyo, Japan, or Nacalai Tesque Inc., Kyoto, Japan.he molecular structures of these harmful compounds are shown

n Scheme 1. Solvents were used an analytical grade in all the experi-ents described. Ultra-pure water (Millipore Corporation, Billerica,A) was used in this research.

.2. Preparation of DNA-inorganic hybrid-immobilized glasseads

The hydrophilic glass beads was treated the by the following pro-edure: glass beads (500 mg) were heated in 2 mL of 30% hydrogeneroxide water and concentrated sulfuric acid (30:70, v/v) at 70 ◦C

or 30 min [27,28]. The glass beads were rinsed with distilled water5 mL × 10 times) and dried at room temperature for 24 h.

Aqueous double-stranded DNA solution (1 mL, 10 mg mL−1) andilane coupling reagents SiNSi (3 �L) were vigorously mixed in aicrotube. This mixing ratio of DNA and SiNSi is 23 wt% and the

haracteristics of this material have been reported [26]. The treatedlass beads (500 mg) was added in this DNA-SiNSi solution, vigor-usly mixed by the touch mixer for 20 s, and then dried on Petriishes at room temperature overnight. The glass beads were rinsedith distilled water (10 mL × 7 times) to remove non-immobilizedNA. The amount of immobilized DNA was determined by the

ollowing procedure [18,19,26,29]: DNA-immobilized glass beads100 mg) were hydrolyzed with 1 M hydrochloric acid (5 mL) at00 ◦C for 1 h, and then the amount of DNA in the aqueous solutionas determined from the absorbance at 260 nm.

The stability in an aqueous solution of the DNA-immobilizedlass beads was confirmed by the following method: the DNA-lass beads (100 mg) were incubated in ultra-pure water (20 mL)or various time intervals. The absorbance at 260 nm of the solu-ion was measured, and the eluted DNA from the DNA-glass beadsas determined.

.3. Preparation of DNA-inorganic hybrid-immobilized glass beadolumn

The DNA-column was prepared by the previous report [19].NA-immobilized glass beads (500 mg) were packed in a Pasteur

ipette (Ø 5 mm, Iwaki Glass Co. Ltd., Tokyo, Japan). The lengthf the mobile phase in DNA-column was 60 mm. The flow rate ofhe DNA-immobilized glass bead column was 0.5 mL min−1. ThisNA-column was washed in water (ca. 100 mL) and then used forxperiments.

ica Acta 647 (2009) 249–254

2.4. Removal of harmful compounds by the DNA-inorganichybrid-immobilized glass beads

Dibenzo-p-dioxin, dibenzofuran, biphenyl, bisphenol A, anddiethylstilbestrol were used as model harmful compounds, suchas endocrine disruptors. The aqueous harmful compound solutionswere prepared by the reported method [19]. The concentrationsof aqueous dibenzo-p-dioxin, dibenzofuran, biphenyl, bisphenolA, and diethylstilbestrol solutions are 0.68, 0.72, 0.53, 13, and1.4 �M, respectively. The DNA-inorganic hybrid-immobilized glassbeads (500 mg) were incubated in the respective aqueous harmfulcompound solution (10 mL) for 24 h at room temperature. The DNA-glass beads were then separated from the aqueous solutions. Theamount of accumulated compounds was determined by the absorp-tion spectra of the aqueous solutions in the absence or presenceof the DNA-glass beads. The aqueous ethidium bromide (10 �M)solution was used as the intercalating reagent into double-strandedDNA [6,7].

2.5. Removal of harmful compounds by the DNA-inorganichybrid-immobilized glass bead column

The accumulation of harmful compounds by a DNA-inorganichybrid-immobilized glass bead column was examined by thefollowing procedures. The aqueous solution (10 mL) of harmfulcompounds was applied to the DNA-immobilized glass bead col-umn and the eluted solution was reapplied. This process wasrepeated 10 times. The accumulative ratio of these compounds wasdetermined by the absorption spectra of the eluted solution and thestarting solution. The reusability of DNA-column was demonstratedby the applying of ethanol solution (10 mL).

3. Results and discussion

3.1. Preparation of DNA-inorganic hybrid-immobilized glassbeads

DNA-inorganic hybrid-immobilized glass beads were preparedby the following procedure: DNA aqueous solution and silane cou-pling reagents SiNSi were mixed. This aqueous DNA-23 wt% SiNSisolution was mixed with hydrophilized glass beads and dried atroom temperature. The amount of immobilized DNA on glass beadswas hydrolyzed by 1 M HCl and was quantitated by the absorbanceat 260 nm. The amount of the immobilized DNA onto glass beadswas 3.0 mg per gram of glass beads. The amount of the immobi-lized DNA onto the glass bead without hydrophilization was 2.2 mgand was lower than that of glass bead with the hydrophilization.Generally, the trimethoxysilyl group, –Si(OCH3)3, hydrolyzes step-wise in water to give the corresponding silanols, which ultimatelycondense to siloxanes [30]. In this research, since the glass beadswith the hydrophilization have many silanol groups on the sur-face, these beads reacted with the trimethoxysilyl groups of SiNSimolecules. These results indicated that the hydrophilization of glassbeads were effective for the immobilizing of DNA-inorganic hybridonto beads. On the other hand, when the aqueous DNA-23 wt%SiNSi solution was mixed with the smaller glass bead (particle size:0.177–0.250), the amount of immobilized-DNA onto glass beadsincreased. This phenomenon is due to the increase of surface area.

Next, we examined the properties of the double-strandedDNA structure in DNA-inorganic hybrid-immobilized glass beads.

Fig. 1 shows the fluorescence image of DNA-inorganic hybrid-immobilized glass beads with UV irradiation of 254 nm in aqueousethidium bromide solution. The ethidium bromide molecules,which intercalated into the double-stranded DNA, indicate thestrong fluorescence under UV irradiation [6,7,18,19]. In fact, each

M. Yamada, A. Hamai / Analytica Chimica Acta 647 (2009) 249–254 251

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Scheme 1. Molecular structu

NA-immobilized glass bead showed the strong fluorescence underV irradiation. In addition, this fluorescence was obtained at all of

he part. These results suggested that the DNA in DNA-inorganicybrid-immobilized glass beads have maintained the double-tranded structure and the DNA functions as an intercalation.urthermore, since the preparation of DNA-inorganic hybrid glasseads does not use the high power UV lamp, such as UV irradiation-ethod [18,19], this immobilization-methods of double-strandedNA is convenient and safety.

.2. Stability of DNA-inorganic hybrid-immobilized glass beads inater

We examined the stability of the DNA-inorganic hybrid-mmobilized glass beads in water. The amount of eluted DNA fromhe DNA-immobilized glass beads was determined by absorbance

ig. 1. Fluorescence image of DNA-inorganic hybrid-immobilized glass beads withV irradiation of 254 nm.

various harmful compounds.

at 260 nm. Fig. 2 shows the stability of DNA-immobilized glassbeads with a different mixing ratio of SiNSi molecules in water. Theamount of eluted DNA from the DNA-glass beads increased with theincubation time and reached a constant value at 2 h. This amountof eluted DNA decreased with the increase of the mixing ratio ofthe silane coupling reagents. At the 23 wt% SiNSi, approximately20% of the DNA on the glass beads eluted into water. The amount ofeluted DNA did not change for more than 1 week. Therefore, DNA-23 wt% SiNSi hybrid-immobilized glass beads were stored in waterfor more than 24 h to remove the non-immobilized DNA and thenused for experiments.

3.3. Accumulation of harmful compounds by DNA-inorganichybrid-immobilized glass beads

Harmful compounds with a planar structure, such as dioxin- andPCB-derivatives and benzo[a]pyrene, have been shown to be DNA-

Fig. 2. Stability of DNA-inorganic hybrid-immobilized glass beads in water: (�)DNA-3 wt% SiNSi hybrid glass beads; (�) DNA-10 wt% SiNSi hybrid glass beads; (©)DNA-23 wt% SiNSi hybrid glass beads. Each values in represents the mean of threeseparate determinations ± standard deviations. Triplicate experiments gave similarresults.

2 ca Chimica Acta 647 (2009) 249–254

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before (a) and after (b) the application to the DNA-column.When aqueous ethidium bromide solution was applied to theDNA-column, ethidium bromide was completely bound to the DNA-immobilized glass bead column and the absorption spectra of

52 M. Yamada, A. Hamai / Analyti

ntercalating compounds [19,31,32]. Bisphenol A, diethylstilbestrol,nd benzophenone without the planar structure do not inter-alate into double-stranded DNA [19,26,33]. Previously, we alsoeported the selective accumulation of planar structure-containingarmful compounds, such as dioxin- and PCB-derivatives, by theNA-inorganic hybrid film [26] or UV-irradiated DNA material [19].owever, the accumulative ratio of the harmful compound by theNA-inorganic hybrid film was approximately 40% and was not

o high. Here, the accumulation of harmful compounds by DNA-norganic hybrid-immobilized glass beads was demonstrated. TheNA-inorganic hybrid-immobilized glass beads were incubated

or 24 h in aqueous solutions of the harmful compound, and themounts of compound were then determined by measuring thebsorption spectra of the solutions.

Fig. 3(A) shows the absorption spectra of aqueous dibenzo-uran solution, one of the endocrine disruptors with the planartructure, in the absence (a) and presence (b) of DNA-inorganicybrid-immobilized glass beads. When the DNA-glass beads weredded to the aqueous dibenzofuran solution, the absorbance of theolutions decreased. Namely, approximately 25% of dibenzofuranas accumulated by the DNA-glass beads. Similar results, such as

he accumulation of harmful compounds with the planar structure,btained at the dibenzo-p-dioxin, biphenyl, and ethidium bromidesee the diagonal bar in Fig. 4). Fig. 3(B) shows the absorption spec-ra of aqueous bisphenol A solution without the planar structure inhe absence (a) and presence (b) of DNA-glass beads. In this case,he decrease of absorbance did not observe. Similar result, suchs non-accumulation of the harmful compounds without the pla-ar structure, obtained at the diethylstilbestrol (see the diagonalar in Fig. 4). These results suggested that the DNA-immobilizedlass beads could selectively accumulate the harmful compoundsith the planar structure. Additionally, these phenomena with theolecular selectivity coincide with the previous reports and the

ccumulative mechanism of these harmful compounds is due tohe intercalation of chemicals into double-stranded DNA. On thether hand, normal glass bead without the immobilization of DNAid not show the accumulation of harmful compounds.

ig. 3. Absorption spectra of harmful compounds in the absence (a) and presenceb) of DNA-inorganic hybrid-immobilized glass beads: (A) aqueous dibenzofuranolution; (B) aqueous bisphenol A solution.

Fig. 4. The accumulative ratio of harmful compounds by the DNA-glass beads andDNA-column. The amount of DNA-glass beads is 500 mg.

3.4. Accumulation of harmful compounds by DNA-immobilizedglass bead column

Since DNA-inorganic hybrid-immobilized glass beads couldaccumulate approximately 25% of the endocrine disruptorswith the planar structure from aqueous solutions, we preparedDNA-inorganic hybrid-immobilized glass bead column to moreeffectively accumulate endocrine disruptors. Fig. 5(A) showsthe absorption spectra of aqueous ethidium bromide solution

Fig. 5. Absorption spectra of harmful compounds before (a) and after (b) applica-tion to DNA-inorganic hybrid-immobilized glass bead column: (A) aqueous ethidiumbromide solution; (B) aqueous dibenzofuran solution; (C) aqueous bisphenol A solu-tion.

a Chimica Acta 647 (2009) 249–254 253

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he elution disappeared. Additionally, DNA-glass beads were dyeded by the intercalation of ethidium bromide (data not shown).hese results suggested that DNA-inorganic hybrid-immobilizedlass bead columns were effective methods to accumulate thearcinogenic DNA-intercalating compounds, such as ethidium bro-ide.

Fig. 5(B) shows the absorption spectra of dibenzofuran before (a)nd after (b) the application to the DNA-column. The absorbancef dibenzofuran decreased by the application to the DNA-columnnd the accumulative ratio was approximately 70%. This value wasigher than that of DNA-glass beads methods. Similar results werelso obtained at the model endocrine disruptors, such as dibenzo--dioxin and biphenyl, and the accumulative ratio of DNA-columnas higher than that of DNA-glass beads methods (see the closed

ar in Fig. 4). In contrast, Fig. 5(C) shows the absorption spectraf bisphenol A, which does not have a planar structure, before (a)nd after (b) the application to the DNA-column. In this case, thebsorbance did not change and bisphenol A molecules did not bindhe DNA-inorganic hybrid-immobilized glass bead column. Sim-lar results were also obtained at the diethylstilbestrol (see thelosed bar in Fig. 3). These results suggested that the DNA-inorganicybrid column is effective methods to accumulate and separate thearmful compounds with the planar structure, such dioxin- andCB-derivatives.

Next, we calculated the maximum accumulative amount oflanar structure-containing harmful compounds into DNA-beadsnd DNA-column. As a result, in our experimental condition, theaximum accumulative amounts of dibenzo-p-dioxin, dibenzo-

uran, biphenyl, and ethidium bromide per gram of DNA-beadsere 0.53 �g (3.1 nmol), 0.56 �g (3.0 nmol), 0.31 �g (2.0 nmol), and

9 �g (20 nmol), respectively. Additionally, the maximum accumu-ative amounts of dibenzo-p-dioxin, dibenzofuran, biphenyl, andthidium bromide per gram of DNA-column were 1.2 �g (7.3 nmol),.8 �g (9.8 nmol), 0.88 �g (5.7 nmol), and 79 �g (20 nmol), respec-ively. These results suggested that the method of DNA-columnas more effective than that of DNA-beads for the accumula-

ion of planar structure-containing toxic compounds. In contrast,he amount of DNA in DNA-immobilized glass beads (1 g) is.0 mg and has the intercalative site of 4.7 �mol. This valueas extremely higher than the total accumulation amount ofarmful compounds. These phenomena have been reported34] and these are due to the equilibrium constant betweenhe double-stranded DNA and the planar structure-containingarmful compounds, such as dibenzo-p-dioxin or dibenzofu-an.

.5. Reusability of DNA-inorganic hybrid-immobilized glass beadolumn

Finally, we demonstrated the reusability of DNA-inorganicybrid-immobilized glass bead column. When the ethanol solutionas applied into the dibenzofuran-accumulated column, diben-

ofuran molecules were released to ethanol solution from theNA-immobilized glass beads (data not shown). These releasesf dioxin-derivatives to organic solvent have been reported [34]nd this is due to the conformational change of double-strandedNA. So, after the application of ethanol solution, aqueous diben-ofuran solution was reapplied into the DNA-immobilized column.hese cycles were repeated nine times. Fig. 6 shows the accumu-

ation percentage of dibenzofuran versus the number of recyclingrocess. Although the DNA-immobilized glass bead was recycled

ine times by the application of ethanol solution, the accumula-ive ratio was maintained at more than 60% and did not appearo be decreasing. These results suggested that the DNA-columnould be recycled by the washing with organic solvent, such asthanol.

[

Fig. 6. Reusability of the DNA-inorganic hybrid-immobilized glass bead column.Dibenzofuran molecule was used for model harmful compounds with the planarstructure. Used DNA-column was recycled by the washing with ethanol solution. Theaccumulation percentage was determined by the absorbance of the eluted solution.

4. Conclusion

We prepared the DNA-immobilized glass bead by the mix-ing of DNA-23 wt% SiNSi solution and glass beads. The DNA-glassbeads effectively bound the harmful compounds with the planarstructure, such as dioxin- and PCB-derivatives. Bisphenol A anddiethylstilbestrol, which lack a planar structure, were not accumu-lated by the DNA-glass beads. The accumulative ratios of harmfulcompounds were improved by the preparation of DNA-inorganichybrid-immobilized glass bead column. Furthermore, this DNA-immobilized column could recycle by the application of commonorganic solvent, such as ethanol. Therefore, DNA-inorganic hybridcolumn is convenient and effective methods to accumulate theDNA-intercalating compounds and harmful compounds with theplanar structure, such dioxin- and PCB-derivatives. Although theaccumulated percentage of the harmful compound by the DNA-inorganic hybrid column was approximately 50–70% and was notso high, these accumulation percentages might be improved byan extension of mobile phase in DNA-immobilized column or thecontrol of flow rate from the DNA-column. DNA-inorganic hybrid-immobilized glass bead column may have a potential for not onlyenvironmental application but also medical, and engineering appli-cations, such as the accumulation of useful and trace compounds.

Acknowledgements

This work was supported by the Grants-in-Aid for ScientificResearch from MEXT (Ministry of Education, Culture, Sports, Sci-ence and Technology of Japan) (no. 19750187), by matching fundsubsidy for private universities, and by the Yakumo Foundation forEnvironmental Science.

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