Xinmei Zhao,Huanhuan You,Faming Gao*

Key Laboratory of Applied Chemistry,Department of Applied Chemistry,Yanshan University,Qinhuangdao 066004,China

Keywords:DNA-assistance BaF2 Morphology Linear arranged nanoparticles Erythrocyte-shaped structure

ABSTRACT The synthesis of inorganic materials with special morphologies with the assistance of biological molecules is a potential development in the field of controllable growth and assembly of nanomaterials.In this paper,BaF2 nanocrystals in patterns of well-defined linear and erythrocyte-shaped structure were synthesized with the assistance of Escherichia coli DNA.Morphology and the arrangement of BaF2particles on DNA were controllable by altering the reaction condition.Square nanoparticles arranged inlinearchains were gained with theassistanceof normal DNA;while,erythrocyte-shaped BaF2nanospheres were synthesized with the assistance of denatured DNA.Besides,the Influences of solvent,reaction temperature,concentration of reactants and the heating time on the morphology of the BaF2particles were studied.

1.Introduction

As an acknowledged ideal fast scintillator,Barium fluoride is one of the dielectric fluorides(CaF2,SrF2,and BaF2)tha thaveawiderange of potential applications in microelectronic and optoelectronic devices, such as wide-gap insulating overlayers, gate dielectrics, insulators and buffer layers in semiconductor-on-insulator structures, and more advanced three dimensional structure devices [1,2]. So far, hydrothermal method, chemical surface modification, precipitation method, and spark plasma sintering were mostly used to synthesize BaF2 [3–6].While, the disadvantages of methods mentioned above are obvious, for example, higher temperature and sophisticated devices are needed, et al. If a new facile synthesis method can be developed, it may be popular.

With the intercrossing and in filtrating of Biology and Nano-chemistry,morphology-controlled synthesis of inorganic nanostructure has drawn significant interests[7–12].With the advantages of simple installation,facile reaction condition and morphologies easy to control,it'sapotential development in the field of controllable synthesis of micro/nano materials.Besides,biological molecules are diverse,renewable and ecofriendly[13].Among these ideal biotemplates,Deoxyribonucleic acid(DNA)was one of the earliest used bio-templates[14–17]and it's also a potentially ideal template to dictate the precise positioning of molecules into any deliberately designed structure due to its remarkable molecular recognition properties and structural features[18–21].Kinds of noble metallic nanowires[22–25]and semiconductor nanowires[26,27]have been prepared using DNA as the bio-template,while BaF2nanostructure synthesized with the assistance of DNA was rarely reported[28].

In our study,we report a facile method for efficiently attaching BaF2nanocrystalswithwell-definedlinear and erythrocyte-shaped structure to DNA skeleton respectively.Morphologies and the arrange manner of BaF2particlesonDNA dependonthereaction condition.Possiblemechanism of different morphologies associated with the DNA's conformational changes is discussed brie fly in the end.

2.Experimental

2.1.Chemicals

All reagents used here were analytical reagent and used as received without further purification.Barium nitrate and ammonium fluoride were purchased from Tianjin Guangfu fine chemical industry research institution.Absolute ethanol,sodium dodecyl sulfate(SDS)and DMSO were purchased from Tianjin Kermel Chemical Reagent Co.,Ltd..Aqueous stock solution of the Escherichia coli(E.coli)B genomic DNA was freshly prepared.Doubly distilled water was used in this work.

2.2.Synthetic protocol

2.2.1.Extraction of E.coli B genomic DNA

To get the biotemplates,a stock solution of E.coli B genomic DNA was firstly prepared.E.coli B cells were cultured overnight in 50 ml of LB medium.Then,the cells were collected by centrifugation and resuspended in TE buffer[10 mmol·L?1Tris(pH 8.0),1 mmol·L?1EDTA(pH 8.0)].Then,10%SDS and 10 mg·ml?1proteinase K were added to adequately disassemble the cells in a 50°C water bath for 30 min.The mixture was extracted with equal volume phenol:chloroform:isoamyl alcohol(25:24:1)and centrifuged at 5000 r·min?1for 10 min.The aqueous supernatant was transferred to a new tube.The extraction process can be repeated if it's necessary.Then 2-fold volume absolute ethanol was added to the supernatant.Then it was placed under room temperature for 30 min and centrifuged at 12000 r·min?1for 10 min.The DNA precipitate was washed with 70%ethanol more than twice.Finally DNA was dissolved in deionized water.The nucleic acid and protein analysis was used to check the purity of DNA.

2.2.2.Preparation of BaF2nanocrystals with DNA assisted

In this work,we used DNA with high purity(OD≈1.8)as biotemplate to design and assemble a series of BaF2nanocrystals with different sizes and morphologies.The basic protocol for the synthesis of BaF2nanocrystal is described as follows: firstly,aqueous solution of Ba(NO3)2(50 μl,0.1 mol·L?1)was added to the solution of E.coli B genomic DNA(1.2 μg·μl?1)as prepared above and the solution was mixed thoroughly and incubated for 5 h at 6 °C;secondly,aqueous solution of NH4F(50 μl,0.2 mol·L?1)was dropped.The solution was mixed thoroughly again and incubated for another 3 h at 6°C;In the end,the mixture was heated and kept at 40°C for 2 h.All the samples as prepared were stored at 4°C for further study.

2.3.Characterization

The morphologies of the samples were observed by transmission electron microscope(TEM).A droplet(20 μl)of the samples was dropped onto a 300-mesh carbon-coated copper grid and then airdried be fore TEM observation.It was carried out ona JEM-2010electron microscope instrument operated at an accelerating voltage of 200 kV.The chemical composition and crystal structure were established by energy-dispersive X-ray spectroscopy(EDXS)and selected-area electron diffraction(SAED)respectively.

3.Results and Discussion

The E.coli B Genomic DNA is a circular double-stranded DNA(dsDNA)with 1.3 mm in length and it contains 4.6 Mb base pairs.DNA's remarkable molecular recognition properties and structural features make it one of the most promising templates to pattern materials with nanoscale precision[29].DNA strands can offer a variety of binding sites for metal ions such as Ba2+.

Fig.1 shows the typical features of DNA-assisted BaF2nanocrystals.Samples were synthesized with equal volume(50 μl)of 0.1 mol·L?1Ba(NO3)2and 0.2 mol·L?1NH4F solutions and incubated at 40 °C.The heating time were 0.5 h(S1,shown in Fig.1a),1 h(S2,shown in Fig.1c),2h(S3,shown in Fig.1d)respectively.All samples were directly characterized by TEM without any negative staining.As shown in Fig.1a,irregular ring structures with several microns in size were observed in the product of S1heated for0.5h,and Fig.1 bshows the higher magnified TEM image.With the extension of heating time,the ring structures of S2 heated for1 h becameapprox imate circles with a diameter of2.5μmaround(asshownin Fig.1c).However,thering structures blasted to linear chains gradually when it was heated for 2 h(as shown in Fig.1d).

The formation mechanism of the ring structures can be explained vividly by Fig.2.And similar ring structure was obtained in our previous report[30].In order to investigate which kind of bases played a major role on the formation of nanocrystal,Li used Oligo(dT),Oligo(dA),Oligo(dG)and Oligo(dC)as the template respectively.And it revealed that the phosphate and possibly the amino moiety binding site on adenine are the favorable targets to feed nanoparticle growth[31].

Fig.1.TEM images of DNA-assisted BaF2nanocrystals incubated at 40 C with different heating time respectively:(a)0.5 h,(c)2 h.(b)shows the higher magnified TEM image of S1.

Fig.2.The formation mechanism of the ring structures.

To investigate the metallization process further,we studied the effect of reagent concentration,taking the linear-chain structure for example.Samples of S4,S5and S6 were prepared with the concentration ratios of Ba(NO3)2/NH4F(0.1 mol·L-1:0.2 mol·L-1,0.2 mol·L-1:0.4 mol·L-1and 0.3 mol·L-1:0.6 mol·L-1,respectively).20 μl Ba(NO3)2and NH4F solution were added successively.The morphologies of the BaF2nanocrystals aggregated on DNA were revealed by TEM images,as shown in Fig.3.BaF2nanoparticles attached to the strand of DNA were arranged in linear structure discontinuously.The average particle size(equivalent diameter)of S4 in Fig.3a was 58 nm(20particles were used to cal culate the averagevalues and the standard deviation was 21 nm).The average particle size of S5 was 102 nm(20 particles were calculated and the standard deviation was 11 nm),and the dispersion of the particles were uneven obviously,compared to S6(equivalent diameter was about 400 nm).It demonstrated that the size of BaF2nanoparticles could be tuned by altering the amount of Ba(II).

Fig.3.(a–c)TEM images of BaF2nanocrystals aggregated on DNA linear chains with different concentration ratios of Ba(NO3)2/NH4F.(a)S4 0.1 mol·L-1:0.2 mol·L-1,(b)S5 0.2 mol·L-1:0.4 mol·L-1and(c)S6 0.3 mol·L-1:0.6 mol·L-1,respectively.(d)SAED pattern taken on the BaF2nanoparticle linear chains shown in(a).(e)EDXS of the DNA-assisted BaF2nanoparticles.

The concentrationof DNAsolutionwas closedforthesamples,while excess of Ba(II)made the square nanoparticles bigger and continuously bind to the strand of DNA(shown in Fig.3c).Fig.3d shows the selected areaelectrondiffraction(SAED)patternrecordedfromBaF2nanocrystal of S4.Throughcalculating,we foundthat diffractionringscorresponded to(111),(220),(311),(422)and(440)planes of cube structure of BaF2respectively.It proved that BaF2was well crystallized.Fig.3e shows the patterns of EDXS.Peaks for the elements of Ba,F,O,C and Cu were observed.Cu and C peaks were due to the carbon-coated copper grid and O peak may arise from DNA.It's con firmed by SEAD and EDXS that we successfully synthesized cubic BaF2nanocrystals with the assistance of DNA.

We also studied the solvent Influence on the morphologies and size of BaF2nanoparticles aggregated on DNA.Sample S7 was prepared using the mixture of water and ethanol(1:2)as solvent and reacted under the same conditions with S4.Then samples were directly detected under TEM and the images gained were shown in Fig.4.

Insets of Fig.4(a)and(b)are highermagnification images of sample S4 and S7.By contrasting,we found square nanoparticles in S4 and nano spheres in S7(with 74 nm in diameter,20 particles were used to calculate the average values and the standard deviation was 23 nm).We propose that:when using pure water as solvent,there is maybe an obvious preferential growth phenomenon along some crystal faces,so crystals grow faster in these directions,and slower in the others,thus making it easier to form square nanoparticles.Meanwhile,with the adding of ethanol,the preferential growth of the particles is gradually weakened,eventually making the growth speeds along different crystal faces closed,thus making it easier to form nanospheres.

DsDNA molecule is stabilized by base stacking and the interaction can be disturbed relatively easily by thermal fluctuations[32].DNA has various conformational transitions via its double-helix twisting or folding.Double strands even can be separated into single lines when it's heated to 80°C(denaturation temperature),creating nano pair-linear arrays.Herein,we also investigated a simple method for the preparation of different morphologies of BaF2nanocrystals using denatured DNA(heated to 90°C),as shown in Fig.5.S8 was synthesized in the mixed solvent of ethanol and water(2:1).It proves that DNA's conformational change has a dramatic effect on the final products,leading to the formation of erythrocyte-shaped BaF2na no spheres.It was at otally different and novel morphology,unlike the BaWO4nano pair-linear arrays reported by Li et al.

Fig.4.Effect of solvents onproduct's morphologies,(a)S4:wateras the solvent;(b)S7:mixture of water and ethanol(1:2)as the solvent.Insetof(a)show the arresting differenceof BaF2 nanocrystals taken on a higher magnification.

Fig.5a shows the monodisperse,size-uniformed and erythrocyteshaped BaF2nanospheres.And Fig.5b shows orthograph of the erythrocyte-shaped nanospheres observed under higher magnification.These erythrocyte-shaped nanospheres were about 400 nm in outer diameter with a hollow center.Moreover,the patterns of EDXS shown in Fig.5c also indicated that we synthesized erythrocyte-shaped BaF2nanocrystals assisted by DNA successfully.

The mechanism of DNA assisted synthesis of BaF2is showed in Fig.6 as follows.When Ba2+was added to DNA solution,it attached to DNA skeleton by electrostatic force and combined with phosphate groups on DNA.F?reacted with DNA-Ba2+compound and crystal nucleus of BaF2was formed,then square or spherical particles were fabricated along DNA chain with different size.The mechanism of the formation of the erythrocyte-shaped nanospheres was not so clear and more study will be done in our further work.

4.Conclusions

Fig.5.TEM images of erythrocyte-shaped BaF2nanospheres prepared with denatured DNA treated at 90 C.(b)A higher magnified TEM image of S8.(c)SAED pattern taken on the BaF2 nanospheres shown in(a).(d)EDXS of the DNA-assisted BaF2nanospheres.

Fig.6.Schematic diagram of DNA-assistant synthesis of BaF2.

Assisted by normal E.coli B circular dsDNA,we present an effective and efficient method for preparation of BaF2square nanocrystals arranged in irregular rings,circles to linear chains.These morphology differences areaffected byconformational changes of the dsDNA superhelix,which can be disturbed relatively easily by thermal fluctuation.Thus,we can easily get various nano-patterns by regulating the heating time and DNA's conformation.An interesting result shows that mixed solvent of ethanol and water can obviously make the BaF2square nanocrystals become round.Based on this,we rationally designed one more kind of amazing nano-patterns,erythrocyte-shaped BaF2nanospheres,by using the denatured DNA.

Different morphologies of BaF2with the assistant of DNA templates were synthesized in our study,and even the linear-arrangement nanocrystals and erythrocyte-shaped nanocrystals of BaF2were synthesized for the first time.We expect that erythrocyte-shaped BaF2crystals,asanewmorphology,mayhavethepotentialapplicationinthefuture.So erythrocyte-shaped nanocrystals with uniform size,morphologies,good optical and mechanical properties is being studied in our present work.It'ssignificanttothesynthesisofBaF2withidealapplicationperformance.