PAPER www.rsc.org/faraday_d | Faraday Discussions

Nanostructured wrinkled surfaces for templatingbionanoparticles—controlling and quantifyingthe degree of order

Anne Horn,a Heiko G. Schoberth,a Stephanie Hiltl,a Arnaud Chiche,b

Qian Wang,c Alexandra Schweikart,a Andreas Fery*a

and Alexander B€oker*ad

Received 9th February 2009, Accepted 16th March 2009

First published as an Advance Article on the web 22nd July 2009

DOI: 10.1039/b902721a

We present a novel method to align the tobacco mosaic virus (TMV) on

topographically structured surfaces. In order to gain defined patterns we use

wrinkled polydimethlysiloxane (PDMS) sheets as templates. We aligned the virus

with a simple spin-coating procedure on the PDMS sheet. The concentration of

the virus solution and the spin speed are varied in order to identify ideal

conditions for the arrangement of the viruses on the wrinkled templates. Here, we

establish a simple analytical approach which allows quantifying the degree of

order of the patterns, which is the basis for a quantitative discussion of

templating efficiency. Furthermore, we discuss the role of dewetting processes for

the particle assembly. TMVs can be used as reactive nanoparticles due to their

well-defined surface chemistry. They can as well serve as a model system for

alignment of anisotropic particles via spin coating from solution.

1 Introduction

The arrangement of colloidal particles is of common interest in technology and scienceas well. Potential applications in the field of data storage devices1 and nano-elec-tronics2 were investigated by several groups. Therefore, the use of topographicallystructured substrates to align colloids gained increasing attention in the recent years.3

Until now lithographic methods were the common techniques of obtaining thosestructures.4,5 An alternative approach to generate patterned surfaces is controlledwrinkling or buckling of an elastomeric substrate with a hard top layer. Templatedself-assembly of synthetic colloids like polystyrene, silica or gold particles has beenexplored recently.3,6–8 However, anisotropic particles open a wider range of potentialapplications due to their unique electrical and optical properties. Rod-shaped biomol-ecules combine anisotropy and monodispersity along with chemical addressabilityand can serve as efficient templates for various reactions e.g. metallization or miner-alization.9–12 Hence, arrays of well-defined tobacco mosaic virus (TMV) on wrinkledsubstrates is an excellent system to study the self-assembly of anisotropic colloids on

aLehrstuhl f€ur Physikalische Chemie II, Universit€at Bayreuth, D-95440 Bayreuth, Germany.E-mail: Andreas.Fery@uni-bayreuth.debDSM-Material Science Centre, P.O. Box 18, 6160, MDGeleen, The NetherlandscDepartment of Chemistry and Biochemistry and Nanocenter, University of South Carolina,Columbia, South Carolina, 29208, USAdLehrstuhl f€ur Makromolekulare Materialien und Oberfl€achen and DWI an der RWTH Aachene.V., RWTH Aachen University, D-52056 Aachen, Germany. E-mail: Boeker@dwi.rwth-aachen.de

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structured surfaces. In particular, we paid special attention to developing quantitativemeasures for the particle order, which allows optimizing assembly parameters.

In the work reported here, we developed a method yielding excellent control overTMV alignment on a large scale using wrinkled polydimethylsiloxane (PDMS)substrates. Via a simple spin-coating technique we are able to fill the grooves ofthe wrinkles with the virus and achieve patterns with high aspect ratios. The advan-tages of this approach are that it is not necessary to functionalize either the virus orthe surface chemically and that there is no need of external physical forces other thanthose present during the spin-coating process in order to direct the self-assemblyprocess. Additionally the PDMS substrates are produced in an easy, cheap andlithography-free way. Hence, we found a simple procedure to obtain highly alignedTMV strings with dimensions on the order of several microns in length but onlya few nanometres in width on a cm2 substrate.

2 Experimental

2.1 Production of wrinkled PDMS substrates

We prepared the PDMS by mixing Sylgard 184 Base (Dow Corning) and Sylgard184 Curing Agent (Dow Corning) in a mass ratio of 10 : 1. The mixture was cast(3 mm in height) in a petri dish and heated to 80 �C for 24 h. Pieces of 6 mm widthand 30 mm length were positioned in a custom-made stretching apparatus13 andextended to 130% of the original length. These substrates were oxidized between40 s and 120 s in an air plasma (1 mbar, 18 W, PDC-32 G, Harrick) in the stretchedstate. After relaxing the wrinkled samples could be used for further application.The application of wrinkles for templating has recently been reviewed.14

2.2 Alignment of TMV on wrinkled substrates

A TMV stock solution (10 mg ml�1, 0.1 M potassium phosphate buffer, pH 7.8) wasdiluted with ultrapure water to the desired final concentration (between 0.2 mg ml�1

and 1.2 mg ml�1). A droplet of 50 ml was spin-cast onto the wrinkled substrates(between 2000 rpm and 4000 rpm). After drying under N2-flow the samples werecharacterized with scanning electron microscopy (SEM) and scanning force micros-copy (SFM).

2.3 Characterization

SEM images were recorded by using FE-SEM (Zeiss 1530) operating at a voltage of0.75 kV and with a working distance between 4 mm and 5 mm. To improve themeasurements samples were sputtered with carbon (approximately 6 nm). SFMimages were performed with a commercial SFM (Dimension 3100, Vecco Instru-ments Inc.) in Tapping Mode�. Si3N4 cantilevers were purchased from Olympus(spring constant �40 N m�1). Transmission electron microscopy (TEM) was per-formed on a Zeiss CEM902 microscope operated at 80 kV.

3 Results and discussion

The preparation of the wrinkled substrates was done using a previously reportedmethod.15–17 In Fig. 1a, a scanning force microscope (SFM) image is shown repre-senting the topography of the obtained structures where the height profile illustratesthe sinusoidal shape of the wrinkles. In order to verify this structure an epoxy resinreplica of the wrinkled surface was made and imaged with transmission electronmicroscopy (TEM). The TEM cross section of the epoxy replica is shown inFig. 1b. The desired wavelength l can be tuned easily by the plasma exposuredose (in the range between 130 nm and 1 mm, see ref. 18) as it is dependent on thethickness of the hard oxide layer generated by the plasma. The amplitude A is

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Fig. 1 (a) SFM height image (z-range¼ 0–60 nm) of wrinkled PDMS substrate (l¼ 324� 22 nm;A¼ 58� 2 nm). The white line represents the position of the height profile shown on the right side.(b) TEM image showing a cross section of an epoxy replica of wrinkled PDMS (l¼ 232� 24 nm;A ¼ 59� 8 nm).

dependent on the applied strain and the present wavelength but the aspect ratio islimited to 0.1 for the conditions used here. Therefore it is possible to generate definedsubstrates with variable dimensions.

The aim of our study is to find optimum conditions to arrange the TMV in thegrooves of the wrinkles. The rod-shaped virus is 300 nm long and 18 nm in diameter.To ensure comparable results we always used wrinkled templates with a wavelengthl of 300 � 15 nm and an amplitude A of 30 � 3 nm. Fig. 2 shows SFM images ofaligned TMV on the wrinkled substrates. The viruses are mainly found in thegrooves of the substrate. Previous studies have shown that, due to discontinuousdewetting, TMV particles selectively place at the base of relief structures.10 Thedistances between the single virus strings are predetermined by the wavelength ofthe wrinkles. Therefore, the spacing of the strings can be controlled by adjustingthe wavelength of the substrate. Furthermore it is possible to generate structuraldefects on the wrinkled supports. By regulating the release rate of the stretchedPDMS the defect density can be controlled15 and more complex geometries can beobtained.19,20 Indeed, TMV is flexible enough to bend which means that it can beplaced in e.g. a Y-junction (see Fig. 2b). As shown in Fig. 2c we can distinguishbetween one-fold and multiple adsorption by an SFM height profile. The heightitself is the same in both cases but the shape of the topography is different. In thecase with only one virus string lying in the groove the peak is much sharper thanfor multiple adsorption.

In the following, we focused on exploring the parameter range of the alignmentconditions. Therefore we used SEM as our measuring technique, which allowsone to examine larger regions to obtain statistically relevant information. We variedthe concentration of the TMV solution (0.2 mg ml�1, 0.4 mg ml�1, 0.9 mg ml�1,1.2 mg ml�1) while keeping the spin speed, the wavelength of the substrate and theamplitude constant at 3000 rpm, 300 nm and 30 nm, respectively. For every samplefour images (2 � SFM, 2 � SEM) at different sample spots (a representative selec-tion of SEM images is shown in Fig. 3a) were evaluated to calculate the virus occu-pancy parameter U (Here, we note that the following parameters Vk, Wk andOk were extracted from the images by manually measuring the respective lengths):

U ¼ 1

Atot

X4

k¼1

Ak

Vk

Wk

(1)

where Ak is the evaluated area of the image k, Atot¼P

4k ¼ 1Ak is the total area of

all images (Atot $ 770 mm2), Vk represents the total length of virus-filled grooves of

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Fig. 2 (a) SFM phase images (z-range¼ 0–40�) of aligned TMV on wrinkled PDMS substrateshowing multiple adsorption of the virus in the grooves (l ¼ 294 � 21 nm; A ¼ 32 � 1 nm).(b) SFM phase image (z-range ¼ 0–100�) of aligned TMV on a wrinkled PDMS substrateshowing a Y-junction (l ¼ 286 � 20 nm; A ¼ 29 � 1 nm). (c) SFM height image (z-range ¼0–50 nm) with corresponding height profile showing one-fold (solid arrow) and multipleadsorption (dashed arrow) of the virus in the grooves (l ¼ 299 � 18 nm; A ¼ 27 � 1 nm).

the image k and Wk is the total length of the grooves of the image k. The virus occu-

pancy parameter characterizes how many viruses are in the grooves of the wrinkles

ranging from 0 / 1 (where 1 represents 100% occupied grooves) but it does not take

into account the viruses found outside the grooves. Therefore another parameter is

necessary, namely the virus deviator parameter F:

F ¼ 1

Atot

X4

k¼1

Ak

Ok

fk Vk þOk

(2)

where Ok is the total length of viruses outside the grooves of image k and fk is the

multiple adsorption factor of image k. We extracted fk by evaluating the averaged

number of adsorption (one-fold, two-fold.) of the viruses from SFM images

with higher magnification (e.g. 2 mm � 2 mm). The results of varying the concentra-

tion of the virus solution by keeping all other parameters constant are shown in

Fig. 3b. The virus occupancy parameter increases with higher concentration. It rea-

ches almost full groove occupancy (U ¼ 1) at 1.2 mg ml�1 which means that all the

wrinkles are filled with the virus. As the virus deviator parameter is a relative value

which compares how many viruses are out of the grooves to the total number of

viruses on the sample it is obvious that the error bar at 0.2 mg ml�1 is very high.

The number of viruses in the grooves at this concentration is rather low but

there are always some viruses which stick directly to the surface. Relative to the

total number of viruses the number of viruses outside the grooves decreases with

increasing concentration. Hence, the virus deviator parameter decreases with

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Fig. 3 (a) SEM images of TMVs aligned on wrinkled PDMS substrates prepared from virussolutions of different concentrations. (b) Plot of virus occupancy parameter (U) and virus devi-ator parameter (F) versus concentration (dashed line is a guideline for the eyes).

increasing concentration till reaching its minimum at 0.9 mg ml�1. When the virus

concentration reaches a certain value where nearly all the wrinkles are filled, the

virus deviator parameter increases again. Obviously, in this case, the viruses have

no space in the grooves any more and adsorb outside. Therefore, we find an

optimum concentration of 0.9 mg ml�1 where the virus deviator has its minimum

while the virus occupancy parameter has a high value. Nearly 90% of the wrinkles

are filled with TMV and only a few adsorb unaligned on the surface.

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Another important parameter which determines the TMV alignment is thespin speed. Therefore, we varied the spin speed (2000 rpm, 3000 rpm, 3500 rpm,4000 rpm) by keeping the concentration constant at 0.9 mg ml�1, the wavelengthof the substrate at 300 nm and the amplitude at 30 nm. We evaluated the data inthe same way as for the concentration dependency. Fig. 4a shows the dependenceof the virus occupancy (U) and virus deviator (F) parameters on the spin speed.At a spin speed of 2000 rpm the value of U is around 0.5. By increasing the spinspeed to 3000 rpm the value increases to approximately 0.9 which means that90% of the wrinkles are filled with TMV. Further raising the spin speed leads toa decrease of the occupancy parameter again. This behaviour may be explainedby the thickness of the water film. When starting from a continuous film it startsto dewet at certain areas of the substrate. At low spin speeds (e.g. 2000 rpm) thewater film is rather thick and randomly formed droplets are generated. This leadsto a discontinuous distribution of TMV on the substrate. There are areas with novirus (holes of the water film) and areas with a rather high concentration of virus(droplets). When the water is fully evaporated we have, on the one hand, regimeswhere no virus is found at all, and on the other hand regimes where the virus

Fig. 4 (a) Plot of the virus occupancy parameter (U) and virus deviator parameter (F) versusspin speed (dashed line is a guideline for the eyes). (b) SEM image of TMVs aligned on wrinkledPDMS (l ¼ 324 � 22 nm; A ¼ 58 � 2 nm) with optimum conditions (spin speed ¼ 3000 rpm,c ¼ 0.9 mg ml�1).

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concentration is so high that TMV not only fills the grooves but is also a part of thesurface. This leads to a virus deviator parameter of around 0.2. The maximum of thevirus occupancy parameter is at 3000 rpm and was found to be the optimum spinspeed for the alignment of TMV. Here, the water film is thinner than in the casedescribed before. Hence, it starts dewetting on the top of the wrinkles which meansthat the virus concentration is locally raised in the grooves and lowered to nearlyzero on the top. Therefore, TMV is forced to align in the grooves and only a fewviruses are found outside. For spin speeds faster than 3000 rpm the water film istoo thin to result in a sufficient solvent evaporation time allowing for controlledfilling of the grooves. The film thickness may be thinner than the amplitude ofthe wrinkles. Consequently the top of the wrinkles does not lead to a controlled‘‘breaking’’ of the water film any more. Thus holes occur everywhere in the waterfilm. As a consequence, with increasing spin speed the viruses directly stick to thesurface widely spread over the sample. This effect can be seen in the graph inFig. 4a where the virus occupancy parameter decreases from 3000 rpm to 4000 rpmwhile the virus deviator parameter—which is a relative value—first increases signif-icantly and then remains constant within the error range. The SEM image in Fig. 4bshows a sample prepared at optimum conditions (spin speed¼ 3000 rpm, c(TMV)¼0.9 mg ml�1). The TMVs are ordered parallel to each other with a regular distancedetermined by the wavelength of the substrate. Some of the viruses are disorderedwhich is an effect of the above-mentioned phenomenon.

In summary, we present an effective technique for aligning anisotropic particlesover large areas on predefined substrates. Using wrinkled PDMS substrates to directthe self-assembly process we create highly uniform TMV arrays. The control ofalignment is achieved using a simple spin-coating technique. An important aspect,and not only for this particular case of particle templating, is the development ofquantitative measures to reveal the order of the templated particles or templatingefficiency. For this purpose, we introduce simple parameters and use them fora quantitative discussion of the influence of process conditions. We varied theconcentration of the virus solution and the spin speed to determine the optimumconditions for the virus assembly. We found a concentration limit above which addi-tional TMVs are increasingly deposited outside rather than in the grooves. Ourresults identify the spin speed as a critical parameter for structure formation. Thiscan be explained by a dewetting-assisted structure-formation mechanism. Whilethe particular concentration and speed range is characteristic for the particularcombination of solvent, particle geometry and wetting properties of particle andsubstrates, we believe that this mechanism can be of interest for alignment of parti-cles from solution and further theoretical work for a quantitative modelling of theeffect is currently pursued. Until now, several groups used the chemical address-ability of viruses to build silica-coated nanoparticles,21 metal cages22,23 or metalwires.24,25 With these modified viruses, and by using the approach presented inthis paper, it becomes feasible to develop metal wires where the distances are prede-fined by the wavelength of the wrinkled substrate. Furthermore, induced structuraldefects of the substrate open the possibility of producing branched virus assembliesand therefore branched metal wires. To conclude, we generated well-defined TMVarrays which may serve as metallisation or mineralisation templates for future appli-cations and as a model system for the alignment of anisotropic nanoparticles.

Acknowledgements

The authors thank Stephan Herminghaus and Markus Hund for helpful discussions,and Benjamin Goßler and Carmen Kunert for help with the SEM and TEMpictures. This work is supported by the Lichtenberg-Program of the Volkswagen-Stiftung and the Sonderforschungsbereich 481 funded by the German ScienceFoundation (DFG). QW is partially supported by US NSF, US DoD and theW. M. Keck Foundation.

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