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Microelectronic Engineering 85 (2008) 1143–1146 www.elsevier.com/locate/mee
Chemical engineering of silicon oxide surfaces using micro-contact printing for localizing adsorption events of nanoparticles, dendrimers and bacteria Jean-Christophe Cau *, Aline Cerf, Christophe Thibault, Mike Genevie`ve, Childe´rick Se´verac, Jean-Pierre Peyrade, Christophe Vieu LAAS-CNRS, Toulouse University, 7 Avenue du Colonel Roche, Toulouse 31077 Cedex 4, France Received 27 September 2007; received in revised form 21 January 2008; accepted 27 January 2008 Available online 10 February 2008
Abstract A simple surface chemical bi-functionalization procedure, involving micro-contact printing of silane molecules is presented. The printing process has been optimized for obtaining high quality layers of good homogeneity. The produced chemical templates are used in order to spatially localize the adsorption of different entities diluted in liquid solution. By combining optical and Atomic Force microscopy we confirm the selective adsorption of gold nanoparticles, dendrimers and bacteria. ! 2008 Elsevier B.V. All rights reserved. Keywords: Biochip; Surface chemistry; Dendrimers; Chemical patterning; Soft-lithography
1. Introduction Micro-contact printing (lCP) is an easy and low-cost patterning technology. Here, it is used to engineer the surface of biochips with chemical patterns capable of tuning the adsorption of different entities in solution. Chemical patterns are usually composed of molecules which interact with the targeted nano-objects. In order to increase the selectivity of the adsorption, a complementary functionalization of the surface is often necessary in order to repulse the nano-objects from the zones in-between the attractive areas. Some previous works showed the efficiency of this kind of chemical patterns, coupled with combing techniques, for localizing adsorption events [1–5] but pointed out the poor definition of the octadecyltrichlorosilane (OTS) patterns obtained by lCP [6]. Here the optimization of the printing process of OTS molecules is presented with the development and the use *
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[email protected] (J.-C. Cau).
0167-9317/$ - see front matter ! 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mee.2008.01.103
of a bi-functionalization to spatially localize the selective adsorption onto molecular micrometric patterns.
2. Experimental procedure The first step consists in generating a silicon master. It is achieved by UV photolithography and pattern transfer by deep Reactive Ion Etching (dRIE). The targeted etch depth is fixed to 5 lm. To enable simple demolding of this master, an anti-adhesive treatment is carried out by a well established process using OTS (octadecyltrichlorosilane) coating in liquid phase. The final step consists in curing the PDMS pre-polymer solution containing a mixture (10:1 mass ratio) of PDMS oligomers and a reticular agent from Sylgard 184 Kit (Dow Corning) on the silicon master at a temperature of 60 "C during hours. The silicon mold contains microscale patterns of different geometry such as 10 lm size ovals, 5 lm and 10 lm disks, and 20 lm size ‘W’, which corresponds to trenches in the master. After printing, the whole
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surface is coated with the OTS molecules while the micrometric patterns remain free of deposition. The silicon oxide substrates (menzel-glaser cover glass 24 ! 32 mm) are cleaned with trichloroethylene, acetone, ethanol, deionized water. Then, oxygen plasma treatment was used in order to activate the surfaces (800 W, 5 min, and 1000 mL/min). Initially, OTS solution is diluted to 0.4% (v/v) with trichloroethylene. A volume of 100 lL OTS solution is deposited on the stamp during 30 s. The drop is removed thanks to the spin coater (speed 3000 rpm, acceleration: 5000 rpm/ s, time 1 min). The stamp is dried with nitrogen flow during 1 min to evaporate trichloroethylene and then manually printed onto silica substrates for 3 min. The first bi-functionalization is realized with a amino terminated silane: 3-aminopropyltriethoxysilane (APTES) (Sigma-Aldrich) For that, the OTS-stamped substrates are treated with a 1% (v/v) solution of APTES in 99% ethanol for 15 min at room temperature (bi-functionalization named OTS/APTES). The second bi-functionalization was realized with a epoxide terminated silane: 3-glycidoxypropyltrimethoxysilane or GPTS (Sigma-Aldrich). For this purpose, the OTS-stamped substrates are treated with a 2.5% (v/v) solution of GPTS in 97.5% ethanol for 1 h at room temperature (bi-functionalization named OTS/GPTS). Au nanoparticles (40 nm) electrostatic adsorption is realized by incubation in 9 " 1010 nanoparticles/mL (BBInternational, negatively charged) during 1 h and rinsed with deionized water. Dendrimers chemisorptions are realized by incubating the solution onto chemical templates. For aldehyde terminated dendrimers [7] the incubation time is 24 h in 54 lM solution and the OTS/APTES template is used. While for G4-PAMAM dendrimers (Sigma-Aldrich), the incubation time is reduced to 1 h in 60 lM solution and the OTS/ GPTS template is used. After incubation, the different samples are dried with nitrogen flow. Bacteria adsorption (Escherichia coli) is realized by incubating OTS/GPTS templates during 20 min with the bacteria solution. The sample is then rinsed with PBS (phosphate buffer saline) solution. 3. Results and discussion 3.1. Control of OTS patterning Micro-patterning of OTS through micro-contact printing was demonstrated in previous works [6] but some defects were reported. Indeed, some isolated ‘islands’ of OTS on the non-stamped areas were observed. This detrimental effect was attributed to the diffusion of OTS molecules on the surface mediated by the solvent which penetrates the stamp. These islands are groups of OTS molecules that have polymerized. In preliminary experiments (data not shown) we have observed the same phenomenon. The drying step presented in Fig. 1 was then introduced in
Fig. 1. Schematic procedure of OTS micro contact-printing.
our process to prevent the penetration of a large amount of solvent molecules inside the elastomer. This drying step turned out to be crucial for obtaining high quality prints because it improves the quality of the OTS layer and the definition of the patterns. A very uniform OTS layer of 2.5 nm is observed which corresponds to a monolayer. The roughness is close to 3 nm which indicates that the OTS monolayer is covered by islands of a second monolayer. Voids in the OTS deposit are not observed. This result is crucial for the success of a bi-functionalization of the surface. 3.2. Electrostatic adsorption of Au nanoparticles The functionality of our bi-functionalization protocol is proved thanks to electrostatic bindings between APTES molecules and negatively charged Au nanoparticles. As can be seen in the AFM image of Fig. 2A, the nanoparticles are found principally in the central region of the attractive patterns. We think that this effect is related to the spatial distribution of the electrostatic potential created by the positive surface density of charges of the APTES zones. 3.3. Chemisorption of dendrimers presenting aldehyde-group on chemical templates The covalent binding between dendrimers (aldehydegroup) and APTES relies on the strong interaction between aldehyde and amino-group. Dendrimers are selectively deposited on the APTES regions. AFM imaging revealed that the dendrimers agglomerated inside the APTES patterns (Fig. 2B but formed thick 3D islands with typical heights of 100 nm (±40 nm). No trace of self-organization of dendrimers could be observed due to the strong interaction between the dendri-
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Fig. 2. AFM topographical image (tapping mode) (A) of electrostatic adsorption of 40 nm Au nanoparticles (negatively charged) (dashed line represents the border between OTS and APTES)(B) with a cross section of dendrimers (aldehyde-group) on OTS/APTES chemical templates (the APTES region corresponds to the dark area), (C) with two cross sections of PAMAM dendrimers on OTS/GPTS chemical templates (the GPTS zones correspond to the dark areas), (D) of bacteria adhesion on OTS/GPTS chemical templates, (E) a zoom on a single pattern showing the adsorption of a single bacterium.
mers and the APTES molecules, preventing their rearrangement on the surface, associated with the strong inter molecular interactions between these hydrophobic dendrimers. 3.4. Chemisorption of dendrimers presenting amino-group on chemical templates The selective deposition of amino-terminated dendrimers (G4 PAMAM) onto GPTS/OTS chemical templates was also investigated. We observe a good selectivity of the adsorption with thick deposit onto the GPTS zones and an absence of adsorption of the dendrimers on the OTS zones. The morphology of the deposit as seen by AFM (Fig. 2C indicates the presence of 3D thick islands (thickness included between 150 nm and 250 nm). 3.5. Selective adsorption of bacteria on OTS/GPTS chemical templates The bi-functionalization OTS/GPTS enables to make an interface between life related entities (cells, bacteria, virus, proteins, DNA, . . .) and inorganic parts (silica). For that, the selective adsorption of bacteria (E. coli) on OTS/GPTS chemical patterns is investigated. Bacteria are incubated on OTS/GPTS chemical templates. There is a selective bacteria adsorption on epoxyde zone (composed by GPTS). We think that epoxide surface groups react with the amino-group found in the membrane
proteins of the bacteria resulting in the selective adsorption on the GPTS zones. This is confirmed by the AFM images of Fig. 2D and E where it is shown that the fixation of individual bacteria occur on the GPTS micrometric patterns. Such assembly of individual bacteria can be controlled over surfaces of 1 mm2. In such patterns, 2500 sites of selective adsorption are created (20 lm pitches). A systematic characterization has shown that all of them are equipped with single bacteria. 4. Conclusion OTS micro-contact printing process on glass surfaces has been optimized by introducing a drying step after stamp inking. This major improvement increases drastically the edge definition of the patterns due to a limited surface diffusion of the OTS molecules. A simple and low-cost way to create two kinds of bi-functionalization OTS/ APTES and OTS/GPTS was developed. It was applied to the adsorption (electrostatic or covalent) of nano-objects (Au nanoparticles, dendrimers). Adsorption mechanisms are complex, depending on the nature of the interactions between the nano-objects inside the colloidal solution and the surface as well as on the interaction between the nano-objects themselves. In the case of Au nanoparticles, selective adsorption has been obtained without self-assembly due to the strong interacting forces with the surface. For dendrimers, the intermolecular interactions impose a 3D growth mode with a very high selectivity. Finally, it was shown that these chemical templates printed by
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lCP can be used for the selective fixation of individual bacteria. Acknowledgments This work was supported by the EC-funded project NaPa (Contract No. NMP4-CT-2003-500120). The authors would like to thank Emmanuelle Tre´visiol (Biochips Platform Genopole Toulouse UMR-CNRS 5504 & INRA Toulouse, France) for providing the dendrimers solution.
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