MECHANISM OF DNA COMBING THROUGH RECEDING MENISCUS ASSEMBLY ON MICROSTRUCTURED SUBSTRATE

B. Charlot1*, F. Bardin1,2, N. Sanchez3, P. Roux3, S. Teixeira3 and E.Schwob4 1IES CNRS, Institut d’Electronique du Sud, Université Montpellier 2, France

2Université de Nîmes, France 3SANOFI, exploratory unit Montpellier, France

4IGMM CNRS, Institut de Génétique Moléculaire de Montpellier, France

ABSTRACT This work concerns the deposition and combing of DNA filaments by forced dewetting. This technique involves

scanning a droplet of DNA solution held between a movable blade and a PDMS substrate containing an array of microwells. The deposition and elongation appears when the receding meniscus dewets the microwells, the latter acting here as a perturbation in the dewetting line. This technique, based solely on a physical phenomenon, can deposit molecules of DNA in an orderly manner and most of all in an elongated conformation, which allows observing uncoiled DNA molecule in all its length. However, the exact mechanism that governs the deposition of DNA strands are not well understood. This paper is an analysis of the physical phenomenon occurring in the deposition process and is based on observations made with the use of high frame/second rate video microscopy. KEYWORDS: DNA, combing, dewetting, pdms, capillary deposition, meniscus

INTRODUCTION

Molecular combing [1][2] is a method used to stretch and deposit DNA molecule on substrates in order to visualize them in an elongated conformation. This technique allows observing events occurring along DNA fragment such as replication sites with ad hoc fluorescent markers. The standard technique for DNA combing uses the dewetting of a silanized glass from an aqueous solution of DNA. The solution is treated to lower the pH at a value of 5.4. This allows DNA strands ends to open and induces a negative electric charge density located at the ends of the molecule. The electrostatic attraction between DNA ends and positively charged silanized glass surface will generate an anchoring of DNA molecule on the substrate. Thus, the dewetting of the substrate will uncoil molecules anchored by their end. The drawback of this method is the randomness of the localization of molecule deposition and some other effects such as U-shaped deposition that occurs sometimes when both ends are anchored at different locations.

More recently, a new method for DNA[3][4][5] or chromatin[6] combing has appeared using a combination of liquid dewetting and micro structured substrates. This technique produce ordered arrays of elongated DNA strands, however the physics behind the deposition process is not yet clearly understood. Liquid dewetting is a technique that found several applications in nanotechnology[7]

THEORY

The capillary deposition technique uses the forced dewetting of a partially wetting liquid on a micro structured sub-strate. Microstructures play the role of perturbation in the dewetting process leading to the uncoiling and deposition of DNA pellets. A droplet of DNA solution is drained on the surface of the substrate by a glass blade. The motion of the droplet is achieved by moving the blade with a piezoelectric linear motor. The droplet is spread at the surface of a PDMS bloc containing an array of micro wells: 5µm diameter, 5µm deep, and regularly spaced with a pitch ranging from 20 to 200µm.

Figure 1: Side view schematic and microphotograph of the principle of the capillary assembly of DNA strands on a

microstructured PDMS substrate. The presence of wells will perturb the dewetting line and create a pinching at the edge of the well separating the liq-

uid in two bodies, one part left in the well, the other in the droplet. The presence of a DNA molecule at this exact place will lead to its elongation by retention forces and consequently the deposition of the molecule on the surface of PDMS. We show that, for a given concentration (10ng/ml), static contact angle (35°) and temperature (25°C) the deposition starts

978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 823 17th International Conference on MiniaturizedSystems for Chemistry and Life Sciences27-31 October 2013, Freiburg, Germany

to be efficient for a given draining speed (200µm.s-1 in our case). In this case, DNA strands are elongated and deposited at the surface of the PDMS, perpendicular to the dewetting line, as seen by fluorescence imaging. The DNA are 48Kbase long λ phage DNA tagged with YOYO and can be observed in Figure 3.

When a liquid is forced to dewet a moving solid, several phenomenon occurs depending on wetting conditions. In the case of a hydrophilic solid, when the liquid completely wets the solid, the moving solid drains the liquid with a uniform liquid film. This has been modeled by Landau and Levich[8][9]. The liquid coats the solid with a thickness that is given by the model :

�

h = 0,94λCa2 / 3 = 1,1µm with

�

Ca = ηU /γ = 1,3.10−5 . Ca being the capillary number and

�

λ =γρg

= 1,7mm , the

capillary length, η the viscosity, U the draining speed (400µm.s-1) and γ the surface tension (30.10-3N.m-1). This liquid film thickness of 1,1µm is a rough estimation since the model accounts for fully wetting liquids, that is not the case here. It is however in the order of magnitude of what has been observed.

When the droplet, caught between the driving blade and the substrate is moved at a speed U, as depicted in figure 1(A), a dynamic equilibrium settles, resulting from the competition between different forces. The contact surface between the liquid and the air will be deformed and a meniscus will appear. The surface force induced by the increase of surface in the meniscus drives the liquid tail. The viscous stress within the narrowing wedge of liquid will grow and produce a viscous force that opposes to the draining. Finally a frictional force arises at the location of the triple line and also tends to opposes the dewetting of liquid adsorbed on the substrate. As a results a thin liquid film will appear at the end of the meniscus with close to zero apparent angle but a microscopic contact angle is present at the dewetting line location, the exact value of this angle is still debated [8]. When the draining speed increases again, a transition appear from a steady dewetting line to a dispersive regime with apparition of rivulets and droplets, and a serrated dewetting line, as shown in Figure 2(B).

When micro wells are dewetted, fluid movements appear in wells and coiled trapped DNA escape with the shear flow and are drawn by the pinching of the liquid. Fast rate video microscopy (1000 fps) (Figure 4) showed that the pinching appears always at the edge of wells with no formation of liquid channel [10] or satellite droplets. When the draining speed increases, the balance between friction forces and surface tension decreases the dynamic contact angle down to a minimum value forming a thin liquid film[8]. When draining speed increases more, a transition appears, the dewetting line becomes serrated and finally leads to a dispersive regime (as seen in Figure 2(B))

EXPERIMENTAL

The receding meniscus has been observed with fast videomicroscopy up to 1000 frame per second (Figure 2(A)). The shape of the dewetting line, is deformed when passing over wells and a pinching point appears at the edges of wells. The liquid breaks in a region close to the wells without formation of a sharp channel, this observation is valid for draining speed up to the transition speed. This transition speed has also been observed and it results in effective DNA deposition but shows a dendritic shape deposition pattern with several DNA strands deposited at the same location (Figure 2(C)). This phenomenon is even enhanced with temperature and evaporation rate.

Figure 2: (A)Partial screenshot of 1000 fps microscope video capture of the movement of the dewetting line at the

surface of patterned PDMS, the liquid is drained at 300µm.s-1. 40ms between first and last frame (B) low surface tension liquid (triton X 10%) transition between steady and dispersive regime and (C) dispersive regime deposition producing

dendritic shapes (triton X 10%).

A flow analysis has been carried out using a solution of diamond nanoparticles (70nm mean diameter) using the exact same procedure (surfactants, draining speed, temperature) as for DNA combing (Figure 3(C)). The results show a uniform displacement of particles in the direction of the dewetting with no retrograd flow. The liquid tail is composed of a thin liquid film that is out of the convection cells. We notice that a depletion of particles appears near the dewetting line. This tends to validate the assumption that only the DNA pellets that are actually trapped in microwells are involved

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in the deposition process. This is also confirmed by the fact that each wells where a DNA strand is attached contains DNA pellets at their bottom. DNA pellets, trapped at the bottom of microwells, 5µm below the surface can be seen tagged in red in the composite image of figure 3(A).

Figure 3: (A) Composite fluorescence imaging of capillary force DNA combing on a patterned PDMS substrate. In

green the GFP fluorescence of YOYO tagged lambda phage DNA and in red the DNA pellets trapped in the bottom of microwells (5µm below the surface)(B) detail and (C) fast videomicroscopy screenshot of dewetting line, 100nm diamond

nanoparticle solution for flow visualization. CONCLUSIONS

The receding meniscus assembly technique allows to deposit elongated and ordered DNA strands on top of a microstructured PDMS substrate. This technique is efficient for a liquid draining speed below the transition speed. Experiments with fast rate videomicroscopy show that the breakup of liquid appears near the edge of micro wells and that the region close to the dewetting line is mostly depleted. These observations tend to confirm that only DNA molecules trapped in microwells prior to dewetting accounts for the uncoiling phenomenon that leads to deposition of elongated DNA strands. This technique needs to be improved in order to succeed in the deposition of longer DNA molecules. REFERENCES [1] X. Michalet et al., Dynamic Molecular Combing: Stretching the Whole Human Genome for High-Resolution

Studies, Science 277 5331 (1997) [2] E. Schwob, C. de Renty, V. Coulon, T. Gostan, C. Boyer, L. Camet-Gabut, and C. Amato, Use of DNA combing

for studying DNA replication in vivo in yeast and mammalian cells, Methods Mol Biol . 521 673-87 (2009) [3] J. Guan and L.J.L ee, Generating highly ordered DNA nanostrand arrays, PNAS 102 51, (2005) [4] A. Cerf, X. Dollat, J. Chalmeau, A. Coutable and C. Vieu, A versatile method for generating single DNA molecule

patterns: Through the combination of directed capillary assembly and (micro/nano) contact printing, Journal of Materials Research, 26 2 (2011)

[5] A. Cerf, C. Thibault, M. Geneviève, C. Vieu, Ordered arrays of single DNA molecules by a combination of capillary assembly, molecular combing and soft lithography, Microelectronic Engineering 86, 1419 (2009)

[6] A. Cerf, H.C. Tian and H.G. Craighead, Ordered arrays of native chromatin molecules for high-resolution imaging and analysis, ACS Nano 6(9), 7928 (2012)

[7] D. Gentili, G. Foschi, F. Valle, M. Cavallini, F. Biscarini, Applications of dewetting in micro and nanotechnologyChem. Soc. Rev. 41, pp.4430–4443 (2012)

[8] E. Bertrand, T.D. Blake, J. De Coninck, Dynamics of Dewetting, Colloids and Surfaces A: Physicochemical and Engineering Aspects, 369 (2010)

[9] L.D. Landau. and V. G. Levich, Dragging of a liquid by a moving plate, Acta Physicochim.USSR 17, pp.42-54, (1942)

[10] C.H Lin et al., Experimental and numerical analysis of DNA nanostrand array formation by molecular combing on microwell-patterned surface, J. Phys. D: Appl. Phys. 42 025303, (2009)

CONTACT *B. Charlot, tel: +33-476143785; benoit.charlot@um2.fr

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