s8_Stretching and Immobilization of DNA Molecules in Si Channels with Integrated Electrodes

Stretching and Immobilization of DNA Molecules in Si Channels with Integrated Electrodes

S. W. Pang, V. R. Dukkipati, J. H. Kim, R. G. Larson

University of Michigan, Ann Arbor, Michigan 48109-2122, USA

Low temperature Si to glass bonding using polymethylmethacrylate (PMMA) as an adhesive layer is developed to integrate electrodes with Si channels. The integrated microsystem contains channels dry etched in Si with widths ranging from 3 to 100 µm and depths ranging from 100 nm to 30 µm. The channels are bonded to a 100 µm thick glass consisting of 600 nm thick patterned PMMA and 20/50 nm thick Cr/Au electrodes, with PMMA as an adhesive layer. The typical bond strength is 3 MPa, obtained by bonding at 110 °C with 600 nm thick PMMA. Fluidic flow studies are carried out in channels that are 50 and 100 µm wide with a depth of 100 nm. De-ionized water flows through the sealed Si channels due to capillary pressure with an initial velocity of 0.65 mm/ s for 50 µm wide and 100 nm deep channels. Electric fields are used to induce deoxyribonucleic acid (DNA) motion with velocities from 2.4 to 14.5 µm/ s in 100 µm wide and 20 µm deep channels. The forces generated by the fields and the fluid flow are also used to stretch the tethered DNA molecules up to 15 µm long in the microchannels.

A technique called “protein-assisted DNA immobilization” (PADI) is developed to immobilize and stretch, but not overstretch, DNA molecules inside a micro/nanochannel with limited surface interactions while maintaining continuous hydration at physiological pH. The biological activity of the immobilized DNA molecules is confirmed by digesting the DNA with restriction enzymes in the microchannel. Single-molecule transcription, which has stringent requirements on the immobilized DNA with respect to surface interactions and stretched lengths, is also successfully demonstrated on DNA molecules immobilized by PADI. In addition to arraying DNA molecules for study of DNA-protein interactions, the immobilization method could be used to construct DNA-templated nanoelectronic devices.

Control over the placement of stretched DNA molecules in a microfluidic system is a critical requirement for molecular nanotechnology. A technique is developed where a large number of DNA molecules can be immobilized specifically at one end to the electrode tip and stretched in a microchannel using high frequency ac fields. λ-DNA molecules are immobilized and stretched using 100 kHz ac fields in a 100 µm wide and 75 µm deep Si microchannel. Using a floating electrode in between two biased electrodes, stretched T2 DNA molecules are immobilized across a 5 µm wide electrode gap by electric field and hydrodynamic flow.

Figure 1 is an optical micrograph of an integrated fluidic system with two intersecting channels. The system consists of electrodes on a 100 µm thick glass aligned and bonded to a Si substrate containing channels perpendicular to each other.

Figure 1. 20/50 nm thick Cr/Au electrodes on 100 µm thick glass bonded to 30 µm deep, 3 and 10 µm wide Si channels.

Bond strength is measured to optimize the bonding conditions using PMMA as an adhesive layer. Figure 2(a) shows the variation of the bond strength with temperature. Figure 2(b) shows the variation of bond strength for different PMMA thicknesses.

Figure 2. Dependence of bond strength on (a) temperature and (b) PMMA thickness. Bonding is carried out at 0.4 MPa pressure, and 75 Torr pressure for 15 min. The PMMA thickness for (a) is 600 nm and the temperature for (b) is 110 °C.

At lower voltages, the DNA molecules in the vicinity of the electrodes are influenced by the dielectrophoretic and electrothermal forces. Beyond a threshold voltage, the torque and electrothermal forces on the DNA molecules become dominant and they together induce a motion on the DNA molecules that would move the molecules away from the electrodes. Figure 3 shows the velocity of the DNA molecules at the center of the channel for various applied voltages and at a distance of 1110 µm away from the center of the electrode gap.

Figure 3. Velocity of DNA molecules in Si microchannels for different applied ac voltages.

When the DNA-protein complex reaches the channel surface, the proteins on the DNA adsorb to the surface, resulting in immobilization of the stretched DNA molecule inside the channel. We use a hydrophobic polymethylmethacrylate (PMMA) surface inside our microchannel, leading to a large number of immobilized DNA molecules, as shown in Figure 4.

Figure 4. Images of λ-DNA (5.5 pM) stretched onto a PMMA-coated glass in the microfluidic device (100 µm width, 1 µm depth) in the presence of T7 RNAP (10 nM) (a) at 200 µm from the inlet and (b) near the inlet. The direction of flow is from right to left.

DNA are immobilized to Au electrodes irrespective of the fluid flow direction. In order for the attached DNA to be stretched outward from the electrode edge, the electrothermal induced flow should cause the fluid to flow away from the electrode edges. Figure 5 shows DNA stretching in a 75 µm deep microchannel at different voltages.

Figure 5. DNA stretching for different voltages at 100 kHz in a 100 µm wide and 75 µm deep Si microchannel. (a) DNA stretching starts at the tip of the pointed electrode at 8 V. (b) Larger number and area for stretched DNA molecules near the tip of the pointed electrode at 12 V. (c) DNA molecules stretched at both the straight edge and pointed electrodes at 16 V.

References

Last Updated: November 19, 2007

E-Mail: pang@umich.edu

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