DNA SEPARATION IN A PLASMA-THINNED NANOPOST ARRAYJ. Ou and K.D. DorfmanUniversity of Minnesota, USA

ABSTRACTThis paper reports a method to fabricate a sparse nanopost array using standard photolithography equipment. The fabri-cation method does not require any nanopatterning steps. The sparse post array produced by this fabrication method was used to separate long DNA in a microfluidic electrophoresis device.

KEYWORDS: DNA, Electrophoresis, Nanofabrication

INTRODUCTIONElectrophoretic separation, in particular DNA electrophoresis, is a long-standing topic of interest in the microfluidics

community. The separation of long DNA (tens of thousands of base pairs) has drawn particular attention due to the limi-tations of agarose gel electrophoresis, the standard laboratory approach. While an agarose gel is an inexpensive and sim-ple medium for separating short DNA, the orientation of longer DNA with the electric field in the gel leads to all of the DNA moving at the same rate [1]. While these DNA can be separated by periodically changing the direction of the elec-tric field, such pulsed-field gel electrophoresis is very slow.

Prior work in the microfluidics community has demonstrated that long DNA can be separated using dense arrays of nanoposts [2-4] or sparse, disordered arrays of micron-sized posts [5-6]. These separations take minutes, making them an order-of-magnitude faster than pulsed-field gel electro-phoresis. However, the fabrication methods used in these experiments are not optimal, requiring nanopatterning [2-4] or the use of an electromagnet [5-6].

We have recently shown through experiments [7] and simulations [8] that sparse, ordered arrays of microposts can separate large DNA, ranging in size from 10 kilobase pairs (kbp) to 97 kbp. We believe that the separation in a sparse, ordered array can be further improved by shrinking the size of the posts so that even small DNA can form a rope-over-pulley.

Our goal here was to fabricate an array of 400 nm di-ameter posts, with a 3 µm center-to-center spacing, over the entire length of a 15 mm separation channel. Such posts cannot be fabricated using conventional photolithog-raphy equipment, whose features are limited to 1 µm. At the same time, it would be very expensive to create such a large pattern using electron beam lithography. In this pro-ceedings, we present a method to create large arrays of sparse nanoposts and the application of the device to the separation of long DNA.

EXPERIMENTALTo construct the sparse nanopost array, we employed

the plasma-thinning fabrication procedure in Fig. 1. This method provides reliable results using standard photo-lithography and etching equipment that are widely avail-able in industry and academic research facilities. We be-gan by growing an oxide layer on a silicon wafer; the ox-ide layer serves as the mask for a subsequent deep trench etching step. Using standard photolithography and image reversal, we created a 15 mm long by 50 µm wide array of 1 µm circles with a 3 µm spacing in a hexagonal pattern in positive photoresist (S1804, Microposit) on the oxide layer. The pattern itself is inside a 50 µm wide, shifted-T microchannel, which is on the same mask as the circle array. The circle diameter is at the resolution limit of the MA-6 contact aligner available in our clean room and rep-resents a conventional limit for optical photolithography. We then used an oxygen plasma, created in a reactive ion etcher, to etch the resist. The plasma etching acts in both the vertical and horizontal directions, and we have cali-brated the process so that the remaining photoresist is thick enough to transfer the pattern of circles and the mi-crochannel to the oxide layer by wet etching in BOE. We then transferred the thinned post pattern into the silicon

Figure 1. Schematic illustration of the method for producing nanopost arrays.

978-0-9798064-3-8/µTAS 2010/$20©2010 CBMS 1028 14th International Conference onMiniaturized Systems for Chemistry and Life Sciences

3 - 7 October 2010, Groningen, The Netherlands

using a slow deep trench etching step to minimize the scalloping of the vertical wall. The fluid reservoirs were drilled into the chip and then the whole chip was electri-cally insulated by creating a 300 nm silicon dioxide layer using thermal oxygen atmosphere reaction. The com-pleted chip was sealed with a glass coverslip using UV sensitive optical adhesive [9].

For the electrophoresis experiments, we partially digested λ-DNA with the XhoI restriction enzyme, which cuts the 48,502 base pair λ-DNA at the 33,498th base pair. Follow-ing digestion, the DNA were dyed with YOYO-1. The re-sulting ternary mixture was injected using the shifted-T geometry and the fluorescence intensity was measured at 10 mm downstream from the injection point using photom-ultiplier tube (Hamamatsu) and an epifluorescence micro-scope (Leica DMI-4000). The electrophoresis buffer was TBE 5x supplemented with an oxygen scavenger (100 mM DL-dithiothreitol).

RESULTS AND DISCUSSIONThe finished array is shown in Fig. 2. Using profilometry on the open channel region, we determined that the channel

is 3.5 µm deep. The 420 nm post diameter was obtained from a top-down scanning electron microscope (SEM) image. The SEM images in Fig. 2 were taken with a slight tilt of the chip to image simultaneously the top and the sides of the post. As seen in the inset, there is a slight scalloping of the posts from the deep-trench etching step. We do not believe that the scalloping has a significant effect on the DNA electrophoresis, since the posts represent a steric obstacle to the DNA. The main image illustrates the overall uniformity of the nanopost array. We have a very high yield of posts from the fabrication procedure, although several posts typically col-lapse from excessive scalloping during the fabrication process. Given the size of the array (>104 posts), the yield is satisfactory for our applications.

To demonstrate the utility of the nanopost array, we separated

a partial XhoI digest of λ-phage DNA (15, 33.5 and 48.5 kbp). Figure 3 shows an electropherogram of the separation. The black lines correspond to the raw data obtained from the photomulti-plier tube. We can clearly identify three peaks in the output, cor-responding to each species, but they are not baseline resolved.

To be more quantitative, we computed the separation resolu-tion. Since the peaks are overlapping, we first fit the electro-pherogram with the sum of three Gaussian functions using Ori-gin. The individual Gaussians are included in the figure, along with their sum (the red line). From the fitting, we computed the resolution as the difference between the peaks divided by twice the sum of their standard deviations. The resulting resolutions were 1.18 (15/33.5 kbp), 2.29 (15/48.5 kbp) and 0.76 (33.5/48.5 kbp).

CONCLUSIONWe have developed a robust method to shrink micron-scale features using conventional photolithography equipment.

This method was applied to construct a post array for separating long DNA. The first results obtained in the device are promising, but the optimization of the device requires further experimentation.

ACKNOWLEDGEMENTSThis material is based upon work supported by the DARPA YFA program under Award No. N66001-09-1-2103. Any

opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of DARPA.

Figure 2. Scanning electron microscopy (SEM) image of the post array following the thermal oxidation step in Fig. 1.

Figure 3: Electropherogram of the XhoI digest of λ-DNA at an electric field of 10 V/cm after 10 mm of the nanopost array.

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[7] J. Ou, K. D. Dorfman, in preparation.[8] J. Cho, K. D. Dorfman, “Brownian dynamics simulations of electrophoretic DNA separations in a sparse ordered

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CONTACT*K.D. Dorfman, tel: +1-612-624-5560; dorfman@umn.edu

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