Fabrication and characterization of solid-state nanopore arrays for high-throughput DNA sequencing

Abstract

We report the fabrication and characterization of uniformly sized nanopore arrays, integrated into an optical detection system for high-throughput DNA sequencing applications. Nanopore arrays were fabricated using focused ion beam milling, followed by TiO(2) coating using atomic layer deposition. The TiO(2) layer decreases the initial pore diameter down to the sub-10 nm range, compatible with the requirements for nanopore-based sequencing using optical readout. We find that the TiO(2) layers produce a lower photoluminescence background as compared with the more widely used Al(2)O(3) coatings. The functionality of the nanopore array was demonstrated by the simultaneous optical detection of DNA-quantum dot conjugates, which were electro-kinetically driven through the nanopores. Our optical scheme employs total internal reflection fluorescence microscopy to illuminate a wide area of the TiO(2)-coated membrane. A highly parallel system for observing DNA capture events in a uniformly sized 6 × 6 nanopore array was experimentally realized.

Figure 1

Schematic of the nanopore array fabrication process. Low Pressure CVD (LPCVD) process was employed to deposit a low-stress nitride layer above the bulk silicon substrate. A free-standing SiN membrane is fabricated using photolithography. Nanopore arrays were drilled through the SiN membrane using FIB milling. The pore size was measured using TEM. Reduction of the nanopore diameters to sub-10 nm range was achieved using ALD.

Figure 2

Pore size as a function of FIB dwell time for a 30 nm-thick SiN membrane, fabricated at three different probe currents.

Figure 3

TEM micrographs of FIB-drilled nanopores pre- and post-ALD coating with TiO₂ deposited at different temperatures as indicated.

Figure 4

A. TEM image of an 8×8 array of nanopores, fabricated using a FIB current of 2 pA. B. TEM image of the same membrane post ALD deposition of TiO₂. The pore size histograms are shown for pre- and post-ALD (C and D, respectively). Solid lines represent Gaussian fits to the distributions.

Figure 5

A. Photoluminescence spectra of TiO₂-coated membranes and a Silicon Nitride membrane, showing the low photoluminescence at the visible wavelength. B. XPS spectra of a TiO₂-coated membrane. C. Optical background comparison between TiO₂ and Silicon nitride membranes when excited by a 488 nm laser under TIRF illumination and with the emission band at 650–700 nm. The inset shows the enhancement in image contrast of the single quantum dots on a TiO₂-coated membranes versus a Silicon Nitride membrane (scale bars equal 5 μm).

Figure 6

A. Schematic illustration of the TIRF based imaging of the nanopore array and Qdot-DNA conjugates. Bottom: Optical images of the membrane before (B) and after application of a positive voltage (C). The discrete fluorescent spots arise from the Qdots-DNA conjugates, exhibiting partial occupancy of a 6×6 nanopore array when a positive voltage is applied.