Magnetic Assembly of High-Density DNA Arrays for Genomic Analyses

Abstract

A method for rapidly assembling high-density DNA arrays with near-perfect order is described. Photolithography is used to generate a wafer-scale array of microwells in a layer of photoresist on a chemically functionalized glass coverslip. The array is enclosed within a microfluidic device, and a suspension of superparamagnetic microbeads conjugated to DNA molecules is introduced into the chamber. A permanent magnet is used to direct the rapid assembly of the beads into the wells, with each well containing a single bead. These beads are immobilized on the glass surface via affinity binding, and excess beads can be recycled or washed away. Nonspecifically bound beads are removed by dissolving the photoresist. The result is a high-density array of beads with virtually no background. This method can be used to produce protein arrays for chip-based assays and DNA arrays for genotyping or genome sequencing.Some of the greatest breakthroughs in biomedical research can be attributed to the development of the numerous high-throughput technologies for quantitative measurements of biomolecules. Many of these technologies are made possible by microfabrication techniques commonly used in the semiconductor industry. For example, DNA and protein arrays fabricated by robotic printing and photolithographic methods have enabled extremely large-scale surveys of biomolecules. [1][2][3][4] The emerging "next generation" genome sequencing technologies, many of which utilize massive parallelization and miniaturization to achieve unprecedented multiplexing, throughput and cost reductions, [5][6][7][8][9][10] promise to revolutionize biomedical research and enable personalized healthcare. However, some of these technology platforms utilize randomly distributed DNA-conjugated microbeads or clones on a glass slide within a reaction chamber. The random arrangements of the beads or clones result in low throughput and imaging efficiency, complicated image processing, and high reagent costs. 6-10 One approach to dramatically improve these devices involves the use of microfabricated arrays to eliminate overlap and to minimize the area between the beads or clones.Such arrays can be generated by depositing samples onto glass slides using robotic contact printing, 2 microcontact printing, 11-13 or dip pen lithography. 14 These arrays can also be generated by assembling beads onto microfabricated arrays of wells on glass or silicon substrates [15][16][17][18] or in etched wells on the face of a fiber-optic bundle. 19,20 Since bead assembly will not occur in an efficient and reliable manner if the process depends solely upon gravitational forces and Brownian motion, this process is typically achieved via solvent evaporation or dewetting. [17][18][19][20][21] However, these approaches are not suitable when rapid assembly is required or sample drying is undesirable. Other groups have employed electric 22 and magnetic [23][24][25] assembly methods to overcome these issues, but these active approaches require multistep fabrication processes and...

Transport of biomolecules to binding partners displayed on the surface of microbeads arrayed in traps in a microfluidic cell

“…In other studies, wells at the floor of the microchannel are used to sequester and array microbeads to construct the probe library. 17,18,[22][23][24] In the well geometry, target only streams over the top part of the microbead, and at high Pe thin boundary layers cannot encircle a large portion of the microbead. As a result, diffusive transport is hindered, and one solution which is proposed is to create drains in the wells to enhance convection around the microbead.…”

“…21 Well deposition has also been enhanced by using electric and magnetic fields to assist in the capture, [22][23][24] or by using holes placed in the well and connected to a drain to provide fluid suction (see McDevitt et al 25,26 and Ketterson 27 ). The use of traps to array microbeads follows from the studies of arraying cells, or droplets containing cells (e.g., Refs.…”

Temperature-controlled microintaglio printing for high-resolution micropatterning of RNA molecules

“…However, the microarray format is advantageous to the batch format for the screening of biomolecules (Emili and Cagney, 2000), and therefore the screening of DNA aptamers using a DNA microarray has been attempted by several groups (Asai et al, 2005;Knight et al, 2009). Since the massively parallel DNA arraying method employing the DNA-immobilized magnetic beads used in this advanced TC-mIP enables the arraying of over 10 8 variants (Barbee and Huang, 2008), the combination of this DNA arraying method, in situ RNA synthesis, and TC-mIP will enable the microarray format screening of RNA aptamers and ribozymes in addition to DNA aptamers.…”

A large-area hemispherical perforated bead microarray for monitoring bead based aptamer and target protein interaction

“…16 Although the previous microbead array formats are adequate for sensitive biochemical analysis in a high-throughput manner without cross-contamination between beads, most of their designs were restricted to microwell structure. [8][9][10][11][12][13][14][15][16][17][18] Such a microwell structure mainly depends on the passive immobilization of the beads into each microwell, which is time-consuming, and suffers from the bead aggregate formation, leading to the non-uniform distribution of single beads in each chamber. In addition, the recovery of the immobilized beads is very difficult using the microwell array.…”