Electric Field Directed Assembly of High-Density Microbead Arrays Kristopher D. Barbee, Alexander P. Hsiao, Michael J. Heller & Xiaohua Huang Department of Bioengineering, Jacobs School of Engineering, University of California, San Diego Overview Results Microbead-based platforms have become a popular technology for many high-throughput biological assays such as A B C genotyping, DNA sequencing, and protein detection due to the ease in which they enable multiplexing and miniaturization. Many of these assays involve the immobilization of microbeads onto solid supports by capturing or assembling them using evaporation, gravity, centrifugation, and magnetic and electric fields. While all of these methods have been been successfully utilized, they typically suffer from one or more limitations pertaining to assembly speed, filling efficiency, scalability and control over microbead number, position and order. We present a method for rapid assembly of high-density microbead arrays using an electric field. Our approach offers many advantages over other microbead assembly strategies in that it is fast, efficient, scalable, automatable, capable of producing arrays of single microbeads with near perfect order and is compatible with microfluidics, biological assays and real-time epifluorescence microscopy. Photolithography is used to create arrays of wells in an epoxy-based photoresist on gold-coated wafers. Thin gaskets are used to form microfluidic chambers between the wafers and glass coverslips with thin metal lines or an ITO coating. SEM images of arrays of streptavidin-conjugated polystyrene microbeads assembled within Wafer-scale arrays of streptavidin-coated microbeads are assembled and captured within the wells by applying low photolithographically defined wells via an electric field. A. An array of 1 µm beads at a 2.4 µm pitch. B. An array of voltage, 1 Hz electrical pulses. Microbead binding is robust and occurs through gold-protein interactions, allowing 1 µm beads at a 1.2 µm pitch. C. An array of 0.5 µm beads at a 1.2 µm pitch. excess beads to be washed away or recycled. The well and bead sizes are chosen such that each well can capture just one bead. We have demonstrated that: 1) The assembly process can be completed in as little as 30 seconds; 2) Filling D E F efficiencies as high as 99.9% can be achieved; 3) Arrays with densities as high as 69 million beads/cm2 can be assembled using 0.5 and 1.0 µm beads. Potential applications for this technology include the assembly of DNA arrays for genome sequencing and antibody arrays for proteomic studies. This device may also be used to enhance the concentration-dependent processes of various assays through the accelerated transport of molecules using electric fields. Device Fabrication and Assembly 24 µm 24 µm 32 µm A B C Fluorescent micrographs of arrays of streptavidin- and Neutravidin-conjugated polystyrene microbeads assembled using an electric field. D. An array of 1 µm beads at a 2.4 µm pitch. E. An array of 1 µm beads at a 2.4 V µm pitch. F. An array of 0.5 µm beads at a 1.6 µm pitch. a Deposit SiO2 via PECVD G H I on a silicon wafer b Apply low-voltage DC pulses c Deposit Ti and Au films via V sputtering or evaporation d e Remove excess microbeads SEM images of microbead doublets, size variation and alignment. G. An array with a doublet in one well. H. An array showing the size variation among the 1 µm beads. I. An array of 0.5 µm beads assembled into over-sized wells. Spin-coat photoresist and Microbead positioning and filling efficiency may be compromised if the wells are significantly larger than the microbeads. pattern array of wells V Summary We have demonstrated the ability to use electric fields to direct the rapid assembly of arrays of 0.5 and 1 µm protein- conjugated microbeads on photolithographically defined templates. Standard microfabrication procedures are used to generate wafer-scale arrays of wells on gold in a robust, epoxy-based photoresist. Hundreds of millions of microbeads Device fabrication and assembly. A. Fabrication of an array of microwells on a silicon wafer. The photolithography is can be assembled within these wells in 30-45 seconds by applying low-voltage, low-frequency DC electrical pulses. performed on an i-line stepper system, which allows wafer scale arrays of high-resolution wells to be printed in minutes. Each well contains only one microbead and filling rates as high as 99.9% are easily achieved with minimal defects. B. Exploded view of the device. a: ITO-coated glass coverslip; b: silicone gaskets with flow channels; c: silicon wafer Array assembly takes place within a microfluidic device that is compatible with real-time biright-field and epifluorescence with a high-density array of wells in photoresist on a thin layer of gold; d: double-coated adhesive gasket; e: microscope imaging. The methods presented here may be applied to the assembly of arrays of microbeads conjugated to stage insert with fluidic ports. C. Electric field directed assembly of arrays. The protein-conjugated microbeads are antibodies and DNA for use in high-throughput assays. In addition, the use of such a platform may provide a means of directed into the wells and captured onto the gold surface by applying a series of electrical pulses across the chamber. accelerating diffusion-limited assays by actively concentrating molecules of interest via an electric field.
Pages to are hidden for
"Electric Field Directed Assembly of High-Density Microbead Arrays"Please download to view full document