Development of microdevices for applications to bioanalysis

Abstract

The development of microdevices for applications related to bioanalysis is described. There are two types of microdevices involved in this study: DNA (or RNA) microarrays and bead-based microfluidic devices. First, a new method to fabricate DNA microarrays is developed: replication of DNA microarrays. It was shown that oligonucleotides immobilized on a glass master can hybridize with their biotin-modified complements, and then the complements can be transferred to a streptavidinfunctionalized replica surface. This results in replication of the master DNA array. Several innovative aspects of replication are discussed. First, the zip code approach allows fabrication of replica DNA arrays having any configuration using a single, universal master array. It is demonstrated that this approach can be used to replicate master arrays having three different sequences (spot feature sizes as small as 100 [mu]m) and that master arrays can be used to prepare multiple replicas. Second, it is shown that a surface T4 DNA polymerase reaction improves the DNA microarray replication method by removing the requirement for using presynthesizd oligonucleotides. This in-situ, enzymatic synthesis approach is used to replicate DNA master arrays consisting of 2304 spots and arrays consisting of different oligonucleotide sequences. Importantly, multiple replica arrays prepared from a single master show consistent functionality to hybridization-based application. It is also shown that RNA microarrays can be fabricated utilizing a surface T4 DNA ligase reaction, which eliminates the requirement of modified RNA in conventional fabrication schemes. This aspect of the work shows that the replication approach may be broadly applicable to bioarray technologies. A different but related aspect of this project focuses on biosensors consisting of microfluidic devices packed with microbeads conjugated to DNA capture probes. The focus here is on understanding the parameters affecting the hybridization of DNA onto the probeconjugated microbeads under microfluidic flow conditions. These parameters include the surface concentration of the probe, the flow rate of the solution, and the concentration of the target. The simple microfluidic device packed with probe-conjugated microbeads exhibits efficient target capture resulting from the inherently high surface-area-to-volume ratio of the beads, optimized capture-probe surface density, and good mass-transfer characteristics. Furthermore, the bead-based microchip is integrated with a hydrogel preconcentrator enhancing the local concentration of DNA in a icrochannel. The integration of the preconcentrator into the bead-based capture chip allows significantly lower limit of detection level (~10-fold enhancement in the sensitivity of the microbeadbased DNA detection).