Our goal is to design and fabricate an automated sample processor for massively parallel analysis of nucleic acid isolated from blood or bone marrow. It will comprise an array of miniaturized integrated disposable sample preparation devices designed for cell isolation and amplification of DNA or RNA. This processor will provide much needed simplification and automation of sample preparation essential for large-scale cancer studies, and will be part of a larger system containing an array of analytical stems fed by the individual sample preparation devices. Each device will accept a nucleic acid, either directly or after an intermediate amplification step (PCR, RT-PCR) to the detection modules. The complexity of the processing leads up to design the devices in two ways. Firstly, we will stack the individual preparative microstructures into a series of modules that interlock for fluidic connection. Modules can then be assembled for a desired preparative function. In a second and more challenging format, we will integrate all of the preparative functions into a single disposable totally integrated microchip. The microchip modules and the totally integrated microchip will be fabricated from silicon and glass using standard photolithography, wet-chemical etching and anodic bonding, and will contain microfilters, channels and chambers designed for each of the specific functions. Each type of device will contain microfilters, channels and chambers designed for each of the specific functions. Each type of device will be housed in a plastic module that will provide microfluidic connections in and out of the chip for sample application and delivery of processed nucleic acid. A key part of the microfluidics for each the proposed devices, is a new type of membrane valve based on a discontinuous microchannel and a membrane. The microstructured integrated devices will facilitate high throughput, fully automated parallel processing or samples for nucleic acid analysis with reduced sample and reagent consumption, and no cross-contamination. We have selected the liquid cancers as an important and clinically relevant model for assessing the effectiveness of the micro-chip based systems. The system will be optimized for both whole blood and bone marrow samples. The overall goal of this project will be accomplished by the design and optimization of: 1) a microfluidic module for sample application and delivery of processed sample for analysis; 2) a series of six microchips that will isolate human white blood cells, isolate cell sub-sets, isolate DNA or RNA, amplify DNA or RNA; 3) a totally integrated miniature microchip or a series of interlocking microchip modules that can perform combinations of cell isolation, cell selection, nucleic acid isolation and nucleic acid amplification; and finally, 4) system integration to provide a multi-place sample processor.
