Polymer Lab-on-a-Chip with Functional Nano/Micro Bead-Packed Column for Biochemical Analysis

The objective of this research is to develop new functional nano/micro bead-packed columns on polymer lab-on-a-chips (PLOC) using self-assembly microfabrication technologies for practical on-site biochemical analyses or point-of-care clinical diagnostics. In order to achieve the goal of this research, new polymer microfabrication methods such as (a) a multi-chip assembly method for polymer chips using pin-hole pair structure and (b) a self-assembly and packing method for nano/micro beads on polymer chips have been newly developed and characterized. The novel microfabrication methods have been applied for the realization of practical polymer lab chips such as (a) sample preparation for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and (b) on-chip capillary electrochromatography with electrochemical detection for on-site clinical analysis. The state-of-art microfabrication techniques have been demonstrated by development and characterization of high quality disposable polymer capillary electrophoresis (CE) microchips. To realize polymer lab-on-a-chips in a multilayer format, a new assembly technique using pin-hole pair structure has been developed for the fabrication of multilayered polymer chips. This newly developed technique directly addresses the alignment problem that has been considered as one of the most difficult tasks in the bonding assembly of multiple polymer layers for the multi-layered polymer lab chips. Functional on-chip bead-packed columns using a slurry packing method and a selfassembly method have been developed and characterized in this work. An on-chip reversed phase chromatography (RPC) column packed with RPC media (SOURCE 15RPC) has been realized using the slurry packing method, where the geometrical restrictions with precise alignments, which are essential for the packing process, were achieved using the pin-hole pair structure assisted assembly technique. The RPC column integrated with the sample preparation chip for MALDI-MS has been designed, fabricated, and fully characterized. The integrated column has shown the holding capacity of 48.8 ng of peptide and was applied for the sample preparation for MALDI-MS analysis. 5 μg/ml of Neurotensin sample was filtered using the sample preparation chip and the results analyzed through MALDI-MS were comparable to the results from the commercially available ZipTip products. Small volume of sample (less than 1 μl) and parallel processing capability in the sample preparation are benefits derived from the newly developed sample preparation chip. A high quality on-chip capillary electrochromatography (CEC) column has been developed using the self-assembly method of silica colloidal beads in microchannels with a depth of 50 μm and a length of 2 cm. Nano/micro beads (0.8 μm ~ 1.98 μm in diameter) were successfully self-assembled in the microchannels (50 μm ~ 100 μm in width), resulting in a hexagonal crystalline structure. For a point-of-care system for urine sampling and analysis, the self-assembled columns have been developed over the entire channels for both sample injection and separation. This allows the sample to be introduced into the sample injection channel using capillary forces, which eliminates the difficulties associated with the traditional sample introduction techniques such as an external vacuum or applied pressure. In addition, an electrochemical (EC) sensor placed at the end of the separation channel has been implemented and characterized as a CEC device with EC detection, and uric acid and ascorbic acid have been successfully separated and detected using the developed chip. Reproducible results have been achieved and the average migration times from serial injection and separation were 6 ± 0.3 s (n = 5) and 10.8 ± 0.3 s (n = 5) for ascorbic acid and uric acid, respectively. Linear calibration plots between 0.1 mM to 1 mM concentration for uric acid (R = 0.991) and ascorbic acid (R = 0.998) have been achieved, which covers the clinical range of sample concentration from 10-fold dilution of urine samples. In this research, a new alignment and bonding method for multi-layered polymer chips and an on-chip nano/micro bead self-assembly method for functional columns have been successfully developed and fully characterized. These promising microfabrication methods have been applied for various polymer lab-on-a-chips for biochemical analysis or clinical diagnostics, which may enable the practical on-site or point-of-care testings using on-chip capillary electrochromatography.