Abstract
Microfluidics is a technology to manipulate a small amount of fluids by using well-designed microchannels, microchambers and microdevices. It offers a number of advantages, including low sample/reagent consumption, high-resolution separation, fast detection, high sensitivity, low cost and automation. While the micro-fabrication process uses bio-compliable materials, increasing numbers of molecular biological and medical diagnosis could be performed on microfluidic systems for rapid and accurate diagnosis. In this work, we presented the application and screening of molecular biomarkers for disease diagnosis on the microfluidic systems. First, a new integrated microfluidic system for rapid detection of influenza infections was developed, which integrated a suction-type, a pneumatic-driven microfluidic control module, a magnetic bead-based fluorescent immunoassay and an end-point optical detection module. The molecular biomarker used was a protein-based biomarker- the specific mouse anti-influenza nucleoprotein (NP)-A mAb or anti-NP-B mAb. This system could successfully distinguish between influenza A and B using a single chip within 15 minutes automatically. Furthermore, the results of diagnostic assays from 86 patient specimens have demonstrated that this system has 84.8% sensitivity and 75.0% specificity. Then, in order to improve the optical signal of a magnetic bead-based FIA, we produced MnFe2O4 magnetic nanoparticles around 100 nanometer for diagnostic applications. After the nanoparticles were coated with anti-influenza-NP-A mAbs, we further adopted a layer-by-layer surface modification process to significantly reduce the background noise due to non-specific adhesion of nanoparticles. When compared with the 4.5-µm magnetic beads, the optical signals of the MnFe2O4 nanoparticles were twice as sensitive, providing a promising platform for rapidly diagnosing infectious diseases. Because antibodies are relatively expensive, hard to preserve and may vary from batch to batch fabrication, in-vitro screening methods for high-affinity biomarker searching attract intensive attention recently. Among them, systematic evolution of ligands by exponential enrichment (SELEX) has been explored extensively. In this work, we used SELEX for cancer-specific aptamer screening. The different histologically classified ovarian cancer cells, BG-1, TOV112D, IGROV1 and TOV-21G, were applied for aptamer screening. Thirteen OvCa-specific aptamers were successfully selected, including four specific aptamers for the clear-cell-type cell line, seven specific aptamers for the endometrioid-type cell lines, and two specific aptamers for the serous-type cell line. An automatic screening system which enabled one to use multiple cell lines for high-throughput screening work in one integrated microfluidic system was developed. Furthermore, cancer stem cells (CSCs), which are a tiny group of cancer cells having the ability to self-renew and proliferate indefinitely, may play an important role for cancer therapy. In this dissertation, colorectal-CSCs (CR-CSCs) and colorectal cancer (CRC) cells were applied for specific aptamer screening. Eight CR-CSC/CRC-specific aptamers were successfully selected. Three of them showed high affinities towards their target cells with dissociation constants (Kd) of 27.4, 28.5 and 12.3 nM. These selected aptamer biomarkers specifically for CR-CSCs and CRC cells may be further applied for personal medical screening or drug discovery.