서강대학교

초록/요약 도움말

In this study, the simple but sensitive process integration for total analytical micro-fluidic chip to detect biological target is developed for performance of sequential biosensing processes (target concentration, isolation, and sample/signal amplification) in microfluidic single chamber at low concentrations in food, environmental, and clinical samples. The simplification of integrated system was conducted to detection of microbes and animal cells with the highly sensitivity in a large volume sample by performing isolation, amplification, and detection in a single chamber micro fluidic biosensor without transferring samples from one chamber to another. With this advanced technology, I designed and fabricated the integrated system for performing entire process to detect environmental and food samples. First, nucleic acids from E.coli O157:H7 were isolated in the glass micro-beads embedded in a conical polymer tube chamber in a low pH buffer. The mRNA, which was adsorbed to the glass micro-beads, was amplified by Nucleic Acid Sequence Based Amplification in the same chamber at a relatively high pH (pH 8.9). The products amplified were measured using the hair-loop type detection probe with FAM and DABCYL, which was pre-mixed in the NASBA mater mixture. As a result, high sensitivity (100% for rain water and 60% for river water) with less than 10 viable E.coli O157:H7 in 100 ml could be achieved within 4 h using the simple method proposed. Second, a simple and fast isolation tool of E.coli O157:H7 was developed using a magnet nanoparticle embedded silica nanotube (MNSNT) for the detection of E.coli O157:H7 in the sample with nucleic acid based amplification. This method does not require chaotropic salt and sophisticated equipment to isolate bacteria. The E.coli O157:H7 in the sample was effectively bound to the hydrophilic surface of MNSNT in low pH binding buffer containing divalent ions and PEG without the need for expensive biological reagents such as antibodies. This E.coli O157:H7 bound MNSNT was simply isolated by a magnet, prior to adding an amplification mixture to the same micro tube without transferring the sample to another tube. Using this novel method, the detection sensitivities of E.coli O157:H7 (102 cfu/1 g of seed sprout and 102 cfu/5 mL of water) were 80% and 100%, respectively, whereas that was 0% using the commercial method. Third, a simple and effective bacterial isolation and separation method was developed with Gram (+) and Gram (-) bacteria that can be directly used for the detection of pathogenic bacteria. The optimal aquatic buffer conditions for bacteria adsorption on the hydrophilic surface were determined to be 1% polyethylene glycerol (PEG) and 10mM MgCl2 in 100mM phosphate at pH 4 for the Gram (-) bacteria, E.coli O157:H7 and 10mM Na2SO4 in 100mM phosphate in 100mM phosphate at pH 4 for the Gram (+) bacteria, B.cereus. When these divalent cation and anion (MgSO4) containing acidic solutions were used, 40% of both bacteria adsorbed onto the hydrophilic surface at a loading rate of 2 mL/min after introduction of low concentrations of bacteria. This method was directly employed to detect E.coli O157:H7 in beef using a single plastic tube chamber that was partially filled with nickel micro beads coated with TEOS. In this system, E.coli O157:H7 were lysed by induction heating of the nickel micro beads. The extracted mRNA was readily amplified and detected by adding an isothermal amplification solution. As a result, this highly sensitive sensing tool could detect very low concentrations of E.coli [100 CFU/1 g of beef]. Fifth, a fast, simple, and effective tool was developed for the detection of pathogens in large volumes samples by using an automatic system device for adaptive process unification using single plastic tube chamber. The serial processes with including sample loading by pump, heating induction lysis, mRNA amplification by isothermal induction, and signal generation were sequentially performed in a single plastic tube chamber. The induction technology of flash heating for lysis and isothermal heating for amplification of RNA was performed using nickel beads in the single plastic tube chamber. The automatic system device can be highly sensitive while not provide false negative results. Sixth, a simple and efficient tool to isolate epithelial cells from bacteria-contaminated samples was developed using two different micro particles functionalized with chemical molecules. The epithelial cells could be captured simply by biocompatible anchors for membranes (BAM), consisting of PEG functionalized with oleyl-chain-conjugated NHS on glass micro particles, whereas bacteria were adsorbed on amino-functionalized magnetic micro particles. In the case of samples highly contaminated with bacteria, epithelial cells were not isolated successfully by both of the single BAM- and antibody-functionalized micro particles. Therefore, serial isolation steps of these two different chemical functionalized micro particles were introduced. The concentration of bacteria was decreased dramatically by using APTS-functionalized magnetic particles prior to the isolation of epithelial cells by BAM micro particles. With these serial processes, successful isolation of epithelial cells was achieved from bacteria-contaminated epithelial samples. The applicability of this method was verified with bacteria-contaminated intestinal samples biopsied from a BALB/C mouse for primary cell cultivation. Seventh, the integrated system for a signal amplification and quantitative detection was developed to combine nucleic acid amplification tools with marker detection in clinical samples. An Fc-region specific aptamer was introduced as a reporter probe in QIHC, and employed to characterize breast cancer tumor cells, which are already clinically categorized by protein markers (HER2, ER, PR, ki-67). The aptamer is simply amplified by Quantified-NASBA, which is an isothermal, RNA-preferred amplification reaction. Samples are prepared in the form of a paraffin block slide since most clinical tissue samples are frozen and blocked by paraffin for a long storage. The new aptamer-assisted QIHC (AA-QIHC) micro fluidic platform is suitable for paraffin block slides, and is designed, fabricated, and applied to breast tumor samples. These proposed tools are first verified with three different breast cancer cell lines (SK-BR-3, MCF-7, and MDA-MB-231) because it is well known that the lines have totally different characteristics. The verified aptamer QIHC is also tested and compared with real clinical samples. Therefore the simple and easy sensitive sensing tools assisted process integration for total analytical micro-fluidic chip to detect biological target are developed to integrated system for isolation, separate concentration and detection of target at low concentrations in food, environmental, and clinical samples using several isolation process for each sample type and signal amplification methods.