Microfluidic Methods for Single Multidrug Resistance Cell Drug Transport Study, Kras Mutation Analysis and Influenza Antibody Detection Assay
소속 :
연사 : Prof.Paul C.H. Li(Simon Fraser University)
일시 : 2012-09-18 11:00 ~
장소 : 503동 327호
일 시 : 2012년 9월 18일, 11:00 AM
장 소 : 503동 3층 세미나실(327호)
-Abstract-
Long-term monitoring of biochemical changes on a single cell has provided new information about unusual cellular response to reagents. This finding is employed in a cell-based medical diagnosis device for cancer drug resistance. A glass microchip device that was fabricated with a special cell retention chamber allows selection, retention and shuttling of a single biological cell for long-time study and analysis. Excellent optical and fluorescent observation of the single cell have been simultaneously achieved. We previously reported this microfluidic method for the analysis of single muscle cells. Now, this method was employed to test a single cancer cell. We have carried out the analysis on single multi-drug resistance (MDR) leukemia cells because MDR-mediated drug efflux is known to be a major cause of the failure of chemotherapy. The efflux measurement method, previously performed on yeast cells, was used to measure the retention of the chemotherapeutic drug, daunorubicin (DNR) on leukemia cells. We have also conducted the inhibition study of the drug efflux process using common MDR inhibitors, such as verapamil (Ver) and cyclosporine A. To resolve the problem of cellular variation, cell study has been conducted on one and the same cell (i.e. first without Ver, and then with Ver). In this way, the same cell is used as the control cell (without Ver) and the test cell (with Ver). This one-cell measurement method is dubbed as the same-single-cell analysis (SASCA), which takes advantages of tracking one and the same cell over a long period of time.
Other than the cell-based medical diagnosis, the microfluidic chip has also been used for DNA-based diagnostic applications. Usually, DNA microarrays are constructed using expensive methods such as photolithographic synthesis of oligonucleotide probes or by spotting of pre-synthesized probes. In addition, only one sample can be applied on a microarray chip at one time. Here, DNA hybridizations of 96-384 samples with 96-384 probes were carried out in a nanofluidic biooarray (NBA). Small volume (1 μL) and low concentration (1nM or lower) of multiple Kras mutations as well as fungal pathogenic samples were tested all on one 3.5-inch compact disk (CD). Liquid flow was driven by centrifugal force obtained by spinning the CD. The NBA chip includes 2 channel plates and 1 common chip. When channel plate 1 is assembled with and sealed against the common chip, liquid flow inside radial microchannels allows the immobilization of a line microarray of DNA probes. When channel plate 2 is sealed against the common chip, liquid flow inside spiral microchannels allows the formation of a spot microarray with DNA samples after hybridization. Our method allows multiple samples to be tested on a single DNA array chip using microfluidic operation. In addition, the probe immobilization time and sample hybridization time are shortened because the high surface-to-volume ratio achieved in the microchannels facilitates fast surface reaction rates. As enabled by gold nanoparticles, even single base-pair discrimination was achieved without using the temperature stringency method.
The NBA chip is also used for fast detection of antibodies present in biological fluids such as mouse ascites fluid. This capability has applications in diagnosing disease (e.g. influenza), and in monitoring efficacy of immunotherapy or vaccination. To begin designing and testing this system, the synthetic peptide (HA; a 12 amino-acid residue fragment from hemagglutinin A glycoprotein of the influenza virus) was used as a model antigen to be detected by the monoclonal antibody (MAb) produced from the murine 17/9 hybridoma cell line. Several MAb samples were tested by reacting each with multiple antigen probes. Multiple tests were conducted in a single run either on a single sample, or on multiple samples, with sensitivity comparable or better than ELISA, but with faster detection and higher throughput offered by the NBA chip.
장 소 : 503동 3층 세미나실(327호)
-Abstract-
Long-term monitoring of biochemical changes on a single cell has provided new information about unusual cellular response to reagents. This finding is employed in a cell-based medical diagnosis device for cancer drug resistance. A glass microchip device that was fabricated with a special cell retention chamber allows selection, retention and shuttling of a single biological cell for long-time study and analysis. Excellent optical and fluorescent observation of the single cell have been simultaneously achieved. We previously reported this microfluidic method for the analysis of single muscle cells. Now, this method was employed to test a single cancer cell. We have carried out the analysis on single multi-drug resistance (MDR) leukemia cells because MDR-mediated drug efflux is known to be a major cause of the failure of chemotherapy. The efflux measurement method, previously performed on yeast cells, was used to measure the retention of the chemotherapeutic drug, daunorubicin (DNR) on leukemia cells. We have also conducted the inhibition study of the drug efflux process using common MDR inhibitors, such as verapamil (Ver) and cyclosporine A. To resolve the problem of cellular variation, cell study has been conducted on one and the same cell (i.e. first without Ver, and then with Ver). In this way, the same cell is used as the control cell (without Ver) and the test cell (with Ver). This one-cell measurement method is dubbed as the same-single-cell analysis (SASCA), which takes advantages of tracking one and the same cell over a long period of time.
Other than the cell-based medical diagnosis, the microfluidic chip has also been used for DNA-based diagnostic applications. Usually, DNA microarrays are constructed using expensive methods such as photolithographic synthesis of oligonucleotide probes or by spotting of pre-synthesized probes. In addition, only one sample can be applied on a microarray chip at one time. Here, DNA hybridizations of 96-384 samples with 96-384 probes were carried out in a nanofluidic biooarray (NBA). Small volume (1 μL) and low concentration (1nM or lower) of multiple Kras mutations as well as fungal pathogenic samples were tested all on one 3.5-inch compact disk (CD). Liquid flow was driven by centrifugal force obtained by spinning the CD. The NBA chip includes 2 channel plates and 1 common chip. When channel plate 1 is assembled with and sealed against the common chip, liquid flow inside radial microchannels allows the immobilization of a line microarray of DNA probes. When channel plate 2 is sealed against the common chip, liquid flow inside spiral microchannels allows the formation of a spot microarray with DNA samples after hybridization. Our method allows multiple samples to be tested on a single DNA array chip using microfluidic operation. In addition, the probe immobilization time and sample hybridization time are shortened because the high surface-to-volume ratio achieved in the microchannels facilitates fast surface reaction rates. As enabled by gold nanoparticles, even single base-pair discrimination was achieved without using the temperature stringency method.
The NBA chip is also used for fast detection of antibodies present in biological fluids such as mouse ascites fluid. This capability has applications in diagnosing disease (e.g. influenza), and in monitoring efficacy of immunotherapy or vaccination. To begin designing and testing this system, the synthetic peptide (HA; a 12 amino-acid residue fragment from hemagglutinin A glycoprotein of the influenza virus) was used as a model antigen to be detected by the monoclonal antibody (MAb) produced from the murine 17/9 hybridoma cell line. Several MAb samples were tested by reacting each with multiple antigen probes. Multiple tests were conducted in a single run either on a single sample, or on multiple samples, with sensitivity comparable or better than ELISA, but with faster detection and higher throughput offered by the NBA chip.
