Microfluidic High-Throughput Bioanalysis: From Single Cell To Complex Biological Microenvironments

In the postegenomic era with the explosive development of bioinformatics, it is emerging to develop high-throughput analytical techniques. Such high-throughput analytical approaches will facilitate the researches in various area including genomics, proteomics, drug development and stem cell differentiation. However, the challenge remains in obtaining bioinformations from both the single cells and complex cell microsystems in a high-throughput way.The conventional high-throughput analysis is mainly based on flow cytometry, plate reader and liquid dispenser. However, those techniques cannot obtain dynamic informations from individual cells and fails to recapitulate the complex three dimensional cell microenvironments. Recently, the microfluidic chip has received widespread attention, due to adavantages of precise control, high-throughtput and its similar size to cells. And for various applications in biological analysis, microfluidic device is highly desired. In this thesis, for high-throughput analysis of single cells and complex cell microenvironments, we designed three kinds of unique microfluidic chips based on agarose hydrogel, wettability-patterned substrate and PDMS microchannel respectively.Firstly, we demonstrated an ultra-high throughput approach with microfluidic chip to simultaneously interrogate DNA damage conditions of up to ten thousand individual cells (approximately 100 folds in throughput over conventional method) with better reproducibility. Agarose was chosen as the chip fabrication material, which would further act as electrophoretic sieving matrix for DNA fragments separation. Cancer cells (HeLa or HepG2) were lined up in parallel microchannels by capillary effect to form a dense array of single cells. By electrophoresis, the damage conditions of individual cells could be quantified. DNA repair capacity was further evaluated to validate the reliability of this method.Secondly, a multifunctional gradients-customizing microfluidic device was developed for high-throughput single-cell multidrug resistance (MDR) analysis. The gradient profile was determined by the lengths of the distribution microchannels regardless of flow rates and pressure, which provided good stability and remarkably reduced redundancy of microfluidic architecture. The drug of gradient concentrations consecutively stimulate upon the cells in the downstreaming cell cultivation chamber. Time-dependent drug efflux kinetics of HepG2 cells were firstly investigated on our device using both the different-single-cell and the same-single-cell strategies. Furthermore, hepatic polarized HepG2 cells, which collected the secreted cholephilic substances in the apical vacuoles, were used as model to investigate the inhibition of MDR-associated protein with secretion inhibitor cyclosporine A of varied concentrations on single organelle level. Finally, a high-throughput drug screening experiment was conducted to examine both the chemo-sensitizing effect and the cytotoxity of the potential chemo-sensitizing agents.Thirdly, a low-cost, rapid, and portable agarose-based microfluidic device was developed to concentrate biological fluid from micro-to pico-liter volume. Pathogens could be concentrated using this device due to the capillary effect and the strong water permeability of the agarose gel. Results showed that 90% recovery efficiency could be achieved with a million-fold volume reduction from 400?L to 400 pL. For concentration of 1×103 cells mL-1 bacteria, approximately ten million-fold enrichment in cell density was realized with volume reduction from 100 ?L to 1.6 pL. Urine and blood plasma samples were further tested to validate the developed method. In conjugation with fluorescence immunoassay, we successfully applied the method to the concentration and detection of infectious Staphylococcus aureus in clinics.Fourthly, we propose a three-dimensional (3D) microfluidic strategy for generating controllable cell gradients. In this approach, a homogenous cell suspension is loaded into a 3D stair-shaped PDMS microchannel to generate a cell gradient within 10 min by sedimentation. We demonstrate that cell gradients of various profiles (exponential and piecewise linear) can be achieved by precisely controlling the height of each layer during the fabrication. With sequential seeding, we further demonstrate generation of two overlapping cell gradients on the same glass substrate with pre-defined designs. Cell gradient based QDs cytotoxicity assay was also demonstrated showing cell behaviors and resistances were regulated by the chances in cell density.Fifthly, we describe a method to rapidly bioengineer thousands of heterogeneous cell niches in 3D microgel arrays with quantitative controls over physical, biochemical cues and cell populations by using surface-wettability guided assembly. Assembly of cell niches on the wettability patterned surface is demonstrated with chemical gradient, cell density gradient and orthogonal gradients of chemical concentration and cell density over individual microgels. Additionally, we show spatial organization of heterogeneous cell populations in individual microgels and assembly of a hydrogel sheet with spatially-defined chemical distribution. This platform technology potentially facilitates wide applications for tissue engineering, stem cell differentiation and drug discovery.Finally, we introduce the concept of "microearth-on-a-chip", where a wettability guided microdevice reconstitutes the biogeochemical process of soil-microbe complex including soil formation, biophysical and biochemical process and changes of carbon/nitrogen. In soil assembly process, we find most of the soil organic matters (SOM) was deposited on the brim and corners based on the coffee ring effect. By in situ quantitative analysis of the soil using fluorescence imaging and XPS technique, we investigate complex interaction between bacteria and soils on the artificial biogeochemistry interface. Artificial "microearth-on-a-chip" microdevices that precisely assemble soil microenvironment critical to biogeochemical process may therefore revolutionize modeling and investigation of environment issues, geoscience and soil microbiology.In summay, we developed a noval agarose-based microfluidic device enabling automatic assembly of single cell array for high-throughput single cell genomic damage analysis, which was further used for pathogen concentration and detection. Then, we proposed a surface-wettability based microchip for high-throughput assembly of heterogeneous three-dimensional cell microenvironment arrays with precise control, which was further used for rapid reconstitute and analysis of soil-microbe microenvironments. Finally we demonstrated PDMS microchannel guided microfluidic device, which can precisely generate chemical/cell gradients, providing new strategy for high-throughput single-cell analysis of multidrug resistance and nanocytotoxicity. Conclusively, these devices would be widely used in biological high-throughput analysis.