A Novel Approach For Realizing Concentration Gradients On Microfluidic Chip And Its Applications

Organisms can sense a variety of chemical signals surrounding them, and changetheir physiological activities in accordance with the signals’ concentration gradient.Traditional methods for investigating the responses of organisms to the externalenvironment are complicated, time-consuming, and difficult to realize quantitativeanalysis. Microfluidic chip technology allows different operations on a single chip,showing advantages such as miniaturization, integration and automation. Therefore, it isof great significance to use microfluidic-based method to simulate external environment,and establish chemical concentration gradient for studying the responses of organisms tothe external environment at the single-cell level.In this paper, a new concentration gradient microfluidic chip is developed. Differentfrom preveiously reported "Christmas tree" structure, the new microfluidic chip make useof stepped microchannel network to dilute the sample solution, and to generateconcentration gradients.We first used numerical simulations to optimize the design of the microfluidic chipfor generating chemical concentration gradient by serial dilution. The influence ofdifferent microchannel structures on the concentration gradient was investigated. Resultsshowed that the best concentration gradient generating structure was the ladder networkwith the right bifurcation in the lower main channel and the microchannel width of100μm at quantity of flow of0.5μL/min in inlets.In order to verify the results of mathematical modeling, we loaded water andfluorescein into the chip, and studied the generation of concentration gradient byfluorescence imaging. Results showed that water and fluorescein were mixed completely,and a linear concentration gradient was generated at the outlet of the six microchannels. Inthe main channel of the outlet, the six-way concentrations of the solution are broughttogether. The solutions are layered, and last a long distance for keeping stableconcentration gradients apparently. These results were consistent to those obtained fromnumerical simulations.Subsequently, we applied the microfluidic chip to cell analysis. HeLa cells,expressing CD2probes for detecting apoptosis, were loaded into the main channel throughthe outlet of the chip, and attached to the bottom of the microchannel for cell culture. Astable concentration gradient of the anti-cancer drug, cisplatin was established in the main channel. Thus adherent cells could be treated with different concentrations of cisplatinsimultaneously. Fluorescence resonance energy transfer imaging system was used tomonitor the cisplatin-induced apoptosis in real time. Experimental results showed thatcisplatin could induce the apoptosis of HeLa cells, and the apoptosis rate was positivelycorrelated with the cisplatin concentrations. The protease caspase-2mediated theapoptosis induced by cisplatin. All these results suggested that the microfluidicconcentration gradient chip could be used for studying apoptosis.Finally, we used the microfluidic chip to establish concentration gradient forculturing Caenorhabditis elegans (C. elegans), which was difficult to achieve throughtraditional methods. The chemotaxis of C. elegans to NaCl was investigated. We loaded C.elegans to the main channel from the outlet of the microchannel, and the NaClconcentration gradient was generated simultaneously. The distribution of C. elegans in themain channel was investigated to quantify the chemotaxis C. elegans to NaCl. Resultsshowed that the wild-type C. elegans responded to NaCl with a concentration more than20mM. C. elegans showed no chemotaxis to50-100mM NaCl, and hated NaClconcentration higher than300mM. In addition, we have implemented directly the C.elegans with Nacl and with hungry on the same chip. Therefore, the learning training andthe chemotaxis analysis after training were conducted on the same chip. Results showedthat C. elegans could change its chemotaxis to low concentration of NaCl through leaning.Thus, the microfluidic chip provided a useful platform for investigating the chemotaxisand learning behaviors of C. elegans with reduced analysis time and enhanced analyticalefficiency.Above experimental results showed that the novel, simple and efficient microfluidicmethod was able to generate controllable chemical concentration gradients, and it could beapplied to study different model organisms. Except forming concentration gradient, thischip can also form a temperature gradient, which provides a new way for the future studyof reaction of cells and multicellular organisms in different temperature. This new methodwould have potential applications in drug screening, biological examination andenvironmental testing.