Construction And Application Of Microfluidic Concentration Gradient Chip

Chemical concentration gradient plays an important role in life activities,and in vitro simulation of concentration gradient is of great scientific significance for biomedical research.Generation of concentration gradient is useful method to optimize the experimental conditions for scientific research,and it is often used for drug screening,immunoassay analysis,cell stimulation and so on.Due to the advantages of automation,miniaturization,high throughput,ease of adjustment and precise control,microfluidic chips have been widely used for researches involving concentration gradients.However,current studies are limited to the formation of a single concentration gradient of one or multiple substances.In addition,some concentration gradient chips require complex external devices,and many devices suffer from hydrodynamic shear stress that is harmful to cell culture.To overcome these problems,three microfluidic chips of different designs were developed for the construction and application of concentration gradients.(1)For the generation and application of dual concentration gradients,a novel slipchip-based microfluidic system was designed.The slipchip platform consists of two plates of PDMS/glass chips,which are fixed and contacted with each other by using a homebuilt PMMA clip.After loading without interference,samples and reagents could form two separate concentration gradients simultaneously.After one-step sliding,samples and reagents with various concentrations could be mixed for multiplexed reactions.After numerical simulations and experiments validated the developed microfluidic system with food dyes and fluorescein,studies of the reaction conditions involving?-Galactosidase(?-Gal)were demonstrated.These experiments confirmed the reliability of the developed system.The slipchip system has shown advantages of ease of operation and low sample and reagent consumption,allowing rapid establishment of two orthogonal concentration gradients for multiplexed reactions.(2)For the generation and application of dual concentration gradients,a rotatable slipchip-based microfluidic system was proposed.The microfluidic chip consists of two plates of PDMS/glass chips,which were fixed and contacted with each other by a screw.Two reagents could form multi-level concentrations through a simple introduction in each chip,and be contacted and mixed by one-step rotation.As a result,the reaction microarray with orthogonal concentration gradient could be established.We evaluated the system with food dyes,fluorescein and sulforhodamine B.Studies of the conditions for the reaction of?-Gal were further demonstrated.The experimental results showed that the system could be used for various biochemical researches,especially for complex reactions with multi-factors and multi-levels.(3)For the generation and application of single concentration gradients,a shear-free and pump-free microfluidic system was developed.In this chip,no external pump was needed for the formation of concentration gradients.Solutions could flow into the channels under the static stress,and further be merged to generate gradients.By adjusting the ratio of the lengths of the distribution channels,arbitrary gradient profiles could be formed.Ultra-low flow rates ensured the shear-free environment,which provided the cell friendly microenvironment.To further validate this device,five days culturing of NIH-3T3 cells was conducted.The microfluidic gradient generator was further used for the adaptive cytoprotection analysis.The results indicated that the pump-free microfluidic gradient chip generated arbitrary gradients profile with high stability and ultralow shear stress,which provided a new strategy for long-term cell culture and could be further used for the application to adaptive cytoprotection analysis.In summary,three different concentration gradient generators based on microfluidic chip were reported.These chips could form complex dual concentration gradients and a shear-free concentration gradient,which had been used for enzyme reaction and cell research.These works provided a new strategy for concentration gradient research and could be further used for the application to multiple complex reactions and cytological studies.