Various concentration gradient generation methods based on microfluidic systems are summarized in this paper. The review covers typical structural characteristics, gradient generation mechanisms, theoretical calculation formulas, applicable scopes, and advantages and disadvantages of these approaches in detail. According to the type of reagents involved, these methods are classified into mono-phase methods and multi-phase methods, both of which can be implemented by alternative protocols, while the latter methods particularly refer to droplet-based platforms. For mono-phase methods, the shearing effect would be presented if there are flowing streams in the gradient generation channel. Therefore, the generation speed of channels with moving liquids is relatively fast, which is suitable for dynamic gradients but accompanied by shearing as well, while channels without flowing streams would avoid shearing but are prone to static gradient generation determined by the low speed. Newly developed droplet-based generation systems could provide isolated droplets to avoid the disturbances from the outside continuous phase, however, they require precise droplet generation and control modules. Thereby the most suitable platform can be chosen according to the specific application, while the advantages of different methods could be combined to evade the defects and improve the precision of a single structure.
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