The outstanding performance of silk, especially in terms of mechanical properties, can be attributed to the sophisticated silk gland and specialized spinning method that exists in nature. In addition, the gland, with decreasing diameter, has been proven to be an outstanding actuator of the sophisticated spinning system. [5,9] It has been qualitatively demonstrated that a region of contracting geometry of the S-shaped duct of silk gland can induce stretching of protein molecules, phase separation, and self-assembly due to shear and elongation flow. [10][11][12][13][14][15] During the subsequent spinning process in air, the protein ultimately aggregates into fibers with spectacular and distinctive properties. However, the mechanisms within the glands with exponential decaying diameter and their ability to dynamically change the conformation of the proteins is yet to be elucidated. [6] At present, various biomimetic spinning methods have been used to fabricate high-performance artificial silk and reveal the relationship between the spinning channel and silk performance. For example, the microfluidic device with miniaturized channels of designed configurations can easily control the physicochemical microenvironment, while using trace amounts of reagents. [6,10,[16][17][18][19][20] The sophisticated, compact, and highly functional microfluidic devices have been widely used to understand the geometry confinements, control overflow, and synthesis of artificially regenerated silk fibroin (RSF) fibers. [21][22][23][24][25][26][27][28][29][30] For example, Rammensee et al. prepared assembled spider dragline silk fibers through a microfluidic device. [31] Kinahan et al. adopted the same method to fabricate functional fibers with expected properties by controlling flow conditions. [32] Based on these studies, Zhang's group designed microfluidic chips mimicking the first-order and second-order exponential decay function of silk glands and prepared high-performance artificial silk fibers. [24][25][26]33,34] In our previous work, [35,36] statistics of spider's major ampullate (MA) gland of Nephila edulis were extracted from the work of Knight et al., [12] and then a microfluidic chip was prepared which fitted well to the second-order exponential decay function. The bioinspired chip possessed excellent similarity with the MA gland of spider, and was used to prepare RSF/cellulose nanofibers (CNF) hybrid fibers. However, there were some differences between the size of our microfluidic chip and the ampullate gland of spider. First, the actual diameter of lumen in the MA gland of N. edulis is from ≈250 to 8 µm. [12] However, high concentration of RSF/CNF suspensions (30 wt%) cannot flow through Microfluidic spinning has been used to mimic and discover the natural spinning process of silk. However, it is still challenging to understand the orientation and alignment of silk-spinning through microfluidic chips. Here, flow analysis is performed for a bioinspired microfluidic chip mimicking the shape of a spider's major ampullate gland a...