Huanhuan Shi,Kaixuan Nie,Bo Dong,Lemeng Chao,Fengxiao Gao,Mingyang Ma,Mengqiu Long,Zhengchun Liu
The amide condensation reaction is crucial for preparing biological probes with a polypeptide backbone. The traditional condensation reaction requires the monomer and condensing agent to be pre-mixed and activated by physical agitation mixing, which entail long time, require many reagents, and yield several by-products. In microfluidic mixing devices, the high specific surface area of microchannel enables traditional chemical reactions to be accomplished efficiently and quickly using only trace amounts of reagents in real time. This paper presents a serpentine mixing channel for the activation of amino acid monomers. A number of outward convex elliptical structures are introduced to both sides of the improved serpentine microchannel, creating a Dean vortex that increases the mixing efficiency at low Reynolds condition (90% at Re = 40). Effective monomer activation can be realized directly using a micromixing reactor and leads to less by-products as verified by ultraviolet spectroscopy and high-performance liquid chromatography analysis. The practicability of the micromixer is successfully tested by utilizing it as a microreactor for the real-time activation of amino acids for the in situ synthesis of polypeptide array at a flow rate of 300 μL/min ( Re = 40). The proposed micromixer has the potential to be an excellent alternative for conventional flask reactions and integrated in automated miniaturized biochip preparation systems.
Weiwei Tang,Andrew Gordon,Hui-ying Wang,Ping Li,Jun Chen,Bin Li
The development of in situ methods for the analysis and visualization of enzyme activity is of paramount importance in drug discovery, research, and development. In this work, the functionalized and array patterned indium tin oxide (ITO) glass slides were fabricated by non-covalent immobilization of amphipathic phospholipid-tagged peptides encompassing the thrombin cleavage site on steric acid-modified ITO slides. The fabricated peptide arrays provide 60 spots per slide, and are compatible with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) measurement, free matrix peak interference, and tolerance to repeated aqueous washing. The peptide arrays were used for the investigation of thrombin activity and screening for its potential inhibitors. The thrombin activity and its Michaelis-Menten constant (K m ) for immobilized peptide substrate was determined using developed MALDI MS peptide array. To investigate the applicability and effectiveness of peptide arrays, the anti-thrombin activity of grape seed proanthocyanidins with different degrees of polymerization (DP) was monitored and visualized. MALDI MS imaging results showed that the fractions of proanthocyanidins with the mean DP of 4.61–6.82 had good thrombin inhibitory activity and their half-maximal inhibitory concentration (IC 50 ) were below 10 μg/mL. Therefore, the developed peptide array is a reliable platform for the discovery of natural thrombin inhibitors .
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