Berger Brett A, Brown Turner P, Im Wonpil, Wittenberg Nathan J
Department of Chemistry, Lehigh University, 6 East Packer Avenue, Bethlehem, Pennsylvania 18015, United States.
Department of Bioengineering, Lehigh University, 111 Research Drive, Bethlehem, Pennsylvania 18015, United States.
Anal Chem. 2025 Sep 23;97(37):20173-20182. doi: 10.1021/acs.analchem.5c02906. Epub 2025 Sep 9.
Reactive oxygen species (ROS) are responsible for the oxidative truncation of polyunsaturated fatty acids (PUFAs). The products of these reactions have been implicated in many diseases such as cancer and atherosclerosis. As increasing attention is directed toward these oxidized phospholipids (oxPLs), higher throughput methods are needed to examine interactions between oxPLs and scavenger receptors in the immune system. Supported lipid bilayers (SLBs) are a reliable model for lipid membrane-protein interaction studies due to their compositional tunability and ease of integration with a variety of sensing methods. Combining SLBs with microfluidic devices can boost experimental throughput. Herein, we develop a microfluidic gradient generator for creating SLBs that allows for simultaneous analysis of arrayed SLBs while maintaining distinct oxPL concentrations. First, we demonstrate the ability of the system to create linear arrays of SLBs that possess a persistent gradient of oxPL concentrations. The concentration-dependent binding of CD36 scavenger receptor-functionalized vesicles to SLBs containing gradients of the high-affinity CD36 ligand, KDdiA-PC, was observed. Thermal oxidation of the PUFA 1-palmitoyl-2-arachidonoyl--glycero-3-phosphocholine (PAPC) created a mixture of oxidized products to serve as potential CD36 ligands in the assay, with the primary oxidation product being 1-palmitoyl-2-glutaryl--glycero-3-phosphocholine (PGPC). Lastly, simulations showed that the oxidized tails of both KDdiA-PC and PGPC protrude from the membrane and are accessible to scavenger receptor binding. Interestingly, despite its shorter length, the oxidized tail of PGPC extends further from the membrane interface.
活性氧（ROS）会导致多不饱和脂肪酸（PUFA）发生氧化截断。这些反应的产物与许多疾病有关，如癌症和动脉粥样硬化。随着人们对这些氧化磷脂（oxPL）的关注度不断提高，需要更高通量的方法来研究oxPL与免疫系统中清道夫受体之间的相互作用。支持脂质双层（SLB）由于其组成的可调性以及易于与多种传感方法集成，是脂质膜 - 蛋白质相互作用研究的可靠模型。将SLB与微流控装置相结合可以提高实验通量。在此，我们开发了一种用于创建SLB的微流控梯度发生器，该发生器允许在保持不同oxPL浓度的同时对阵列化的SLB进行同步分析。首先，我们展示了该系统创建具有持续oxPL浓度梯度的SLB线性阵列的能力。观察到CD36清道夫受体功能化囊泡与含有高亲和力CD36配体KDdiA - PC梯度的SLB的浓度依赖性结合。多不饱和脂肪酸1 - 棕榈酰 - 2 - 花生四烯酰 - sn -甘油 - 3 -磷酸胆碱（PAPC）的热氧化产生了氧化产物混合物，用作测定中潜在的CD36配体，主要氧化产物为1 - 棕榈酰 - 2 - 戊二酰 - sn -甘油 - 3 -磷酸胆碱（PGPC）。最后，模拟表明KDdiA - PC和PGPC的氧化尾部都从膜中突出，并且可被清道夫受体结合。有趣的是，尽管PGPC的氧化尾部较短，但它从膜界面延伸得更远。
