Quantitative Evaluation of Injected Molecules into Phospholipid-Coated Microdroplets for In situ Biological Reactions

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This paper presents a quantitative evaluation of the amount of biological molecules injected into phospholipid-coated microdroplets. Research on developing an artificial cell model using lipid membrane vesicles has been pursued to determine the function between biomembranes and biological molecules. The method is needed to introduce biological molecules into the biomembrane model and observe their reactions. Conventionally, molecules are introduced into vesicles by hydrating dried lipid films or freeze-dried lipid blocks with a solution already including biological molecules. It is difficult, however, to observe reaction in real time and step-by-step for different types of biological molecules, because the reaction has already started by the time vesicles are formed. Our proposal uses micro/nanopipettes based on micromanipulation. It is demonstrated that the injection of different types of biological molecules into a phospholipid-coated microdroplet. Biological molecules, such as F-actin, heavy meromyosin (HMM), and adenosine triphosphate (ATP), were introduced into a phospholipidcoated microdroplet in sequence, and these reactions were observed inside the microdroplet. The amount of molecules introduced into the microdroplet was evaluated quantitatively from the intensity of fluorescent labels through our microchannel calibration method.