103123-LNC-2D-Me3__PePr_peaks.bedLossless Altered Histone Modification Analysis System (LAHMAS)

Miniaturized biological assays using microfluidics have the potential to enhance assay sensitivity, reduce reagent consumption, and increase throughput. However, challenges to miniaturization include increased platform complexity and increased surface to volume ratio leading to risk of evaporation and analyte loss through surface binding. Exclusive Liquid Repellency (ELR) enables open microfluidic systems that minimize these challenges through an oil phase that protects small aqueous volumes from temperature fluctuation and evaporation while eliminating surface fouling that lead to sample loss. Here we report a novel microfluidic platform leveraging ELR and Exclusion-based Sample Preparation (ESP) for the miniaturization of CUT&Tag, a complex multistep biological assay. The resultant Lossless Altered Histone Modification Analysis System (LAHMAS) employs a PDMS-silane treated glass surface immersed in silicone oil to facilitate lossless liquid handling and prevent sample evaporation. The device design, compatible with standard laboratory equipment, allows for effective processing of cell inputs as low as 100 cells with higher specificity than macroscale CUT&Tag facilitating accurate chromatin profiling of low input and rare cell samples.采用微流控技术的微型化生物测定法，有望提升测定灵敏度、降低试剂消耗量并提高通量。然而，微型化过程面临诸多挑战：平台复杂度提升，且表面积体积比增大，易引发蒸发风险以及因表面结合导致的分析物损失。排他性液体排斥技术（Exclusive Liquid Repellency, ELR）可构建开放式微流控系统，其通过油相保护小型水相体积免受温度波动与蒸发影响，同时消除引发样品损失的表面污染，从而最大限度缓解上述挑战。本研究报道一种新型微流控平台，该平台结合ELR技术与基于排斥作用的样品制备（Exclusion-based Sample Preparation, ESP）技术，用于复杂多步骤生物测定法CUT&Tag的微型化。由此构建的无损组蛋白修饰变异分析系统（Lossless Altered Histone Modification Analysis System, LAHMAS），采用经聚二甲基硅氧烷-硅烷（PDMS-silane）处理的玻璃表面，并将其浸入硅油中，以实现无损液体操作并防止样品蒸发。该设备设计兼容标准实验室仪器，可有效处理低至100个细胞的输入样本，且其特异性优于宏观尺度CUT&Tag技术，能够实现低输入量与稀有细胞样本的精准染色质图谱分析。