Dataset_EV layerparameter

Extracellular vesicles (EVs) have been recognized as nanocarriers of disease biomarkers. Large EVs (lEV) and small EVs (sEV) are distinct EVs with different size ranges, biogenesis pathways, and biomarker cargoes. Current methods for EV size measurement, including nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), have limitations in cost, time, and sample handling. This study introduces a novel approach using a shear horizontal surface acoustic wave (SH-SAW) biosensor to estimate the average EV size and the abundance of lEV, independent of the EV concentration.  As the fractional changes in attenuation and velocity for SAW have different scalings with respect to the EV size but the same with respect to concentration, a layer parameter (lp) corresponding to the ratio of amplitude attenuation-to-velocity fractional change (V0ΔA/A0ΔV), is used to remove the EV concentration effect and isolate the dependence on the average size. Uniformly sized Au nanoparticle suspensions are used to establish the linear scaling of lp with respect to particle size, such that lp can estimate the average nanoparticle size within 5 nm. For EV suspensions with complex size distributions, an EV size resolution of about 10 nm is estimated from lp measurements. Separate pulldowns with sEV tetraspanin marker CD9 and generic sEV/lEV marker GPC1 show significant differences in lp for adipose-derived stem cells (ADSCs) and Wharton’s jelly mesenchymal stem cells (WJMSCs), consistent with their distinctly different unimodal and bimodal size distributions from NTA estimates. WJMSC EVs are hence predicted to overexpress lEV. A key to our protocol is to avoid tangential flow filtration, which introduces cell culture-dependent viscosity change that affects SAW characterization.  These results suggest that the upregulation of lEV can be detected with our SH-SAW approach, independent of the EV concentration.

细胞外囊泡（Extracellular Vesicles, EVs）已被证实为疾病生物标志物的纳米载体。大尺寸细胞外囊泡（lEV）与小尺寸细胞外囊泡（sEV）是两类具有不同尺寸范围、生物发生途径及生物标志物载荷的细胞外囊泡。当前用于EV尺寸检测的方法，包括纳米颗粒追踪分析（NTA）与透射电子显微镜（TEM），在成本、耗时及样本处理方面均存在局限性。本研究提出一种全新方案：利用剪切水平表面声波（SH-SAW）生物传感器，无需依赖EV浓度即可估算EV平均尺寸与lEV丰度。
由于表面声波（SAW）的衰减与速度分数变化，其与EV尺寸的缩放关系存在差异，但与EV浓度的缩放关系一致，因此本研究采用层参数（lp）——即振幅衰减与速度分数变化的比值（V0ΔA/A0ΔV）——以消除EV浓度的影响，分离出与平均尺寸相关的依赖关系。
研究使用尺寸均一的金纳米颗粒悬浮液，建立了lp与颗粒尺寸的线性缩放关系，据此可将纳米颗粒平均尺寸的估算误差控制在5 nm以内。对于具有复杂尺寸分布的EV悬浮液，通过lp测量可实现约10 nm的EV尺寸分辨率。分别针对sEV四跨膜蛋白标记物CD9与通用型sEV/lEV标记物GPC1开展亲和下拉实验，结果显示脂肪来源干细胞（ADSCs）与沃顿胶间充质干细胞（WJMSCs）的lp值存在显著差异，这与二者经NTA估算得到的单峰与双峰尺寸分布差异相符，据此可预测WJMSC EVs中lEV的表达水平更高。
本实验方案的核心在于规避切向流过滤操作——该操作会引入依赖于细胞培养体系的黏度变化，进而干扰SAW检测结果。上述结果表明，本SH-SAW方法可在无需考虑EV浓度的前提下，实现对lEV表达上调的检测。