血栓剖析测定：基于微流控的平台，用于全面表征生物力学血栓形成
January 9th, 2026
这项工作描述了一种利用剪切应力触发血液血栓形成的微流控测定法，并通过多维读数表征血栓。该检测方法可以测量血液样本的促血栓倾向，因此在疾病诊断、药物发现以及血栓形成相关的基础机制研究中非常有用。
我们开发了一种新方法，能够充分捕捉血栓的生物力学特性，并检测人体的促血栓异常。首先，使用硅晶圆根据设计通过标准光刻制作SU8光刻胶主模具，并用胶带将主模具固定在15厘米培养皿底部。通过将硅胶弹性体套件中的预聚合物基底和固化剂混合，以10比1的重量比制备聚二甲基硅氧烷（PDMS）混合物。
将PDMS混合物倒入主模上。然后将装有PDMS混合物的板放入真空干燥器中脱气。通过将PDMS混合物放入设定在75摄氏度的培养箱中，进行两小时的热固化。
从培养箱取出样品后，仔细从主模具中切割固化后的PDMS，并将固化后的PDMS切入单个芯片单元。用探针在指定位置打孔，形成进出口。在化学罩中，使用75%乙醇湿工作湿巾清洁玻璃玻片并使其干燥。
接着，使用高频发生器，处理玻璃玻片30秒，PDMS芯片处理20秒。将每个芯片与玻片对齐，轻轻将芯片压在玻璃滑片表面以固定。然后将设备放入设定为150摄氏度的烤箱中加热15分钟，进行热结合。
粘结后，将设备存放在室温且无尘的环境中。然后，用钳子去除探针的塑料部分，将金属管弯曲至90度，形成微流控器件的连接器。在用Alexa Fluor设置好结合纤维蛋白原和所需抗体的反应后，将纯化塔在1,100克离心两分钟以去除储存缓冲液。
丢弃流动后，将反应溶液装入安装在兼容收集瓶中的纯化塔上，并以1,100克离心机加热五分钟，以收集所需的混合物。使用分光光度计测量蛋白质的吸光度和染料信号。计算蛋白质浓度和荧光与蛋白质摩尔比。
在黑暗中储存在摄氏4度。将肝素加入泰罗德缓冲液，最终浓度为每毫升0.32单位，每10毫升血液收集，然后在pH 7.4下抽取500微升泰罗德缓冲液，准备注射器。通过含肝素注射器静脉穿刺采集血液后，将血液转移到15毫升离心管中，并保持在37摄氏度。
将冯·维勒布兰德因子单体稀释至PBS中每毫升两微克。在微流控器件上涂上稀释的冯·维勒布兰因子单体，并在室温下培养一小时。现在用荧光传感器1号或2号在室温下孵育10分钟。
接着，将微流控装置与进出口连接器及管路连接。将入口连接器的另一端连接到含有PBS的管道上，然后将出口连接器的另一端连接到注射器。将注射器安装在注射器泵上，微流控装置安装在倒置显微镜上。
手动操作显微镜台以定位狭窄部位。使用注射器泵以每分钟0.5毫升的流量注入PBS液体，注入微流控通道和管道。检查设备，确保狭窄部位周围没有气泡。
将进气管连接到血样，并用注射器泵以每分钟0.018毫升的流量通过微流控通道灌注血样。在倒置显微镜中，在软件中为每个荧光通道设置曝光时间和增益。通过在四个通道间交替进行实时多色荧光成像，并一次激发一个氟-4。
调整对焦平面对应血栓，并在狭窄部位记录荧光信号15到30分钟。数据分析时，使用ImageJ 1.53版本打开数据文件。使用SZ 22 Fit C通道来识别血栓轮廓。
然后利用多边形选择，大致选择血栓区域。对于每个通道，通过选择图片、调整，然后选择阈值来消除背景信号。最后，选择“分析”并选择测量，测量血栓内的区域和平均信号强度。
代表性快照显示，在健康血液狭窄通道内形成血栓，使用传感器组1可视化血小板、纤维蛋白原、冯·威尔布兰因子和P-选择蛋白，以及用传感器组2观察的血小板、磷脂酰丝氨酸、E阳性整合蛋白α IIb β 3（激活整合素α IIb β 3）。通过选择血栓区域、去除背景信号，测量信号区域和平均亮度，进行单一荧光通道图像处理，以便进行定量分析。对荧光信号强度随时间的持续分析，生成了血小板形成过程中血小板积累的信号-时间曲线。
对于每个时间点，传感器组一中四个生物标志物和传感器组二中四个生物标志物的总信号强度均被获取。通过计算血栓大小并生成了七维血栓谱。目前尚无生物测定方法评估人类患者的促血栓倾向，我们的工作试图填补这一空白。
通过检测血液样本的生物力学活性，我们的检测方法能够全面评估受试者的促血栓特性。我们的检测方法可开发为检测患者促血栓倾向的临床测试，也可用于筛选下一代抗血栓药物。
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Overview
This article presents a novel thrombus profiling assay that integrates microfluidics with multi-color fluorescence imaging to comprehensively characterize biomechanical thrombogenesis. The method enables detailed analysis of thrombus formation under arterial shear conditions, providing seven distinct readouts related to thrombus size, composition, and platelet activation. This assay addresses the need for a robust bioassay to evaluate prothrombotic tendencies in humans and assess the efficacy of anti-thrombotic agents.
Key Study Components
Area of Science
- Hematology
- Bioengineering
- Microfluidics
- Thrombosis research
Background
- Thrombosis involves abnormal accumulation of platelets and clotting factors in blood vessels.
- Arterial thrombosis is often linked to elevated shear stress due to arterial stenosis.
- Biomechanical factors, such as blood flow, play a critical role in thrombus formation.
- Existing assays have not fully captured the biomechanical aspects of thrombogenesis.
Purpose of Study
- To develop an assay that profiles biomechanical thrombogenesis in detail.
- To enable comprehensive evaluation of prothrombotic tendencies in human blood samples.
- To provide a platform for testing anti-thrombotic agents and understanding arterial thrombosis mechanisms.
Methods Used
- Fabrication of microfluidic devices using PDMS and photolithography.
- Surface coating of microchannels with von Willebrand factor.
- Perfusion of heparinized human blood labeled with fluorescent biomarkers through stenotic microchannels.
- Real-time multi-color fluorescence imaging to monitor thrombus formation at the site of stenosis.
- Quantitative image analysis using ImageJ to measure thrombus area, biomarker signal intensity, and generate seven-dimensional thrombus profiles.
Main Results
- The assay successfully visualized and quantified thrombus formation under biomechanical (shear) conditions.
- Seven key parameters, including thrombus size and multiple biomarker signals, were measured simultaneously.
- Distinct profiles were generated for platelet accumulation, fibrinogen, von Willebrand factor, P-selectin, phosphatidylserine, and integrin activation.
- The method enabled continuous monitoring and quantitative analysis of thrombus development over time.
Conclusions
- This thrombus profiling assay fills a critical gap in evaluating biomechanical thrombogenesis.
- It offers a comprehensive tool for assessing prothrombotic risk and screening anti-thrombotic drugs.
- The approach can be further developed for clinical diagnostics and mechanistic studies of arterial thrombosis.
Frequently Asked Questions
Executive Industry Relevance
Biomechanical thrombogenesis presents a critical challenge in cardiovascular drug discovery, as traditional assays often fail to capture the complexity of thrombus formation under arterial shear conditions. The microfluidics-based thrombus profiling assay enables comprehensive, quantitative characterization of thrombus size, composition, and platelet activation, directly supporting predictive confidence in early-stage anti-thrombotic agent evaluation. This platform advances portfolio decision-making by providing mechanistic insights and robust risk assessment for candidate therapies targeting arterial thrombosis.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of biomechanical pathways driving arterial thrombosis.
- Supports functional validation of targets involved in platelet activation and aggregation.
- Facilitates mechanistic de-risking by quantifying multiple thrombus biomarkers simultaneously.
- Improves predictive confidence for triaging anti-thrombotic candidates.
Screening & Assay Development
- Prepares validated microfluidic systems for high-content screening of anti-thrombotic compounds.
- Delivers standardized, reproducible, and quantitative multi-parametric readouts.
- Enables scalable, platform-based evaluation of compound efficacy under physiologically relevant shear conditions.
- Supports reliable comparison of compound effects on thrombus formation and biomarker expression.
Translational & Preclinical Research
- Aligns in vitro thrombus formation with disease-relevant arterial shear profiles.
- Provides translational biomarker data for continuity from discovery to preclinical validation.
- Enables risk-adjusted advancement decisions based on comprehensive thrombus profiling.
- Supports mechanistic studies of arterial thrombosis relevant to human pathology.
Pipeline & Workflow Integration
This assay integrates into the discovery-to-preclinical continuum, bridging early mechanistic studies with translational biomarker validation for anti-thrombotic drug development.
- Discovery Biology: Quantifies biomechanical triggers and pathways of thrombogenesis for hypothesis testing and biological de-risking.
- Screening: Provides reproducible, quantitative outputs for compound evaluation in a physiologically relevant context.
- Analytics: Generates seven-dimensional thrombus profiles, enabling robust statistical comparison across conditions and compounds.
- Translational Research: Aligns in vitro findings with disease-relevant arterial thrombosis mechanisms for preclinical continuity.
- Enterprise Reuse: Establishes a reusable microfluidic and imaging platform adaptable to diverse anti-thrombotic R&D programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in anti-thrombotic target validation.
- Operational Value: Standardizes and scales multi-parametric thrombus analysis for cross-program comparability.
- Strategic Value: Enables informed go/no-go decisions and capital-efficient portfolio advancement.
- Portfolio Impact: Supports risk-adjusted prioritization of anti-thrombotic candidates based on comprehensive mechanistic data.
Implementation Considerations
- Requires expertise in microfluidics, fluorescence imaging, and quantitative image analysis.
- Demands access to specialized instrumentation, including microfabrication and multi-channel fluorescence microscopy.
- Necessitates cross-team standardization of assay protocols and data analysis workflows.
- May require adaptation for different blood sources or disease models to ensure translational relevance.
- Dependent on robust statistical analysis to interpret multi-dimensional thrombus profiles.
