Hepatocellular carcinoma (HCC) is the most common form of liver cancer, with cirrhosis being a major risk factor. Traditional blood markers like alpha-fetoprotein (AFP) demonstrate limited efficacy in distinguishing between HCC and cirrhosis, underscoring the need for more effective diagnostic methodologies. In this context, extracellular vesicles (EVs) have emerged as promising candidates; however, their practical diagnostic application is restricted by the current lack of label-free methods to accurately profile their molecular content. To address this gap, our study explores the potential of mid-infrared (mid-IR) spectroscopy, both alone and in combination with plasmonic nanostructures, to detect and characterize circulating EVs. EVs were extracted from HCC and cirrhotic patients. Mid-IR spectroscopy in the Attenuated Total Reflection (ATR) mode was utilized to identify potential signatures for patient classification, highlighting significant changes in the Amide I-II region (1475-1700 cm). This signature demonstrated diagnostic performance comparable to AFP and surpassed it when the two markers were combined. Further investigations utilized a plasmonic metasurface suitable for ultrasensitive spectroscopy within this spectral range. This device consists of two sets of parallel rod-shaped gold nanoantennas (NAs); the longer NAs produced an intense near-field amplification in the Amide I-II bands, while the shorter NAs were utilized to provide a sharp reflectivity edge at 1800–2200 cm for EV mass-sensing. A clinically relevant subpopulation of EVs was targeted by conjugating NAs with an antibody specific to Epithelial Cell Adhesion Molecule (EpCAM). This methodology enabled the detection of variations in the quantity of EpCAM-presenting EVs and revealed changes in the Amide I-II lineshape. The presented results can positively impact the development of novel laboratory methods for the label-free characterization of EVs, based on the combination between mid-IR spectroscopy and plasmonics. Additionally, data obtained by using HCC and cirrhotic subjects as a model system, suggest that this approach could be adapted for monitoring these conditions.

中文翻译：

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通过红外光谱和等离激元学的细胞外囊泡表征探索肝癌的新型循环生物标志物


肝细胞癌（HCC）是最常见的肝癌形式，肝硬化是主要危险因素。甲胎蛋白 (AFP) 等传统血液标记物在区分 HCC 和肝硬化方面的功效有限，这凸显了对更有效诊断方法的需求。在这种背景下，细胞外囊泡（EV）成为了有希望的候选者。然而，由于目前缺乏准确分析其分子内容的无标记方法，它们的实际诊断应用受到限制。为了解决这一差距，我们的研究探索了中红外（mid-IR）光谱单独或与等离子体纳米结构结合检测和表征循环电动汽车的潜力。 EVs是从HCC和肝硬化患者中提取的。利用衰减全反射 (ATR) 模式的中红外光谱来识别患者分类的潜在特征，突出显示酰胺 I-II 区域（1475-1700 cm）的显着变化。该特征显示出与 AFP 相当的诊断性能，并且当两种标记物组合时超过了 AFP。进一步的研究利用了适合该光谱范围内超灵敏光谱的等离子体超表面。该装置由两组平行的棒状金纳米天线（NA）组成；较长的 NA 在 Amide I-II 波段产生强烈的近场放大，而较短的 NA 用于在 1800–2200 cm 处提供尖锐的反射率边缘，用于 EV 质量传感。通过将 NA 与上皮细胞粘附分子 (EpCAM) 特异性抗体结合，靶向临床相关的 EV 亚群。 这种方法能够检测 EpCAM 呈现的 EV 数量的变化，并揭示 Amide I-II 线形的变化。所提出的结果可以对基于中红外光谱和等离激元学相结合的电动汽车无标记表征的新型实验室方法的开发产生积极影响。此外，通过使用 HCC 和肝硬化受试者作为模型系统获得的数据表明，这种方法可以适用于监测这些状况。