关键词: Digital microfluidics EGFR amplification Fluorescence in situ hybridization

Mesh : Humans Carcinoma, Non-Small-Cell Lung / pathology Lung Neoplasms / pathology Gene Amplification ErbB Receptors / genetics metabolism In Situ Hybridization, Fluorescence / methods Microfluidics Gene Dosage Mutation

来  源:   DOI:10.1016/j.talanta.2023.125444

Abstract:
Signal transduction mediated by epidermal growth factor receptor (EGFR) gene affects the proliferation, invasion, metastasis, and angiogenesis of tumor cells. In particular, non-small cell lung cancer (NSCLC) patients with increased in copy number of EGFR gene are often sensitive to tyrosine kinase inhibitors. Despite being the standard for detecting EGFR amplification in the clinic, fluorescence in situ hybridization (FISH) traditionally involves repetitive and complex benchtop procedures that are not only time consuming but also require well-trained personnel. To address these limitations, we develop a digital microfluidics-based FISH platform (DMF-FISH) that automatically implements FISH operations. This system mainly consists of a DMF chip for reagent operation, a heating array for temperature control and a signal processing system. With the capability of automatic droplet handling and efficient temperature control, DMF-FISH performs cell digestion, gradient elution, hybridization and DAPI staining without manual intervention. In addition to operational feasibility, DMF-FISH yields comparable performance with the benchtop FISH protocol but reducing the consumption of DNA probe by 87 % when tested with cell lines and clinical samples. These results highlight unique advantages of the fully automated DMF-FISH system and thus suggest its great potential for clinical diagnosis and personalized therapy of NSCLC.
摘要:
表皮生长因子受体(EGFR)基因介导的信号转导影响细胞增殖,入侵,转移,和肿瘤细胞的血管生成。特别是,EGFR基因拷贝数增加的非小细胞肺癌(NSCLC)患者通常对酪氨酸激酶抑制剂敏感.尽管是临床上检测EGFR扩增的标准,荧光原位杂交(FISH)传统上涉及重复和复杂的台式程序,这不仅耗时,而且需要训练有素的人员。为了解决这些限制,我们开发了一个基于数字微流体的FISH平台(DMF-FISH),自动实现FISH操作。本系统主要由DMF芯片进行试剂操作,用于温度控制的加热阵列和信号处理系统。具有自动液滴处理和高效温度控制的能力,DMF-FISH执行细胞消化,梯度洗脱,无需人工干预的杂交和DAPI染色。除了操作的可行性,DMF-FISH产生与基准FISH方案相当的性能,但是当用细胞系和临床样品测试时,将DNA探针的消耗减少了87%。这些结果突出了全自动DMF-FISH系统的独特优势,因此表明其在NSCLC的临床诊断和个性化治疗中的巨大潜力。
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