基于塑料薄片建立一步式心血管损伤标志物即时检测免疫分析系统
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(1.天津医科大学生理学与病理生理学系 天津医学表观遗传学协同创新中心,天津市 300070;2.首都医科大学附属 北京安贞医院 北京市心肺血管疾病研究所血管生物研究室 教育部重塑相关心血管疾病重点实验室 心血管重大疾病防治协同创新中心,北京市 100029;3.北京化工大学生物医用材料北京实验室,北京市 100029)

作者简介:

薛冰洁,硕士研究生,研究方向为生物学,E-mail为F14SH_0528@163.com。通信作者杜杰,教授,博士研究生导师,研究方向为心血管病因学及转化医学,E-mail为jiedu@yahoo.com。

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国家自然科学基金项目(81870341)


Establishment of an one-step point-of-care immunoassay system of cardiovascular biomarkers based on plastic sheets
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1.Department of Physiology and Pathophysiology of Tianjin Medical University & Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin 300070, China;2.Beijing Anzhen Hospital Affiliated to Capital Medical University & Department of Vascular Biology, Beijing Institute of Heart, Lung and Blood Vessel Disease & Key Laboratory of Remodeling-related Cardiovascular Diseases, Ministry of Education & Collaborative Innovation Center for Cardiovascular Disorders, Beijing 100029, China;3.Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029, China)

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    摘要:

    目的 建立一种简便快速的一步式心血管疾病标志物蛋白质即时检测(POCT)芯片系统。方法 利用光化学反应,在环烯烃共聚物薄片(COC)上合成具有抗非特异性吸附的聚乙二醇甲醚甲基丙烯酸酯(PEGMEMA)层,在接枝后的表面进行非接触式的喷墨打印,形成抗体微阵列。使用紫外光进行引发,探索了不同光照时间和不同聚合物单体浓度下改性COC薄片表面的接触角角度以及聚合物接枝密度的变化。利用原子力显微镜和红外光谱仪表征了光化学反应改性后COC薄片表面的微观形貌和化学结构,并对比了光化学反应改性前后薄片表面的非特异性蛋白吸附情况。进一步探索了合适的抗体喷墨打印浓度及辅剂条件,并在接枝后的表面打印了心血管损伤生物标志物生长刺激表达基因2蛋白(ST2)的固定型捕获抗体和荧光标记的可溶解型抗体。结果 当聚合物单体的质量分数为30%,光照时长为4 min时,接触角降到最低为38.5°,接枝密度达到最大为68.47 μg/cm2。原子力显微镜和红外光谱仪表征均证明了PEGMEMA层的成功引入。表面接枝PEGMEMA层降低了COC薄片的非特异性蛋白吸附量。当固定型捕获抗体为80 mg/L时,固定效率最高。与不同相对分子质量的聚乙二醇(PEG)相比,海藻糖作为辅剂时可溶解型检测抗体与固定型捕获抗体的识别效率更高。当ST2为10 mg/L时,可以观察到溶解后固定斑处有检测抗体结合。结论 通过光化学反应,在COC薄片表面接枝了抗非特异性吸附的PEGMEMA,在接枝后的薄片表面喷墨打印检测所需的抗体试剂,用简易快捷的方法制备形成了一种能够实现一步式检测的蛋白质芯片,可灵活应用于多种场景下心血管疾病标志物蛋白的快速检测,助力心血管精准医学发展。

    Abstract:

    Aim To establish a rapid and convenient cardiovascular biomarker protein one-step point-of-care testing (POCT) chip system. Methods A polyethylene glycol methyl ether methacrylate layer (PEGMEMA) with anti-nonspecific adsorption property was synthesized on the cyclic olefin copolymer(COC) sheet by photochemical reaction, and non-contact inkjet printing was performed on the modified surface to form antibody microarrays. The contact angles and grafting densities of the surface were explored with several monomer concentrations after irradiated under UV light for certain time periods. The modified surface structure was characterized by atomic force microscopy and infrared spectroscopy, and the protein adsorption on the surface before and after photochemical reaction was compared. The appropriate antibody inkjet printing concentration and adjuvant conditions were further explored. Finally immobilized antibodies and fluorescently labeled soluble antibodies of the growth-stimulated expression gene 2 protein (ST2), a biomarker of cardiovascular damage, were printed on the modified surface. Results When the PEGMEMA monomer concentration was 30% by mass and the illumination time was 4 min, the contact angle reached the minimum as 38.5°, and the grafting density was up to 68.47 μg/cm2. Atomic force microscopy and infrared spectroscopy showed that carbonyl and ether groups were successfully introduced into the surface. The amount of non-specific protein adsorption was reduced after surface grafting of PEGMEMA. The highest immobilization efficiency was obtained when the immobilized antibody concentration was 80 mg/L.Compared with polyethylene glycol (PEG) with several molecular weights, the effective binding efficiency of soluble antibody onto immobilized antibody microarray was higher when trehalose was used as an excipient. When the ST2 concentration was 10 mg/L, the binding of the soluble antibody on the immobilized antibody microarray after dissolution could be observed. Conclusion The anti-nonspecific adsorption of PEGMEMA was modified on the surface of the COC plastic sheet by photochemical reaction, and physically immobilized antibodies and the fluorescently labeled soluble antibodies were printed on the modified sheet surface, fabricated a protein chip system which is convenient to rapidly detect cardiovascular biomarkers, to advance cardiovascular precision medicine.

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薛冰洁,檀鑫,鄢健,王雪,赵长稳,王媛,杜杰.基于塑料薄片建立一步式心血管损伤标志物即时检测免疫分析系统[J].中国动脉硬化杂志,2022,30(5):395~402.

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  • 收稿日期:2022-01-13
  • 最后修改日期:2022-03-01
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  • 在线发布日期: 2022-05-10