CN104076064B - 用碳纳米管微悬臂梁生物传感器检测浓度范围为0.5-10微克/毫升的凝血酶的方法 - Google Patents
用碳纳米管微悬臂梁生物传感器检测浓度范围为0.5-10微克/毫升的凝血酶的方法 Download PDFInfo
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Abstract
用碳纳米管微悬臂梁生物传感器检测浓度范围为0.5‑10µg/mL的凝血酶的方法,通过构建一种碳纳米管微悬臂梁生物传感器来实现。该生物传感器包括支架、基底材料、碳纳米管、拾取电路,在碳纳米管上面通过π‑π叠加作用修饰有一层核酸适配体。先在碳纳米管微悬臂梁上制作含有凝血酶核酸适配体的检测探针,检测时,将检测探针放入待测样本中,待测样本中凝血酶通过特异性反应与检测探针上的核酸适配体形成复合物并附着在微悬臂梁上;利用该复合物在微悬臂上产生的质量变化引起微悬臂梁挠曲位移或谐振频率的变化关系和该复合物的质量大小与待测样本中凝血酶的浓度呈正相关,从而实现对凝血酶的检测。
Description
技术领域
本发明涉及生物医学工程领域,尤其涉及一种用微悬臂梁生物传感器检测凝血酶的方法。
技术背景
凝血酶是一种多功能丝氨酸蛋白酶,在体内一系列生理和病理过程中起重要作用。它不但参与止血和凝血、炎症、免疫反应、组织修复和创伤愈合,而且可激活正常细胞的致瘤潜能和导致恶性细胞的转移表型。此外,凝血酶还是肿瘤细胞的促有丝分裂剂,能够增强肿瘤细胞对细胞因子的增殖反应,增强肿瘤细胞对血小板的黏附及体外细胞基质的侵袭,促进肿瘤血管形成和肿瘤微环境的组织重建。因此,开展凝血酶的检测,具有十分重要的临床意义。目前凝血酶检测方法主要有有发色底物法和荧光法等。其中发色底物法虽然简单易行, 但灵敏度不高, 不宜进行微量分析。而荧光法则操作步骤繁琐, 荧光背景不易降低, 灵敏度低。难以实现早期疾病的诊断与研究,且无法满足快速检测的需求。“nanomechanical microcantilever oprerated in vibratin modes with use of RNAaptamer as a receptoe molecules for label-free detection of HCV helicase”,KyoSeon Hwang et al.,Biosensors and Bioelectrontics,第23卷第459-465页,20070602,公开了一种用核酸适配体作为受体分子通过微悬臂梁的振动模式来检测HCV解旋酶的方法。发明专利申请CN101935008A公开了一种利用功能化碳纳米管为敏感材料的微悬臂梁传感器的方法。目前需要建立一种凝血酶浓度为0.5-10µg/mL的快速、灵敏、操作简便的检测方法。
发明内容
本发明所要解决的技术问题是提供一种快速准确,凝血酶浓度检测范围为0.5-10µg/mL的凝血酶的测定方法。
为了解决上述技术问题,本发明通过构建一种碳纳米管微悬臂梁生物传感器来实现凝血酶的检测。该碳纳米管微悬臂梁生物传感器包括支架、基底材料、碳纳米管、拾取电路;其中基底材料固定在支架一侧构成微悬臂梁结构,采用低压化学气相沉积法(LPCVD),将碳纳米管生长在基底材料的上面,拾取电路在基底材料的下面,在碳纳米管上面还通过π-π叠加作用修饰有一层核酸适配体。
本发明用的碳纳米管微悬臂梁生物传感器的制备步骤如下:
1、微悬臂梁结构的制造
微悬臂梁是将半导体材料硅为基底材料,加工成微悬臂梁结构。
2、拾取电路的制作
拾取电路是在基底材料下表面,利用微电子工艺制作硅压敏电阻,将四个压敏电阻连接成惠斯通电桥形式。
3、悬臂梁生长和涂敷碳纳米管工艺
对前述步骤中的基底材料的上表面进行清洗处理,分别用丙酮、无水乙醇、去离子水进行超声波清洗,然后用低压化学气相沉积法(LPCVD)生长碳纳米管。用LPCVD法生长碳纳米管质量稳定,不易变形、移位,有利于后面工序将核酸适配体通过π-π叠加作用修饰在碳纳米管上,形成稳定的检测探针。
4、碳纳米管微悬臂梁上核酸适配体的修饰
将核酸适配体通过π-π叠加作用修饰在碳纳米管上,形成一种能特异性识别凝血酶的检测探针,从而构建完成碳纳米管微悬臂梁生物传感器。通过π-π叠加作用修饰,使得核酸适配体在碳纳米管上不容易流失,和LPCVD法生长碳纳米管一起作用,便于后序步骤中凝血酶与核酸适配体形成的复合物在微悬臂梁上的质量效应稳定,从而引起微悬臂梁谐振频率变化的稳定,为实现凝血酶的检测浓度范围为0.5-10µg/mL打下基础。
本发明对凝血酶检测的步骤如下:
(1)在碳纳米管微悬臂梁上先制作含有凝血酶核酸适配体的检测探针;
(2)将检测探针放入待测样本中,待测样本中凝血酶的浓度范围为0.5-10µg/mL,凝血酶通过特异性反应与检测探针上的核酸适配体形成复合物并附着在微悬臂梁上;
(3)所形成的复合物的质量大小与待测样本中凝血酶的浓度呈正相关;
(4)所述复合物在微悬臂上产生的质量变化引起微悬臂梁挠曲位移或谐振频率的变化,从而实现对凝血酶的检测。
附图说明
图1是检测凝血酶用的碳纳米管微悬臂梁生物传感器示意图。
本发明的优点和特点
本发明利用碳纳米管微悬臂梁生物传感器来检测凝血酶,该传感器采用低压化学气相沉积法(LPCVD),将碳纳米管生长在基底材料的上面,在碳纳米管上面通过π-π叠加作用修饰有一层核酸适配体,利用核酸适配体与凝血酶发生特异性识别反应形成复合物,在微悬臂梁生物传感器上产生质量效应,通过质量效应来实现对凝血酶的检测。上述技术特征是互相支持,共同作用,实现了当凝血酶浓度为0.5-10µg/mL时快速准确检测,灵敏度高、操作简便。
具体实施方式
图1是凝血酶检测用的碳纳米管微悬臂梁生物传感器的示意图,包括支架1,基底材料2,碳纳米管3以及拾取电路4。其中基底材料2固定在支架1一侧构成微悬臂梁结构,在基底材料2的上表面分别用丙酮、无水乙醇、去离子水进行超声波清洗,然后用低压化学气相沉积法(LPCVD)生长碳纳米管3;拾取电路4在基底材料2的下面,在碳纳米管3上面还通过π-π叠加作用修饰有一层核酸适配体5。
首先,在碳纳米管3上通过π-π叠加作用修饰对凝血酶有特异性识别的核酸适配体5,形成一种检测探针;
然后,将检测探针放入待测样本中,探针里的核酸适配体5与样本中的凝血酶发生特异性识别反应,形成复合物,该复合物在微悬臂梁生物传感器上产生质量效应,利用这种质量效应来实现对凝血酶的检测。
实施例1
本发明检测凝血酶的步骤如下:
(1)将碳纳米管微悬臂梁置于含有对凝血酶有特异性识别作用的核酸适配体的溶液中,通过超声处理的方法,将核酸适配体通过π-π叠加作用修饰在碳纳米管上,形成一种包含有凝血酶核酸适配体的检测探针;
(2)待测样本中凝血酶的浓度为0.5-10µg/mL时,将待测样本滴加到修饰有核酸适配体的碳纳米管微悬臂梁上,在室温下孵育15分钟,使生物传感界面上的核酸适配体与待测样本中的凝血酶发生特异性识别反应,形成复合物;
(3)所形成的复合物的质量大小与待测样本中凝血酶的浓度呈正相关。
(4)形成的复合物在微悬臂梁上产生质量效应,利用该质量效应来实现对凝血酶的检测。
本实验取样10微克/毫升、1微克/毫升、0.5微克/毫升样品各1mL,形成的复合物在硅微悬臂梁上产生质量效应分别是30.5Hz、2.9 Hz、1.6Hz,凝血酶的检测结果分别为10.2微克/毫升、0.97微克/毫升、0.53微克/毫升。
Claims (1)
1.用碳纳米管微悬臂梁生物传感器检测凝血酶的方法,其特征在于:采用碳纳米管微悬臂梁生物传感器来检测,包括如下步骤
(1)将核酸适配体通过π-π叠加作用修饰在碳纳米管上,形成一种能特异性识别凝血酶的检测探针;
(2)待测样本中凝血酶的浓度为0.5-10μg/mL,将检测探针放入待测样本中,待测样本中凝血酶通过特异性反应与检测探针上的核酸适配体形成复合物并附着在微悬臂梁上;
(3)所形成的复合物的质量大小与待测样本中凝血酶的浓度呈正相关;
(4)所述复合物在微悬臂上产生的质量变化引起微悬臂梁挠曲位移或谐振频率的变化,从而实现对凝血酶的检测;
所述碳纳米管微悬臂梁生物传感器包括支架(1)、基底材料(2)、碳纳米管(3)、拾取电路(4);所述基底材料(2)固定在支架(1)一侧构成微悬臂梁结构,碳纳米管(3)生长在基底材料(2)的上面,拾取电路(4)在基底材料(2)的下面;在碳纳米管(3)上面通过π-π叠加作用修饰有一层核酸适配体(5);
所述碳纳米管(3)采用低压化学气相沉积法(LPCVD)生长在基底材料(2)的上面。
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