CN113189193B - 一种隧穿磁阻生物传感器及其制备方法及应用 - Google Patents
一种隧穿磁阻生物传感器及其制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种隧穿磁阻生物传感器及其制备方法及应用。该生物传感器包括隧穿磁阻多层膜结构、隧穿磁阻生物传感器的反应区域、反应区域内的生物敏感膜、产生信号的磁珠;隧穿磁阻生物传感器的反应区域分为新型冠状病毒IgM抗体检测区域、阴性对照区域和新型冠状病毒IgG抗体检测区域,新型冠状病毒IgM抗体检测区域固定抗人IgM抗体,阴性对照区域修饰牛血清蛋白,新型冠状病毒IgG抗体检测区域固定抗人IgG抗体。本发明的隧穿磁阻生物传感器结果更加准确、灵敏度高,而且操作简便,不需由专业人员操作,检测成本低,适用于社区、机场、家庭等现场检测,为快速筛查新型冠状病毒提供了有效手段。
Description
技术领域
本发明属于生物检测传感器技术领域,特别涉及一种隧穿磁阻生物传感器及其制备方法及应用。
背景技术
目前,新型冠状病毒的主要检测方法是荧光PCR,然而鼻咽拭子的核酸检测阳性率只有30%-50%,存在大量的假阴性结果。荧光PCR的检测原理是将样品中的靶标序列进行扩增,经扩增后,其核酸数量达到足够通过荧光进行检测,因此检测阳性率与核酸数量密切相关。由于不同病程、不同病情的患者体内病毒含量不同;单链RNA的新型冠状病毒容易被RNA酶降解;病毒基因组可能发生突变,不同公司的检测试剂灵敏度和特异度不同,荧光PCR检测受多种因素限制,检出率不高。为了弥补其不足,新型冠状病毒特异性抗体检测被开发。抗体检测存在采样简单、样本更稳定的优点,且IgM和IgG抗体的联合动态检测可以和核酸检测互相补充验证以提高诊断效果。
现有的新型冠状病毒特异性抗体检测方法为胶体金法和磁微粒化学发光法,胶体金法灵敏度不高,容易出现漏检。磁微粒化学发光法则需要专门的化学发光检测设备,不适用于社区、机场庭等现场检测。磁生物传感器具有高灵敏度、便携性、低功耗的优势,能满足即时诊断的需求。与巨磁阻、巨磁阻抗、霍尔电阻等传感器相比,隧穿磁阻传感器可以检测到低至10-9~10-10T的磁场,因此,开发联合检测新型冠状病毒特异性IgM/IgG抗体的高灵敏度高、快速便捷的隧穿磁阻生物传感器。
发明内容
本发明的目的是提供一种隧穿磁阻生物传感器及其制备方法及应用,以解决上述问题。
为实现上述目的,本发明采用以下技术方案:
一种隧穿磁阻生物传感器,包括隧穿磁阻多层膜、保护结构层、生物敏感膜和纳米磁珠;隧穿磁阻多层膜上设置保护结构层,保护结构层上设置生物敏感膜,生物敏感膜上划分为三个反应区域分为两个检测区域和一个阴性对照区域,两个检测区域分别固定抗人IgM抗体和抗人IgG抗体,阴性对照区域修饰牛血清蛋白;纳米磁珠设置在反应区。
进一步的,反应区域由传感器阵列组成;每4个传感器分别组成新型冠状病毒IgM抗体检测区域、新型冠状病毒IgG抗体检测区域、阴性对照区域。
进一步的,隧穿磁阻多层膜为位于硅片上的CoFe/CoFeB/MgO/CoFeB多层膜结构,呈曲折形。
进一步的,保护结构层为保护层Al2O3、保护层和生物结合层SiO2,Al2O3和SiO2的厚度均小于1μm。
进一步的,生物敏感膜为硅烷层,由(3-氨丙基)三乙氧基硅烷包被隧穿磁阻传感器上的SiO2形成的硅烷层,经戊二醛处理接上醛基接头,与抗体进行偶联。
进一步的,纳米磁珠为Fe@Fe3O4核壳纳米球,直径小于1μm。
进一步的,一种隧穿磁阻生物传感器的制备方法,包括以下步骤:
步骤1,在硅片上制作隧穿磁阻CoFe/CoFeB/MgO/CoFeB多层膜,形成隧穿磁阻芯片;
步骤2,在隧穿磁阻芯片上制作保护层Al2O3,Al2O3的厚度小于1μm;
步骤3,在保护层Al2O3上制备保护层和生物结合层SiO2,SiO2的厚度小于1μm,形成隧穿磁阻传感器;
步骤4,在隧穿磁阻传感器上组装生物敏感膜硅烷层,经过戊二醛处理接上醛基接头;
步骤5,利用戊二醛的醛基与氨基结合,将抗体固定在生物敏感膜上的检测区域,将牛血清蛋白修饰在阴性对照区域。
进一步的,步骤4中,硅烷层和戊二醛处理的方法,具体包括:
4)丙酮、甲醇、异丙醇洗涤隧穿磁阻传感器三次,氮气干燥,氧等离子处理30min;
5)2%-5%的(3-氨丙基)三乙氧基硅烷溶液处理30min-120min,无水乙醇清洗三次后,超纯水清洗三次;
6)高温烘烤;
4)5%的戊二醛溶液处理1h-2h,PBS缓冲液清洗三次。
进一步的,一种隧穿磁阻生物传感器的应用,包括:
将无需预处理的血液样品直接滴加到隧穿磁阻芯片上的反应区域,37℃孵育后,PBST缓冲液清洗三次;将Fe@Fe3O4纳米磁珠修饰的新型冠状病毒重组抗原溶液滴加到隧穿磁阻芯片上的反应区域,37℃孵育后,PBS缓冲液清洗三次;如果样本中含有新型冠状病毒抗体,生物传感器上会形成抗人IgM/IgG抗体-新型冠状病毒抗体-磁珠修饰的新型冠状病毒重组抗原,在外加磁场的作用下,磁珠产生的寄生磁场会导致传感器的磁阻发生变化,通过标准曲线将磁阻信号转化成新冠病毒抗体浓度。
与现有技术相比,本发明有以下技术效果:
本发明中,生物传感器为隧穿磁阻传感器阵列,可以进行高通量检测,传感器的两个检测区域可以同时检测新型冠状病毒特异性IgM和IgG抗体,有助于提高新型冠状病毒的检出率;生物样品本身几乎不具有磁性,磁信号不会受到背景干扰,提高了信噪比,相比荧光分子、酶、放射性同位素等,磁珠的磁信号非常稳定,相比Fe3O4纳米磁珠,Fe@Fe3O4核壳纳米磁珠具有更大的磁矩,提高了检测灵敏度;采用血样检测,且血液样品无需复杂的预处理,操作简单,有利于快速便携检测。
本发明的传感器结构:生物传感器为隧穿磁阻传感器阵列,可以进行高通量检测,传感器的两个检测区域可以同时检测,有助于提高疾病的检出率
本发明的制备方法:生物样品本身几乎不具有磁性,磁信号不会受到背景干扰,血液样品无需复杂的预处理,操作简单,有利于快速便携检测。相比荧光分子、酶、放射性同位素等,磁珠的磁信号非常稳定,相比Fe3O4纳米磁珠,Fe@Fe3O4核壳纳米磁珠具有更大的磁矩,提高了检测灵敏度。
附图说明
图1为本发明隧穿磁阻生物传感器的阵列示意图;
图2为本发明隧穿磁阻生物传感器免疫检测示意图。
图中:1、新型冠状病毒IgM抗体检测区域;2、阴性对照区域;3、新型冠状病毒IgG抗体检测区域;4、隧穿磁阻多层膜;5、保护层Al2O3;6、保护层和生物结合层SiO2;7、生物敏感膜;8、抗人IgM抗体;9、抗人IgG抗体;10、牛血清蛋白;11、新型冠状病毒IgM抗体;12、新型冠状病毒IgG抗体;13、新型冠状病毒重组抗原;14、Fe@Fe3O4纳米磁珠。
具体实施方式
以下结合附图对本发明进一步说明:
请参阅图1至图2,本发明一方面提供了一种联合检测新型冠状病毒特异性IgM/IgG抗体的隧穿磁阻生物传感器,包括隧穿磁阻多层膜4、保护层Al2O3 5、保护层和生物结合层SiO26、新型冠状病毒IgM抗体检测区域1、阴性对照区域2、新型冠状病毒IgG抗体检测区域3、生物敏感膜7、Fe@Fe3O4纳米磁珠8;所述隧穿磁阻多层膜4为位于硅片上的CoFe/CoFeB/MgO/CoFeB多层膜结构;所述新型冠状病毒IgM抗体检测区域1固定抗人IgM抗体8;所述阴性对照区域2修饰牛血清蛋白10;所述新型冠状病毒IgG抗体检测区域3固定抗人IgG抗体9;所述生物敏感膜7为硅烷层,经戊二醛处理接上醛基接头,利用戊二醛的醛基与抗体的氨基结合,将抗体固定在检测区域。
一种联合检测新型冠状病毒特异性IgM/IgG抗体的隧穿磁阻生物传感器的制作流程,包括以下步骤:
步骤1,在硅片上制作隧穿磁阻CoFe/CoFeB/MgO/CoFeB多层膜;
步骤2,在隧穿磁阻芯片上制作保护层Al2O3,Al2O3的厚度小于1μm;
步骤3,在保护层Al2O3上制备保护层和生物结合层SiO2,SiO2的厚度小于1μm;
步骤4,在隧穿磁阻传感器上组装生物敏感膜硅烷层,经过戊二醛处理接上醛基接头;
步骤5,利用戊二醛的醛基与氨基结合,将抗体固定在检测区域,将牛血清蛋白修饰在阴性对照区域。
步骤4所述硅烷层和戊二醛处理的方法,具体包括:
7)丙酮、甲醇、异丙醇洗涤隧穿磁阻传感器三次,氮气干燥,氧等离子处理30min;
8)2%-5%的(3-氨丙基)三乙氧基硅烷(无水乙醇)溶液处理30min-120min,无水乙醇、超纯水清洗三次;
9)高温烘烤;
10)5%的戊二醛(PBS)溶液处理1h-2h,PBS缓冲液清洗三次。
第二方面,本发明提供了上述隧穿磁阻生物传感器的使用方法,包括如下步骤:
将无需预处理的血液样品直接滴加到隧穿磁阻芯片上的反应区域,37℃孵育一段时间后,PBST缓冲液清洗三次;将适量Fe@Fe3O4纳米磁珠修饰的新型冠状病毒重组抗原溶液滴加到隧穿磁阻芯片上的反应区域,37℃孵育一段时间后,PBS缓冲液清洗三次;如果样本中含有新型冠状病毒抗体,生物传感器上会形成抗人IgM/IgG抗体-新型冠状病毒抗体-磁珠修饰的新型冠状病毒重组抗原,在外加磁场的作用下,磁珠产生的寄生磁场会导致传感器的磁阻发生变化,通过标准曲线将磁阻信号转化成新冠病毒抗体浓度。
为了更好的理解上述技术方案,下面结合具体的实施例对本发明进行详细的说明,以下实施例仅是本发明的优选实施方式,不是对本发明的限定。
实施例1:
(1)隧穿磁阻传感器芯片由位于硅片上的CoFe/CoFeB/MgO/CoFeB多层膜构成,呈曲折形,通过磁控溅射沉积多层膜结构,其中的CoFe和MgO层通过射频模式沉积。
(2)在隧穿磁阻传感器表面利用原子层沉积法镀保护层Al2O3,Al2O3的厚度为20nm。
(3)在保护层Al2O3上面利用等离子体化学气相沉积法镀保护层和生物结合层SiO2,SiO2的厚度为20nm。
(4)丙酮、甲醇、异丙醇洗涤隧穿磁阻传感器三次,清洗时间为10min,氮气干燥,氧等离子处理30min。处理好的传感器在2%的(3-氨丙基)三乙氧基硅烷(无水乙醇)溶液中处理30min,置于90℃烘箱中烘干1h,无水乙醇、超纯水清洗三次,清洗时间为10min。随后,用5%的戊二醛(PBS)溶液处理传感器30min,PBS缓冲液清洗三次。
(5)在新型冠状病毒IgM抗体检测区域滴加10μL抗人IgM抗体,浓度为0.5mg/mL;在新冠病毒IgG抗体检测区域滴加10μL抗人IgG抗体,浓度为0.5mg/mL;在阴性对照区域滴加10μL牛血清蛋白,浓度为10mg/mL。将传感器置于4℃过夜,PBST清洗芯片,滴加2%牛血清白蛋白溶液,室温孵育1h,PBST清洗芯片。
(6)将10μL血液样品直接滴加到隧穿磁阻芯片上的反应区域,37℃孵育1h后,PBST缓冲液清洗三次;将10μLFe@Fe3O4纳米磁珠修饰的新型冠状病毒重组抗原溶液滴加到隧穿磁阻芯片上的反应区域,37℃孵育30min,PBS缓冲液清洗三次;通过标准曲线将磁阻信号转化成新型冠状病毒抗体浓度。
实施例2:
(1)隧穿磁阻传感器芯片由位于硅片上的CoFe/CoFeB/MgO/CoFeB多层膜构成,呈曲折形,通过磁控溅射沉积多层膜结构,其中的CoFe和MgO层通过射频模式沉积。
(2)在隧穿磁阻传感器表面利用原子层沉积法镀保护层Al2O3,Al2O3的厚度为18nm。
(3)在保护层Al2O3上面利用等离子体化学气相沉积法镀保护层和生物结合层SiO2,SiO2的厚度为20nm。
(4)丙酮、甲醇、异丙醇洗涤隧穿磁阻传感器三次,清洗时间为10min,氮气干燥,氧等离子处理30min。处理好的传感器在5%的(3-氨丙基)三乙氧基硅烷(无水乙醇)溶液中处理1h,置于120℃烘箱中烘干1h,无水乙醇、超纯水清洗三次,清洗时间为10min。随后,用5%的戊二醛(PBS)溶液处理传感器5h,PBS缓冲液清洗三次。
(5)在新型冠状病毒IgM抗体检测区域滴加10μL抗人IgM抗体,浓度为0.5mg/mL;在新型冠状病毒IgG抗体检测区域滴加10μL抗人IgG抗体,浓度为0.5mg/mL;在阴性对照区域滴加10μL牛血清蛋白,浓度为2mg/mL。将传感器置于4℃过夜,PBST清洗芯片,滴加1%牛血清白蛋白溶液,室温孵育1h,PBST清洗芯片。
(6)将10μL血液样品直接滴加到隧穿磁阻芯片上的反应区域,37℃孵育1h后,PBST缓冲液清洗三次;将10μLFe@Fe3O4纳米磁珠修饰的新冠病毒重组抗原溶液滴加到隧穿磁阻芯片上的反应区域,37℃孵育1h,PBS缓冲液清洗三次;通过标准曲线将磁阻信号转化成新型冠状病毒抗体浓度。
实施例3:
(1)隧穿磁阻传感器芯片由位于硅片上的CoFe/CoFeB/MgO/CoFeB多层膜构成,呈曲折形,通过磁控溅射沉积多层膜结构,其中的CoFe和MgO层通过射频模式沉积。
(2)在隧穿磁阻传感器表面利用原子层沉积法镀保护层Al2O3,Al2O3的厚度为20nm。
(3)在保护层Al2O3上面利用等离子体化学气相沉积法镀保护层和生物结合层SiO2,SiO2的厚度为50nm。
(4)丙酮、甲醇、异丙醇洗涤隧穿磁阻传感器三次,清洗时间为10min,氮气干燥,氧等离子处理30min。处理好的传感器在2%的(3-氨丙基)三乙氧基硅烷(无水乙醇)溶液中处理1h,置于110℃烘箱中烘干1h,无水乙醇、超纯水清洗三次,清洗时间为10min。随后,用5%的戊二醛(PBS)溶液处理传感器1h,PBS缓冲液清洗三次。
(5)在新型冠状病毒IgM抗体检测区域滴加10μL抗人IgM抗体,浓度为0.1mg/mL;在新型冠状病毒IgG抗体检测区域滴加10μL抗人IgG抗体,浓度为0.1mg/mL;在阴性对照区域滴加10μL牛血清蛋白,浓度为2mg/mL。将传感器置于4℃孵育4h,PBST清洗芯片,滴加1%牛血清白蛋白溶液,室温孵育2h,PBST清洗芯片。
(6)将10μL血液样品直接滴加到隧穿磁阻芯片上的反应区域,37℃孵育1h后,PBST缓冲液清洗三次;将10μLFe@Fe3O4纳米磁珠修饰的新冠病毒重组抗原溶液滴加到隧穿磁阻芯片上的反应区域,37℃孵育1h,PBS缓冲液清洗三次;通过标准曲线将磁阻信号转化成新型冠状病毒抗体浓度。
Claims (8)
1.一种隧穿磁阻生物传感器,其特征在于,包括隧穿磁阻多层膜、保护结构层、生物敏感膜和纳米磁珠;隧穿磁阻多层膜上设置保护结构层,保护结构层上设置生物敏感膜,生物敏感膜上划分为三个反应区域分为两个检测区域和一个阴性对照区域,两个检测区域分别固定抗人IgM抗体和抗人IgG抗体,阴性对照区域修饰牛血清蛋白;纳米磁珠设置在反应区;
生物敏感膜为硅烷层,由(3-氨丙基)三乙氧基硅烷包被隧穿磁阻传感器上的SiO2形成的硅烷层,经戊二醛处理接上醛基接头,与抗体进行偶联。
2.根据权利要求1所述的一种隧穿磁阻生物传感器,其特征在于,反应区域由传感器阵列组成;每4个传感器分别组成新型冠状病毒IgM抗体检测区域、新型冠状病毒IgG抗体检测区域、阴性对照区域。
3.根据权利要求1所述的一种隧穿磁阻生物传感器,其特征在于,隧穿磁阻多层膜为位于硅片上的CoFe/CoFeB/MgO/CoFeB多层膜结构,呈曲折形。
4.根据权利要求1所述的一种隧穿磁阻生物传感器,其特征在于,保护结构层为保护层Al2O3、保护层和生物结合层SiO2,Al2O3和SiO2的厚度均小于1μm。
5.根据权利要求4所述的一种隧穿磁阻生物传感器,其特征在于,纳米磁珠为Fe@Fe3O4核壳纳米球,直径小于1μm。
6.一种隧穿磁阻生物传感器的制备方法,其特征在于,基于权利要求1至5任意一项所述的一种隧穿磁阻生物传感器,包括以下步骤:
步骤1,在硅片上制作隧穿磁阻CoFe/CoFeB/MgO/CoFeB多层膜,形成隧穿磁阻芯片;
步骤2,在隧穿磁阻芯片上制作保护层Al2O3,Al2O3的厚度小于1μm;
步骤3,在保护层Al2O3上制备保护层和生物结合层SiO2,SiO2的厚度小于1μm,形成隧穿磁阻传感器;
步骤4,在隧穿磁阻传感器上组装生物敏感膜硅烷层,经过戊二醛处理接上醛基接头;
步骤5,利用戊二醛的醛基与氨基结合,将抗体固定在生物敏感膜上的检测区域,将牛血清蛋白修饰在阴性对照区域。
7.根据权利要求6所述的一种隧穿磁阻生物传感器的制备方法,其特征在于,步骤4中,硅烷层和戊二醛处理的方法,具体包括:
1)丙酮、甲醇、异丙醇洗涤隧穿磁阻传感器三次,氮气干燥,氧等离子处理30min;
2)2%-5%的(3-氨丙基)三乙氧基硅烷溶液处理30min-120min,无水乙醇清洗三次后,超纯水清洗三次;
3)高温烘烤;
4)5%的戊二醛溶液处理1h-2h,PBS缓冲液清洗三次。
8.一种隧穿磁阻生物传感器的应用,其特征在于,包括:
将无需预处理的血液样品直接滴加到隧穿磁阻芯片上的反应区域,37℃孵育后,PBST缓冲液清洗三次;将Fe@Fe3O4纳米磁珠修饰的新型冠状病毒重组抗原溶液滴加到隧穿磁阻芯片上的反应区域,37℃孵育后,PBS缓冲液清洗三次;如果样本中含有新型冠状病毒抗体,生物传感器上会形成抗人IgM/IgG抗体-新型冠状病毒抗体-磁珠修饰的新型冠状病毒重组抗原,在外加磁场的作用下,磁珠产生的寄生磁场会导致传感器的磁阻发生变化,通过标准曲线将磁阻信号转化成新冠病毒抗体浓度。
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