CN112630282A - 一种结合微流控夹具的阵列电极芯片的制备以及电化学免疫传感器的构建方法 - Google Patents
一种结合微流控夹具的阵列电极芯片的制备以及电化学免疫传感器的构建方法 Download PDFInfo
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Abstract
本发明涉及一种结合微流控夹具的阵列电极芯片的制备以及电化学免疫传感器的构建方法。其特征在于:一种结合微流控夹具的阵列电极芯片,所述多通道阵列电化学,可用于多通道样品定量自动进样,电极个体差异小,稳定性高,可进行电化学生物功能化修饰、检测体量小,便于高通量多通道的整合;通过化学键作用在电极表面修饰双金属纳米材料,并偶联有机磷农药抗体,可采用生物点样技术逐次滴加或并行滴加,使每个工作电极表面均可构建一个完整的特异性的免疫传感器。此外,利用构建的电化学免疫传感器实现多通道检测有机磷农药,实现有机磷农药的同步多通道检测;经上述步骤制备的基于阵列电极结合微流控夹具等电极芯片及电化学免疫传感器,操作简单,灵敏度高,特异性与稳定性较好,符合现场快速检测的要求。
Description
技术领域
本发明涉及在电化学免疫传感检测技术领域,特别涉及结合微流控夹具的阵列电极芯片的制备以及电化学免疫传感器的构建方法。
背景技术
电化学免疫传感器(Electrochemical immunosensor)是将免疫分析与电化学传感技术相结合而构建的一类生物传感器,应用于痕量免疫原性物质的分析研究,电化学免疫传感器属于生物传感器的一种,是通过将电化学分析方法和免疫技术结合在一起构建而成的器件,电化学免疫传感器具有特异性强,灵敏度高等优点,在生物,环境,医学,食品,农业等领域有着极为广泛的应用,电化学免疫传感器的工作原理和传统的免疫分析方法很相似,它们都是把抗原或者抗体通过物理或化学方法固定到某种界面上,然后用直接法或间接法来检测抗体或抗原等目标物,基于抗原-抗体的特异性作用使得免疫传感器具有高灵敏性高特异性的特点,基于修饰电极的纳米质优良的电化学特征使得检出限大大降低,检测过程时间短检测效率高,便携且操作检测流程简单适于快速现场检测。
微机电系统(MEMS, Micro-Electro-Mechanical System)是在微电子技术(半导体制造技术)基础上发展起来的,融合了光刻、腐蚀、薄膜、LIGA、硅微加工、非硅微加工和精密机械加工等技术制作的高科技电子器件,基于 MEMS器件具有体积小、精密度高和一致性好等优点,通过 MEMS 可以将生物传感器敏感元件三维尺寸充分压缩,提高其检测样品数量和检测速度,因此将电化学技术与MEMS 相结合,制作基于 MEMS 的电化学电极阵列是必要的,目前基于 MEMS 的电极阵列大都是基于硅基底板制作的两电极体系,由于缺少三电极系统的参比电极而导致体系的稳定性下降。
微流控(Microfluidics),是一种精确控制和操控微尺度流体,尤其特指亚微米结构的技术,又称其为芯片实验室(Lab-on-a-Chip)或微流控芯片技术,是把生物、化学、医学分析过程的样品制备、反应、分离、检测等基本操作单元集成到一块微米尺度的芯片上,自动完成分析全过程,同时还有着体积轻巧、使用样品及试剂量少、能耗低,且反应速度快、可大量平行处理等优点。
发明内容
本发明所要解决的技术问题是在电化学微阵列电极制备和修饰领域提供一种结合微流控夹具的阵列电极芯片的制备以及电化学免疫传感器的构建方法,其具有设备制作使用方便简单、耗材成本低、操作方便、检测结果重复性好、该电化学免疫阵列电极可满足多通道微量检测的要求,可以在其阵列电极表面进行广谱有机磷农药抗体的修饰,该阵列电极可实现多种有机磷类农药的同时定量检测,实用性强。
进一步的,进行排布和构建结合微流控夹具的阵列电极芯片,在一个芯片上集成N个电极,其中(N-2)个为工作电极,1个为对电极,1个为参比电极,在(N-2)个工作电极上涂以含有不同生物活性元件的聚亚胺酯膜,可同时测定(N-2)种生物物质。
进一步的,制备所述的阵列传感器的芯片,所述制备方法如下:
步骤一:先将硅片表面清洁干燥后,氧气的气氛中热氧化生成一层约300 nm厚的SiO2薄层;
步骤二:经过涂胶后进行金属图形化标准光刻,镜检后打底膜,溅射上20 nm的钛(作为粘合层)和约100 nm的金,之后进行光刻开窗(刻蚀露出黄色电极区域)即在硅表面形成微电极阵列;
步骤三:经上述操作后,再利用等离子体增强化学蒸镀工艺(PECVD)在非电极变表面生长350 nm厚的SiO2;
步骤四:将硅片在氧气气氛下,500℃退火30分钟,并去胶;
步骤五:电极阵列划片,按照设计尺寸制备电极阵列,整体阵列芯片为9.7 mm ×20 mm,制备好的电极阵列精确切割备用;
步骤六:利用光及活性离子刻蚀技术使金圆盘电极及引线连接区暴露出来。最后进行划片分装,利用压片技术将芯片压制到一个设计好的管芯中,引出导线,利用环氧树脂黏合剂盖住连接导线,保证绝缘密封。
进一步的,进行微阵列传感界面的修饰,利用湿化学合成法合成钯纳米单晶作为种子,诱导金纳米树枝的生长,制备导电性好、生物相容性高的纳米复合材料,借助SEM、TEM、AFM等表征手段对纳米复合材料的表面形态进行表征,并借助循环伏安法对所制备的纳米材料的电化学性能进行分析,为选取性能最佳的纳米复合材料修饰于传感界面提供强有力的数据支撑,借助电化学沉积法或者物理吸附滴涂法修饰纳米复合材料与生物传感界面。
进一步的,采用电化学共聚的方式包埋抗体,通过电化学操作,及其所利用的纳米复合材料良好的生物相容性可以有效地控制所固定抗体的生物活性及其包埋有抗体的电极位置及膜厚,从而可以实现在单个芯片的不同位置定点固定不同的抗体,来达到单个芯片上同时检测多个有机磷农药抗原的目的。
本发明的原理如下
所述电化学包埋法是指把生物分子包埋于电极修饰材料的基底中来实现生物分子在电极表面固载的一种方式,包埋法中生物反应较直接,常无生物分子自身官能团参与共价键合反应,使得高级结构和活性得到最大化保留。
所述电化学聚合法是依据电荷平衡原则通过聚合物膜把生物分子固载到电极上的一种固定化方法,该方法稳定性高,选择性好,线性范围较宽,这种方法可以实现对葡萄糖的快速灵敏的检测,还可以调控负荷的酶量、聚合物膜的厚度和膜的致密程度,但是该方法的可固载的生物识别元件量较低,会浪费大量的电聚合溶液中的生物识别元件,且产生的背景电流相对较大。
所述电化学检测过程中,电化学反应体系在电极表面产生电至氧化-还原反应,可通过对氧化峰和还原峰的电流大小计算得到相应的抑制率参数。
本发明所采用的方法不仅可以提高电化学检测结果的稳定性和灵敏度,使得操作简单易行,提高现场快速检测效率,而且有以下两个方面的优势:
(1)克服了抗原抗体在电极表面固载的稳定性问题;
(2)选取了合适的结合方法提高了电极表面抗原抗体的稳定性。
本发明的有益效果如下
(1)本发明所述多通道阵列电化学,可用于多通道样品定量自动进样,电极个体差异小,稳定性高,可进行电化学生物功能化修饰、检测体量小,便于高通量多通道的整合;
(2)本发明所述阵列电极芯片可实现传统电化学检测及电致化学发光检测,同时由于贵金属电极稳定性好,且具有良好的线性关系,该电极阵列基础性能良好,因电极阵列采用光刻工艺,且选用高纯度Au制备,电极阵列表面的平整度及加工精度均优于丝网印刷技术和普通打磨工艺制备的金电极;
(3)本发明所述该电极芯片设计灵活、可用于现场快速检测,可批量制作。
附图说明
图1所示为多通道阵列电极芯片平面示意图。
图2所示为阵列电极芯片晶圆印制图。
图3所示为微流控夹具组装示意图。
图4所示为微流控夹具纵截面示意图。
图5所示为微流控夹具底视图。
图6所示为所述电化学免疫传感器的构建方法示意图。
具体实施方式
下文将结合具体附图详细描述本发明具体实施例,应当注意的是,下述实施例中描述的技术特征或者技术特征的组合不应当被认为是孤立的,它们可以被相互组合从而达到更好的技术效果,在下述实施例的附图中,各附图所出现的相同标号代表相同的特征或者部件,可应用于不同实施例中。
为使本发明要解决的技术问题、技术方案和优点更加清楚,下面将结合附图及具体实施例进行详细描述。
实验一:配制含2 mM K3[Fe(CN)6]的PBS 缓冲溶液(pH=7.4):NaCl 8 g,KCl 0.2g,Na2HPO4 1.42 g、KH2PO4 0.27 g、K3[Fe(CN)6] 685.5 mg,所有试剂称量后置于 1000 mL烧杯中,向烧杯中加入约 800 mL 的去离子水,充分搅拌溶解后移至于 1000 mL 容量瓶中定容,完全溶解定容后将溶液移至于试剂瓶中。
实验二:配制封闭液,准确称取卵清蛋白 10.00 g,加入 1000 mL 磷酸盐缓冲液,搅拌混匀直至蛋白完全溶解。
实验三:制备氯金酸溶液,称量 1 g HAuCl4·3H2O 粉末溶于 20 mL 水中,配制成50 mmoL/L氯金酸溶液,4 ℃保存备用。
实验三:电极的预处理:将阵列电极活化后在含有 5 mmol/L 的[Fe(CN)6]3-/4- 的PBS(pH 7.0)进行循环伏安法和电化学阻抗谱表征。
实验四:将阵列电极置于含有 1 g/L 氯金酸和1 g/L 的氯钯酸溶液中,采用计时电流法恒电位沉积纳米金和纳米钯,反应完成后进行电化学表征。蒸馏水清洗,氮气吹干后备用。取10 mg 的MWCNTs-COOH和20 mg PVP 加入到20 mL 的N, N-二甲基甲酰胺溶液中,混合超声振荡 20 min,通过生物点样仪将0.8 μL 所述混合溶液滴加到纳米金及纳米钯修饰的阵列电极的各个工作电极表面并充分反应。多壁碳纳米管通过一定的吸附作用和共聚作用稳定的沉积到了纳米金层表面。
实验五:通过生物点样仪对阵列电极表面进行封闭处理。
本发明采用的技术方案为:
(1)在电极阵列中,采用带圆形电极结构使电极表面积最大化并减少尖角效应;
(2)采用抗体作为识别元件修饰电极阵列中的每一个工作电极表面;
(3)使用电化学共聚包埋法在电极表面修饰识别元件,可通过生物点样仪采用逐次加载或并行加载;
(4)对电极进行充分的封闭处理以降低非特异性吸附。
当固定在电极表面的有机磷抗体捕捉住待检有机磷抗原时,电化学反应体系在反应电极表面进行电化学反应,并影响该界面上的氧化还原反应的过程,从而影响循环伏安图的氧化峰和还原峰的峰值。
实施例1 阵列电极芯片所结合的微流控夹具的制备
(1)PDMS芯片制作:用SolidWorks软件绘制微流控夹具的基本图形,用高分辨率激光照排机在照相底片上绘制光刻掩模,其后在硅片上旋涂SU-8光刻胶层,对其进行高温固化处理,盖上光刻掩膜后在光刻机上对已固化的SU-8光刻胶进行曝光,最后在显影液中显影制得硅片阳模模板;
(2)PDMS预聚物和PDMS固化剂按以10:1比例混合,在真空中抽气后将其浇注在制作好的阳模模板上,高温固化后剥离得到具有微结构的芯片,根据图纸打出底液灌注孔和底液排出孔;
(3)将前期制备的阵列电极芯片通过密封垫及M3螺丝固定密封。
实施例2 结合微流控夹具的阵列电极芯片检测有机磷农药
(1)进样前使用PBS清洗通道;
(2)进样:取50μL样品底液,通过进样隔膜泵加入进样孔,隔膜泵以吸程为2m的速度抽取样品,出样口通过废液池收集流出的废液。
本领域技术人员可以理解,尽管已经参照本公开的特定示例性实施例示出并描述了本公开,但是本领域技术人员应该理解,在不背离所附权利要求及其等同物限定的本公开的精神和范围的情况下,可以对本公开进行形式和细节上的多种改变。因此,本公开的范围不应该限于上述实施例,而是应该不仅由所附权利要求来进行确定,还由所附权利要求的等同物来进行限定。
Claims (10)
1.一种结合微流控夹具的阵列电极芯片,涉及在电化学免疫传感检测技术领域中应用的制备方法,其特征在于,用于有机磷类农药广谱性检测,包括:阵列电极开窗引脚,其后通过连接有微电极阵列,其中所述微电极阵列包括微电极被均分为至少四组,每组微电极设有与之对应的参比电极及对电极;所述阵列电化学芯片结合微流控夹具,阵列芯片与电极之间通过密封垫进行密封处理,以达到样品无损耗定量精确进样的目的,所述微电极阵列表面修饰有用于有机磷类农药广谱性抗体检测多种有机磷类农药电化学信号检测的纳米复合材料。
2.根据权利要求1所述的一种结合微流控夹具的阵列电极芯片的制备方法,其特征在于:阵列电极2×2的工作电极阵列,每个工作电极附带一个参比电极和一个对电极,所有工作电极共用一个对电极及一个参比电极,工作电极为相同半径0.7036 mm,厚度为100 nm,材质为金的圆形电极片,参比电极为相同边长0.1759 mm,厚度为100 nm,材质为金的正方形电极片,对电极为内径为1.0554 mm,外径为1.2312 mm,厚度为100 nm的C型电极片,相邻工作电极之间的间距为3.5 mm。
3.根据权利要求1所述的一种结合微流控夹具的阵列电极芯片的制备方法,其特征在于:首先将硅片表面清洁并干燥,在氧气的气氛中热氧化生成一层约300 nm厚的SiO2薄层,随后经过涂胶后进行金属图形化标准光刻,镜检后打底膜,溅射上20 nm的钛作为粘合层,并进一步溅射一层约100 nm的金,之后进行光刻开窗刻蚀,露出金电极区域,即在硅表面形成微电极阵列。
4.根据权利要求1所述的一种结合微流控夹具的阵列电极芯片的制备方法,其特征在于:利用等离子体增强化学蒸镀工艺(PECVD)在非电极变表面生长350 nm厚的SiO2,随后硅片在氧气气氛下,500℃退火30分钟,并去胶。
5.根据权利要求1所述的一种结合微流控夹具的阵列电极芯片的制备方法,其特征在于:利用多通道微流控夹具控制完成自动进样并自动清洗,使用多通道继电器控制隔膜泵实现自动进样。
6.根据权利要求1所述的一种结合微流控夹具的阵列电极芯片的制备方法,其特征在于:所述阵列电化学芯片结合微流控夹具和通过CNC数控钻孔处理后通过M3螺丝固定,阵列芯片与微流控夹具进样池之间通过密封垫进行密封处理,以达到样品无损耗定量精确进样的目的。
7.根据权利要求1所述的一种结合微流控夹具的阵列电极芯片的制备方法,其特征在于:利用构建的电化学免疫传感器实现多通道检测有机磷农药,实现有机磷农药的同步多通道检测,通过修饰双金属纳米材料构建有机磷农药抗体修饰材料,由于采用PDMS材料制备微流控夹具并使用密封垫实现样品间密封处理,减少多种进样通道之间的信号干扰。
8.根据权利要求1所述的一种结合微流控夹具的阵列电极芯片的制备方法,其特征在于:阵列电极接口规格为单个引脚为0.8 mm × 2.9 mm 金属开窗,纵向阵列8个,其引脚间距为1.1 mm,整体阵列芯片为9.7 mm × 20 mm × 0.5 mm,其引脚规格与标准SD卡引脚规格相同,与构建的标准信号放大模块接口规格相同,使其能够紧密契合卡槽。
9.根据权利要求1所述的一种结合微流控夹具的阵列电极芯片的制备方法,其特征在于:通过化学键作用在电极表面修饰双金属纳米材料,并偶联有机磷农药抗体,可采用生物点样技术逐次滴加或并行滴加,使每个工作电极表面均可构建一个完整的特异性的免疫传感器。
10.根据权利要求1所述的一种结合微流控夹具的阵列电极芯片的制备方法,其特征在于:
1)制备聚乙烯吡咯烷酮与纳米石墨炔的悬浊液,对所述的悬浊液进行超声分散;
2)将其超声分散后的悬浊液通过生物点样仪滴到阵列电极活化之后的工作电极表面;
3)利用湿化学合成法合成钯纳米单晶作为种子,诱导金纳米树枝的生长,制备导电性好、生物相容性高的纳米复合材料,将有机磷广谱性抗体修饰到纳米复合材料的表面,通过生物点样仪滴到阵列电极滴加聚乙烯吡咯烷酮与纳米石墨炔附着后的的工作电极表层;
4)通过电化学恒电位仪给予所述微电极一个正负相的三角脉冲波或者阶梯脉冲波,使其在电化学循环伏安法和差分脉冲伏安法下,通过氧化峰及还原峰等电流或者电压的波形变化实现以所述构建的电化学免疫阵列传感器为核心的样品中有机磷农药的多通道检测。
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