CN107219278B - 一种细胞释放物的探测器及其探测方法 - Google Patents
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Abstract
本发明提供了一种细胞释放物的探测器及其探测方法。所述探测器的核心部件为包括柔性GO/rGO阵列电极片,所述电极片的曲率值为0.18;所述柔性GO/rGO阵列电极片由GO基底和嵌于GO基底中的rGO阵列组成,所述rGO阵列由直径为20μm圆柱状的rGO单元构成,相邻两个单元的间距为60μm。本发明研究实现了不同弯曲曲率柔性基底GO/rGO阵列的电化学性能和在不负载酶和纳米催化剂的情况下对双氧水的低浓度检测,并显示了较高的灵敏度,具有很好的应用前景。
Description
技术领域
本发明属于微加工及生物检测领域,涉及一种细胞释放物的探测器及其探测方法。
背景技术
生物传感器具有灵敏度高,选择性好,便携操作性强,成本低,简单,快捷等优点。随着生活水平不断提高,人们对健康的追求越来越高,开发新型检测装置成为热点。微阵列传感器具有高通量、高选择性、高灵敏度,微型化,智能化、集成化,低成本,高稳定,高寿命等优点。
传统的微电极阵列主要以金属、玻璃、硅等刚性材料为基底,通过沉积、光刻、腐蚀等微加工手段在其上形成屏幕或者三维结构的微电极阵列。这些微电极虽然在空间分辨率、耐腐蚀性、灵敏度等方面具有各自的优势,但是由于几乎不具有形变能力,在柔软度匹配、共形贴附、以及动态形变匹配等方面很难满足植入式要求,因此正在逐步被柔性微电极阵列技术所取代。
基于可穿戴设备的研究,具有靶组织接触良好,减免机械损伤,降低免疫排斥反应的柔性微阵列传感器,是当今电子、机械、仪器和医学等领域的研究热点之一,直接关系到机器人、医疗设备、可穿戴设备等载体的智能化和多功能化。
石墨烯良好的生物相容性、优异的导电性和机械性能,以及良好的电化学活性,使其在柔性生物传感应用方面展现出极大的优势。基于石墨烯高柔韧性和机械强度,制备的石墨烯柔性电极,可以使电极与靶组织良好接触,减少电极和生物组织之间的机械强度的不匹配,可以减少临床应用中产生的机械损伤和免疫反应。此外,在神经组织工程中,具有较高电子传导速率的石墨烯还可以作为细胞的刺激电极,在没有太多的化学反应下,加强与分化神经元的电耦合,这使得石墨烯与活细胞的界面研究成为未来神经假体装置研发的热点。
发明内容
本发明的目的在于针对现有技术的不足,提供一种细胞释放物的探测器及其探测方法。
本发明的目的是通过以下技术方案实现的:包括柔性基底,所述柔性基底的曲率值为0.18,还包括负载于柔性基底内凹面的石墨烯膜,所述石墨烯膜由GO基底和嵌于GO基底中的rGO阵列组成,所述rGO阵列由直径为20μm圆柱状的rGO单元构成,相邻两个单元的间距为60μm(两个临边之间的距离)。
进一步地,所述石墨烯膜通过以下方法负载于柔性基底上:
(1)以柔性ITO/PET为基底,控制三次旋涂的转速和时间分别为1000rpm,8000rpm,1000rpm以及5s,10s,5s,旋涂正性光刻胶,然后在60℃烘胶台上烘15min,用直径为20μm,间隔60μm的掩膜曝光20s,后烘10min,显影液中显影8s。
(2)在步骤1处理后的基底上继续旋涂4mg/mL的氧化石墨烯溶液6次,每次旋涂GO过程为800rpm,5s;3000rpm,30s;1000rpm,5s。使得GO浸润到光刻胶凹槽内部,旋涂均匀,得到附着GO的柔性ITO/PET基底。
(3)使用电化学工作站三电极系统还原附着基底表面的GO,恒电位-0.9V,还原溶液体系为0.2M PBS(pH7.4),还原50s。
(4)最后,将电化学还原得到的附着有rGO/GO微电极阵列的柔性ITO/PET基底放在丙酮溶液中冲洗,将光刻胶洗掉,上面的GO薄膜落到柔性ITO/PET基底,即得到图案化rGO/GO微电极阵列。
一种探测器检测细胞释放双氧水的方法,使得细胞贴附于石墨烯膜上;然后进行循环伏安特性的测定,电解液为2.5mM K3Fe(CN)6和0.1M KCl的水溶液。
本发明具有以下优点:实现了不同曲率柔性基底GO/rGO阵列的电化学性能的研究,和传统的负载酶和纳米催化剂的情况下对双氧水的低浓度检测相比,显示了较高的灵敏度,具有很好的应用前景。
附图说明
图1为GO/rGO微阵列电极片的制备流程图;
图2为石墨烯微阵列的形貌结构;
图3为不同曲率的伏安特性曲线;
图4为K<0细胞对双氧水的检测曲线;
图5为内凹面和外凸面示意图。
具体实施方式
本发明提供了提供一种基于柔性基底GO/rGO阵列电极的弯曲曲率对细胞释放物的探测器的制备方法。包括以下内容:
柔性基底上rGO/GO微阵列的制备:以柔性ITO/PET为基底,控制三次旋涂的转速和时间分别为1000rpm,8000rpm,1000rpm以及5s,10s,5s,旋涂正性光刻胶,然后在60℃烘胶台上烘15min,紫外光刻机掩膜曝光20s,后烘10min,显影液中显影8s,得到光刻胶微阵列。接着,在步骤1得到的光刻胶微阵列的基底上继续旋涂4mg/mL的氧化石墨烯溶液6次,每次旋涂GO的转速和时间分别为800rpm,3000rpm,1000rpm以及5s,30s,5s。每次旋涂都将GO在光刻胶表面轻轻吹打,使得GO浸润到光刻胶凹槽内部,旋涂均匀,得到附着GO的柔性ITO/PET基底。使用电化学工作站三电极系统还原附着基底表面的GO,恒电位-1V,还原溶液体系为0.2M PBS(pH7.4),还原50s。最后,将电化学还原得到的附着有rGO/GO微电极阵列的柔性ITO/PET基底放在丙酮溶液中冲洗,将光刻胶洗掉,上面的GO薄膜落到柔性ITO/PET基底,即得到图案化rGO/GO微电极阵列。
将电极片置于十二孔板内,石墨烯膜所在的活性面朝上,灭菌后,放置到十二孔板内,依次用PBS,DMEM清洗,然后使用10%含血清培养液37℃孵化30min,最后接种细胞,接种密度约6000个/mL,37℃,5%CO2细胞培养箱中培养24h。
取电极片两片进行弯曲,弯曲呈曲率0.18,一片的活性面(贴附有细胞的石墨烯面)位于内凹面,另一片位于外凸面。使用药物PMA(15μL 0.5μg/L)刺激两个微电极的表面细胞,通过观察电流的变化,实现GO/rGO电极对细胞释放物的检测。结果发现,相同刺激条件下,内凹面细胞负载具有更高的灵敏度,推测其原因,内凹面更倾向于形成三维环绕,增加接收电信号的能力。
将负载有细胞的电极片弯曲,使得石墨烯膜位于电极片的内凹面,曲率分别为0.06、0.08、0.10、0.12、0.18、0.25。分别进行电极的循环伏安特性的测定,电解质为2.5mMK3Fe(CN)6和0.1MKCl的混合溶液。图3为不同曲率的伏安特性曲线。
将负载有细胞的电极片弯曲,使得石墨烯膜位于电极片的内凹面,曲率分别为0.06、0.1、0.18、0.25,进行释放物检测:使用药物PMA(15μL 0.5μg/L)刺激不同曲率的微电极表面细胞,通过观察电流的变化,实现GO/rGO电极对细胞释放物的检测。测定结果表明,K<0.18情况下,曲率增大效果增强,当k>0.18时,检测效果减弱。分析其中原因,在弯曲情况下,电极表面电场分部发生变化,导致测定结果差异;图4为曲率值K为0.06、0.1、0.18、0.25情况下细胞对双氧水的检测曲线,检测限降低,灵敏度增加,而当K>0.18时,随着曲率值的增大,灵敏度降低。
上述实例用来解释说明本发明,然而并非限定本发明。在本发明的精神和权利要求的保护范围内,对本发明作出的任何修改和改变,都落入本发明的保护范围。
Claims (2)
1.一种细胞释放物的探测器,其特征在于,包括柔性基底,所述柔性基底的曲率值为0.18,还包括负载于柔性基底内凹面的石墨烯膜,所述石墨烯膜由GO基底和嵌于GO基底中的rGO阵列组成,所述rGO阵列由直径为20µm圆柱状的rGO单元构成,相邻两个单元的间距为60µm;所述石墨烯膜通过以下方法负载于柔性基底上:
(1)以柔性ITO/PET为基底,控制三次旋涂的转速和时间分别为1000rpm,8000rpm,1000rpm以及 5s,10s,5s,旋涂正性光刻胶,然后在60℃烘胶台上烘15min,用直径为20µm,间隔60µm的掩膜曝光20s,后烘10min,显影液中显影8s;
(2)在步骤1处理后的基底上继续旋涂4mg/mL的氧化石墨烯溶液6次,每次旋涂GO过程为800rpm,5s ;3000rpm,30s;1000rpm,5s;使得GO浸润到光刻胶凹槽内部,旋涂均匀,得到附着GO的柔性ITO/PET基底;
(3)使用电化学工作站三电极系统还原附着基底表面的GO,恒电位-1V,还原溶液体系为pH7.4的0.2M PBS,还原50s;
(4)最后,将电化学还原得到的附着有rGO/GO微电极阵列的柔性ITO/PET基底放在丙酮溶液中冲洗,将光刻胶洗掉,上面的GO薄膜落到柔性ITO/PET基底,即得到图案化rGO/GO微电极阵列。
2.一种权利要求1所述的探测器检测细胞释放双氧水的方法,其特征在于,使得细胞贴附于石墨烯膜上;然后进行循环伏安特性的测定,电解液为2.5mM K3Fe(CN)6和0.1M KCl的水溶液。
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