CN114166727B - 一种用于一氧化氮检测的细胞传感器及其应用 - Google Patents
一种用于一氧化氮检测的细胞传感器及其应用 Download PDFInfo
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Abstract
本发明公开了一种用于一氧化氮检测的细胞传感器及其应用。该细胞传感器包括丝网印刷电极和设置于丝网印刷电极表面的反应池;所述丝网印刷电极包括基片及在基片上印刷的导轨、工作电极、参比电极和辅助电极;所述基片上印刷三条导轨,三条导轨分别连通到工作电极、参比电极和辅助电极;各电极通过绝缘层隔开;所述工作电极为涂覆有四氰基醌二甲烷/碳纳米管/多聚赖氨酸复合材料的碳电极;所述工作电极为待测试细胞供能。该传感器可以用于实时检测细胞释放的一氧化氮。该传感器电化学性能稳定,操作简便,便于商业化应用,具有较大的应用前景。
Description
技术领域
本发明属于电化学测定技术领域,具体涉及一种TCNQ(四氰基醌二甲烷)/碳纳米管(CNT)/多聚赖氨酸(PL)复合材料用于NO检测的细胞传感器。
背景技术
细胞作为电化学活性体,在新陈代谢过程中进行的一系列生化反应都涉及到电子的传递,将细胞固定在传感器上构建细胞传感器,可通过电化学检测方法对细胞中的生物活性小分子。
内皮细胞产生的NO在调节血管紧张度和活动中起着核心作用,传感器检测乙酰胆碱(Ach)刺激下血管内皮细胞(HUVECs)释放的NO可为NO在心血管疾病中的调节作用提供依据。
NO检测的方法众多,其中电化学传感器具有简单、灵敏、成本低、检测快速等优点。
目前报道的NO传感器主要是柱状电极(如GCE),或者碳纤维电极,细胞加入溶液中检测容易失活。
发明内容
针对上述技术问题,本发明的目的在于提供一种TCNQ(四氰基醌二甲烷)/碳纳米管(CNT)/多聚赖氨酸(PL)复合材料构建的细胞传感器及在细胞NO检测中的应用。
本发明采用丝网印刷电极制备传感器,电极表面固定一个PDMS反应池。细胞直接在电极表面的修饰材料上生长。本发明的修饰材料为TCNQ/CNT/PL复合材料,CNT是一种纳米尺度的、具有完整分子结构的碳材料,具有促进电子传递速率的能力,表现出良好的电催化作用;TCNQ作为典型的π电子受体,可在CNT之间或沿着CNT的分布,与CNT形成超分子的排列增强电化学性能;PL是一种生物相容性阳离子聚合物,在生理的溶液中,PL的氨基基团呈质子化,与CNT的π电子云之间的相互作用、静电作用以及疏水力能增强复合物的稳定性。同时,带正电荷的PL能结合带负电的磷脂质,因此可粘附带负电的细胞。因此TCNQ/CNT/PL复合材料不但能有效地吸附和固定细胞,还可实现细胞与电极之间的电子转移。
本发明提供的技术方案如下:
一种用于一氧化氮检测的细胞传感器,包括丝网印刷电极和设置于丝网印刷电极表面的反应池;
所述丝网印刷电极包括基片及在基片上印刷的导轨、工作电极、参比电极和辅助电极;
所述基片上印刷三条导轨,三条导轨分别连通到工作电极、参比电极和辅助电极;各电极通过绝缘层隔开;
所述工作电极为涂覆有四氰基醌二甲烷/碳纳米管/多聚赖氨酸复合材料的碳电极;
所述工作电极为待测试细胞供能。
进一步,所述工作电极由四氰基醌二甲烷/碳纳米管修饰层上涂覆多聚赖氨酸层制得。
更进一步,所述四氰基醌二甲烷/碳纳米管修饰层中碳纳米管与四氰基醌二甲烷的体积比为50-250:1。
更进一步,所述工作电极的制备方法如下:将碳纳米管与四氰基醌二甲烷分散于乙腈中,获得电极修饰溶液;将所述电极修饰溶液涂覆到碳电极上,干燥即得四氰基醌二甲烷/碳纳米管修饰层;多聚赖氨酸用水稀释,在四氰基醌二甲烷/碳纳米管修饰层上涂覆聚赖氨酸溶液,经孵育后洗涤、干燥即得。
进一步,所述导轨由导电银油墨或导电碳油墨印制于基片上形成,为三条平行导轨由宽度不同的两矩形块组成,其中端部较宽的用于连接电化学工作站,中间部分较窄的用于连接各电极。
更进一步,所述工作电极主体为圆形块扩展连接至中间导轨电极较窄部分的末端;
参比电极为印制在左侧导轨电极较窄部分的末端向中间电极延展形成的小段弧形环;
辅助电极为由右侧导轨极较窄部分的末端下部起始,先扩展至导轨电极上,再环绕工作电极,至接近左侧导轨电极较窄部分末端形成的大段弧形环。
进一步,所述参比电极为Ag/AgCl 电极,
进一步,所述辅助电极为碳电极。
进一步,所述反应池由聚二甲基硅氧烷制成。
本发明还提供利用上述的细胞传感器应用于NO测定的方法,包括以下步骤:
(1)将含有待测细胞的溶液加入反应池中,细胞在工作电极上生长;
(2)滴加药物刺激细胞释放NO;
(3)将丝网印刷电极与电化学工作站连接,测定细胞释放NO的含量。
本发明的有益效果如下:
1)TCNQ/CNT/PL不但能作为细胞生长基底,三种组分协同还可以显著增加NO的测定的选择性和灵敏度;
2)测定灵敏度高、准确度高;
3)易操作,简化测试;
4)所提用的传感器可工业化生产,易于推广,应用前景广阔。
附图说明
图1为丝网印刷电极的结构示意图;
图2丝网印刷电极及PDMS反应池的示意图;
图3为实施例3中制备的TCNQ/CNT/PL复合材料的扫描电镜图;
图4为电压范围为-1~1.5V下,实施例2中构建的传感器对NO的循环伏安响应图;
图5为实施例2中构建的传感器在0.8V下测得的对NO的电流-时间响应图;
图6为由图6获得的NO浓度与电流响应之间的关系曲线图;
图7为实施例2中构建的传感器对不同干扰成分选择性测试结果图;
图8为实施例3中生长在TCNQ/CNT/PL复合材料上人脐静脉内皮细胞活性荧光显微镜成像图;
图9为实施例3中构建的传感器对细胞所释放的NO实时检出测试结果图。
附图标记:11-基片,12-导轨,13-参比电极,14-工作电极,15-辅助电极,16-绝缘层;21-反应池。
具体实施方式
下面结合具体实施例对本发明进一步说明,本发明的内容完全不限于此。
实施例1
图1示出了丝网印刷电化学传感器的结构,包括印制电极的基片11,基片上印制三条导轨12、参比电极13、工作电极14、辅助电极15和绝缘层16。
其中:
导轨电极由导电银油墨或导电碳油墨印制于基片上形成的三个并列导轨,由宽度不同的两矩形块组成,其中端部较宽用于连接电化学工作站,中间部分较窄用于连接各电极;优选的,较宽部分2mm×5mm,较窄部分1mm×17mm。
工作电极主体为圆形块扩展连接至中间导轨电极较窄部分的末端;优选的,圆形块直径为2mm;扩展部分1mm×2mm。
参比电极为银/氯化银油墨印制在右侧导轨电极较窄部分的末端向中间电极延展形成的小段弧形环;优选的,参比电极为1/6圆弧,内径为4mm,外径为7mm。
辅助电极为由右侧导轨极较窄部分的末端下部起始,先扩展至导轨电极上,再环绕工作电极,至接近左侧导轨电极较窄部分末端形成的大段弧形环;优选的,主体部分为2/3 圆弧,内径4mm,外径为7mm,扩展部分1mm×5mm。
反应池21由PDMS制得,固定在表面涂覆有TCNQ/CNT/PL复合材料的丝网印刷传感器1表面的反应区域(包含工作电极、辅助电极及参比电极)正上方(图2)。
工作电极为涂覆有四氰基醌二甲烷/碳纳米管/多聚赖氨酸复合材料的碳电极,制备方法如下:
(1)制备TCNQ/CNT修饰液
将20 mg/ml CNT的乙腈溶液和0.13 mg/ml TCNQ的乙腈溶液超声30分钟后,以1:1的比例混合后继续超声30分钟即可。
(2)制备表面涂覆有TCNQ/CNT修饰液的工作电极
将步骤(1)中制得的TCNQ/CNT复合材料分散液涂覆到丝网印刷电极的碳工作电极上,37℃下干燥即可。
(3)制备TCNQ/CNT/PL复合材料修饰的工作电极
PL用水稀释为浓度为0.01%的溶液,在TCNQ/CNT层涂覆PL溶液,孵育10分钟后用水清洗三次,晾干记得。
丝网印刷电极与电化学工作站连接,即可通过电化学方法测定细胞释放NO的含量。
图3为本实施例制备的TCNQ/CNT/PL复合材料的扫描电镜图。
应用实施例1
将一定量的NO溶液加入到实施例1中构建的NO传感器的PDMS反应池21中,在电压范围为-1~1.5V下测试该传感器对NO的循环伏安响应,同时以该传感器对磷酸缓冲液的循环伏安响应为空白对照,结果如图4所示,由图4可知,实施例1中构建的传感器在0.8V时出现NO的氧化峰,说明该传感器对NO有明显的电化学催化氧化能力。
应用实施例2
在循环伏安曲线的峰值电压0.8V下测试实施例1中构建的传感器对NO的电流-时间响应,测试时连续向实施例2中构建的传感器的PDMS反应池21中加入不同浓度的NO溶液,时间间隔为20s,记录响应时间与电流值的关系曲线,即得该传感器对NO的安培响应图,结果如图5所示,由图5可知,加入不同浓度的NO后,该传感器电流响应不断增加并在较快的时间内达到稳态,其响应时间小于3s;由图5获得的NO浓度与电流响应之间的关系曲线图,如图6所示,由图6可知,该该传感器对NO的线性响应范围为0.52~12 µM,其检测限为370 nM。
应用实施例3
将不同物质的溶液依次加入到实施例1中构建的传感器的电解液中,测试该传感器对不同干扰成份的电流-时间响应,测试电压为0.8V,加入不同干扰物的时间间隔为20s,得到该传感器对不同干扰成分选择性测试的安培响应曲线,结果如图7所示,由图7可知,该传感器对NO具有良好的选择性。
应用实施例4
测试人脐静脉内皮细胞释放的NO
使用实施例1构建的传感器进行测试,测试方法如下:
(1)TCNQ/CNT复合材料滴涂至碳工作电极上表面,晾干后在电极表面固定PDMS反应池21,无菌环境中加入PL孵育5min,晾干后形成细胞生长基底;
(2)含有人脐静脉内皮细胞的溶液加在PDMS反应池21中生长;
(3)将丝网印刷电极1与电化学工作站相连接,通过电化学方法测定细胞释放NO的含量。
图8为施例6中生长在TCNQ/CNT/PL复合材料上的人脐静脉内皮细胞荧光显微镜图,从图中可以看出大量的细胞固定在TCNQ/CNT/PL复合材料表面,且细胞有明显的伪足拉伸状态,表明TCNQ/CNT/PL复合材料使细胞的粘附更稳固,并有利于细胞的分裂增值。
将含有人脐静脉内皮细胞的溶液加在实施例1中构建的传感器PDMS反应池里,细胞直接在电极表面的修饰材料上生长,以浓度为0.01M pH=7.4的磷酸缓冲液为电解液,测试细胞在乙酰胆碱(Ach)和硝基左旋精氨酸甲酯(L-NAME)刺激下细胞释放NO的安培响应。结果如图9所示,采用不同浓度Ach刺激细胞释放NO,由图9可见明显的NO峰,且峰电流随着Ach浓度的增加而增高。加入L-NAME抑制Ach后,无NO的峰出现,表明该传感器可对细胞释所的NO实时检出。
以上所述,仅为本发明较佳的具体实施方式,但本发明保护的范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内所做的任何修改,等同替换和改进等,均应包含在发明的保护范围之内。
Claims (10)
1.一种用于一氧化氮检测的细胞传感器,其特征在于:包括丝网印刷电极和设置于丝网印刷电极表面的反应池;
所述丝网印刷电极包括基片及在基片上印刷的导轨、工作电极、参比电极和辅助电极;
所述基片上印刷三条导轨,三条导轨分别连通到工作电极、参比电极和辅助电极;各电极通过绝缘层隔开;
所述工作电极为涂覆有四氰基醌二甲烷/碳纳米管/多聚赖氨酸复合材料的碳电极;
该细胞传感器用于测试人脐静脉内皮细胞释放的NO。
2.根据权利要求1所述的细胞传感器,其特征在于:所述工作电极由四氰基醌二甲烷/碳纳米管修饰层上涂覆多聚赖氨酸层制得。
3.根据权利要求2所述的细胞传感器,其特征在于:所述四氰基醌二甲烷/碳纳米管修饰层中碳纳米管与四氰基醌二甲烷的体积比为50-250:1。
4.根据权利要求2所述的细胞传感器,其特征在于,所述工作电极的制备方法如下:将碳纳米管与四氰基醌二甲烷分散于乙腈中,获得电极修饰溶液;将所述电极修饰溶液涂覆到碳电极上,干燥即得四氰基醌二甲烷/碳纳米管修饰层;多聚赖氨酸用水稀释,在四氰基醌二甲烷/碳纳米管修饰层上涂覆聚赖氨酸溶液,经孵育后洗涤、干燥即得。
5.根据权利要求1所述的细胞传感器,其特征在于:所述导轨由导电银油墨或导电碳油墨印制于基片上形成,为三条平行导轨由宽度不同的两矩形块组成,其中端部较宽的用于连接电化学工作站,中间部分较窄的用于连接各电极。
6.根据权利要求5所述的细胞传感器,其特征在于:所述工作电极主体为圆形块扩展连接至中间导轨电极较窄部分的末端;
参比电极为印制在左侧导轨电极较窄部分的末端向中间电极延展形成的小段弧形环;
辅助电极为由右侧导轨极较窄部分的末端下部起始,先扩展至导轨电极上,再环绕工作电极,至接近左侧导轨电极较窄部分末端形成的大段弧形环。
7. 根据权利要求1所述的细胞传感器,其特征在于:所述参比电极为Ag/AgCl 电极。
8.根据权利要求1所述的细胞传感器,其特征在于:所述辅助电极为碳电极。
9.根据权利要求1所述的细胞传感器,其特征在于:所述反应池由聚二甲基硅氧烷制成。
10.权利要求1-9任一项所述的细胞传感器应用于NO测定的方法,包括以下步骤:
(1)将含有待测细胞的溶液加入反应池中,细胞在工作电极上生长;
(2)滴加药物刺激细胞释放NO;
将丝网印刷电极与电化学工作站连接,测定细胞释放NO的含量。
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