CN115704797A - 基于丝网印刷电极的电化学检测方法 - Google Patents
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Abstract
本发明涉及一种基于丝网印刷电极的电化学检测方法,具体包括以下步骤:S1蛋白结合基团的制备:对丝网印刷电极基片上的工作电极进行处理,粘附有蛋白结合基团;S2基础蛋白反应:加入基础蛋白,所述基础蛋白上的活性基团与所述步骤S1中的蛋白结合基团进行反应;S3封闭基础蛋白:采用封闭液对所述步骤S2中反应后的基础蛋白进行封闭处理,得到待检测丝网印刷电极;S4电化学检测:加入含有目标蛋白的检测试剂,所述基础蛋白与所述目标蛋白进行特异反应,并对反应后的工作电极采集数据进行电化学分析,得出检测结果。采集数据进行电化学分析,根据不同的基础蛋白和目标蛋白即可得出不同检测项目的结果,灵敏度高。
Description
技术领域
本发明涉及电化学检测技术领域,尤其涉及一种基于丝网印刷电极的电化学检测方法。
背景技术
电化学检测是一种利用物质的氧化还原特性,通过测量溶液中的电化学参数的变化,确定待测物质特性信息的方法。
在中国专利文献CN109490399A中,公开了一种电化学检测设备及电化学检测方法,包括使得所述电压扫描单元根据所述扫描波形控制信号产生多种不同波形的电压扫描信号并将所述电压扫描信号施加于所述电极单元;使得所述电极单元在所述电压扫描信号的驱动下参与电化学反应以产生检测电流信号;以及使得所述检测单元从所述电极单元获取检测电流信号。在进行电化学检测时,一般通过一个电极体系向溶液中施加具有一定电流、电位强度的电信号作为激励信号,激发溶液中发生氧化还原反应,使溶液的电化学参数产生相应的变化。通过测定溶液中的电化学参数的变化,电化学检测设备能够精确地测定溶液中待测物质的特性信息。
但在上述技术方案中,对于进行不同项目甚至是多个项目的检测,并没有进行详细的说明。
发明内容
本发明要解决的技术问题是,提供一种基于丝网印刷电极的电化学检测方法,能在复杂体系样品中进行不同的检测项目,灵敏度高。
为了解决上述技术问题,本发明采用的技术方案为:该基于丝网印刷电极的电化学检测方法,具体包括以下步骤:
S1蛋白结合基团的制备:对丝网印刷电极基片上的工作电极进行处理,粘附有蛋白结合基团;
S2基础蛋白反应:加入基础蛋白,所述基础蛋白上的活性基团与所述步骤S1中的蛋白结合基团进行反应;
S3封闭基础蛋白:采用封闭液对所述步骤S2中反应后的基础蛋白进行封闭处理,得到待检测丝网印刷电极;
S4电化学检测:加入含有目标蛋白的检测试剂,所述基础蛋白与所述目标蛋白进行特异反应,并对反应后的工作电极采集数据进行电化学分析,得出检测结果。
蛋白结合基团粘附固定在工作电极表面,当其与基础蛋白上的活性基团进行反应结合时,原有的膜电荷密度将发生改变,最终导致膜电位改变,这样就能表征或者说界定出有多少基础蛋白参与了与蛋白结合基团的反应结合;封闭液的作用是反应没有反应的基础蛋白上活性基团,然后加入渗透保护膜,使“基础蛋白上活性基团与蛋白结合基团结合”更牢固;进行电化学检测时,基础蛋白与目标蛋白进行特异反应,采集数据包括电极+电信号的采集,尤其是针对丝网印刷电极的工作电极及参考电极之间的阻抗或电压差,进行电化学分析,根据不同的基础蛋白和目标蛋白即可得出不同检测项目的结果,灵敏度高。
优选的,在所述步骤S1中,对所述工作电极的表面进行预处理,所述预处理的具体方法为:
S11清洁电极表面:机械研磨,抛光至镜面;
S12鉴定电极表面清洁度:在1×10-3mol/L的K3Fe(CN)6磷酸盐缓冲溶液中扫描,直到出现可逆的阴极和阳极峰,即表面已清洁,否则返回所述步骤S11。
需要注意的是,进行步骤S11抛光时,按粒径降低的顺序进行研磨,抛光后移入超声水浴中清洗,直至干净。
优选的,在所述步骤S1中,所述蛋白结合基团为醛基或羧基或酰基。
醛基可以采用在工作电极基片表面用25%(摩尔浓度)的戊二醛处理作为露出的蛋白结合基团。
优选的,所述步骤S2中的所述基础蛋白和所述步骤S4中的所述目标蛋白是相对应的,用于不同的检测项目。
优选的,所述步骤S2中的所述基础蛋白为肌钙蛋白cTnI抗体;所述步骤S4中的所述目标蛋白是为肌钙蛋白cTnI,用于肌钙蛋白cTnI检测项目。
肌钙蛋白由肌钙蛋白I、T、C三亚基构成,和原肌球蛋白一起通过钙离子对横纹肌动蛋白ATP酶的活性来调节肌动蛋白和肌球蛋白相互作用,当心肌损伤后,心肌肌钙蛋白复合物释放到血液中,4-6小时后,开始在血液中升高,升高的肌钙蛋白I能在血液中保持很长时间6-10天,肌钙蛋白I具有高度心肌特异性和灵敏度,所以肌钙蛋白I已成为目前最理想的心肌梗死标志物。
优选的,所述步骤S2中的所述基础蛋白为肌红蛋白MYO特异性单克隆抗体;所述步骤S4中的所述目标蛋白是为肌红蛋白MYO;用于肌红蛋白MYO检测项目。
MYO方法是基于“夹心”原理的两步法酶免疫测定,样品与包被了肌红蛋白特异性单克隆抗体的二氧化铬颗粒孵育形成颗粒/MYO复合物。颗粒被磁化分离,去除上清液。在第二步,颗粒/MYO复合物与共轭试剂(β-半乳糖苷酶标记的肌红蛋白特异性单克隆抗体)孵育形成颗粒/MYO/共轭物夹心。未结合的共轭物和分析物磁化分离和洗涤去除。夹心结合的β-半乳糖苷酶与生色团底物氯酚红-β-d吡喃半乳糖苷(CPRG),催化底物水解为氯酚红(CPR)。CPR形成在577nm光吸收改变与病人样品中存在的MYO浓度成正比。
优选的,所述步骤S2中的所述基础蛋白为肌酸激酶同工酶CK-MB抗体;所述步骤S4中的所述目标蛋白是为肌酸激酶同工酶CK-MB;用于肌酸激酶同工酶CK-MB检测项目。
肌酸激酶同工酶CK-MB检测项目:肌酸激酶是二聚体酶,其亚基有M型(肌型)和B型(脑型)两种。由三种同工酶的不同组合构成,即CK-MM、CK-MB、CK-BB。CK-MM(CK3)主要存在于横纹肌中,心肌中有少量。CK-BB(CK1)主要存在于脑组织中,其他主要器官如肝、肾、肺、脾和红细胞内含量极微,正常状态下不易检出。CK-MB(CK2)主要存在于心肌中,其他组织几乎不存在。在正常血清中,主要存在CK-MM和少量的CK-MB,CK-BB几乎不存在。血液中CK-MB明显升高提示心肌梗死,它比CK总活性测定更能判断心肌损伤,并具有更高的特异性和敏感性。
正常血清中几乎无CK-BB,抗M亚单位的抗血清可以和CK-MM及CK-MB中的M亚单位形成抗原抗体复合物,从而完全抑制M亚单位的酶活性,将测得的CK活性值乘以2则代表CK-MB的活性。肌酸激酶(CK)活性的检测原理是:CK催化磷酸肌酸与二磷酸腺苷(ADP)反应产生三磷酸腺苷(ATP)与肌酸;偶联己糖激酶(HK)及葡萄糖-6-磷酸脱氢酶(G6PDH)的催化反应。HK催化葡萄糖与ATP反应,形成葡萄糖-6-磷酸,G6PDH催化葡萄糖-6-磷酸氧化,形成6-磷酸葡萄糖内酯及NADPH,NADPH生成的速率代表CK活力。反应机理如下所示:
优选的,所述步骤S2中的所述基础蛋白为N末端脑钠肽前体NT-proBNP抗体;所述步骤S4中的所述目标蛋白是为N末端脑钠肽前体NT-proBNP;用于N末端脑钠肽前体NT-proBNP检测项目。
血液循环中同时存在proBNP、BNP、NT-proBNP。NT-proBNP是一个非活性的氨基酸片段,目前尚未发现明确的生物学功能,左室牵张及室壁张力对NT-proBNP的释放进行调节。正常人NT-proBNP和BNP总是等摩尔分泌,两者血浆浓度基本一致,但在心力衰竭时血浆NT-proBNP的浓度比BNP高2-10倍,半衰期为60~120min,比BNP(20min)长。
N末端脑钠肽前体NT-proBNP检测项目是检测人血液里NT-proBNP浓度,其中NT-proBNP值根据年龄分层的诊断届值如下表1所示:
表1 NT-proBNP根据年龄分层的诊断界值
BNP(type B natriuretic peptide/brain natriuretic peptide)最初从猪的脑组织中分离出来并被称为脑利钠肽,但其合成及分泌主要在心室肌细胞。BNP的基因存在于1号染色体短臂的末端,包括3个外显子和2个内显子,通过mRNA被转录为由108个氨基酸组成的BNP前体(proBNP),存在于心室肌的一些内分泌颗粒中。心室容量负荷或压力负荷增加,使心室肌受到牵张或室壁压力增大时,储存的proBNP即分泌释放,并很快分解为无活性的76个氨基酸组成的氨基末端proBNP(NT-proBNP)和有内分泌活性的32个氨基酸组成的BNP。BNP的作用包括利尿、利钠,扩血管及抑制肾素-血管紧张素-醛固酮系统,抑制促肾上腺皮质激素(ACTH)的释放及交感神经的过度反应,参与调节血压、血容量及盐平衡;最近有研究显示BNP还可抑制心肌纤维化、血管平滑肌细胞增生以及抗冠状动脉痉挛等作用。
优选的,所述步骤S2中的所述基础蛋白为血清淀粉样蛋白A抗体、C反应蛋白抗体;所述步骤S4中的所述目标蛋白是为血清淀粉样蛋白A、C反应蛋白;用于血清淀粉样蛋白A/C反应蛋白检测项目。
SAA:人血清淀粉样蛋白A,组织淀粉样蛋白A的前体物质,属急性时相反应蛋白,正常情况下血浆中的含量极少,当机体收到细菌、病毒、支原体、衣原体等抗原刺激后,肝脏细胞合成分泌大量的SAA进入血液,在5-6h内迅速升高约1000倍。
CRP:C反应蛋白,机体在应激状态下由肝脏合成的急性时相反应蛋白。生理功能包括感染防御及炎症过程中的吞噬和调节作用等,正常情况下血浆中的含量低,当机体收到感染、创伤后5-8h迅速升高,24h达峰值。
病毒感染时,SAA变化比CRP变化更为敏感,因此SAA水平可作为判断感染灵敏而可靠的监测指标。联合监测SAA和CRP,弥补病毒感染时CRP水平无显著变化的劣势。
优选的,所述步骤S2中的所述基础蛋白为降钙素原PCT抗体;所述步骤S4中的所述目标蛋白是为降钙素原PCT;用于降钙素原PCT检测项目。
PCT也叫作血清降钙素,其主要是从甲状腺肿瘤细胞培养液中提取的一种多肽激素,是甲状腺肿瘤血清的肿瘤标志物,因此,正常人的PCT水平指标会较低,在针对诊断感染性疾病的检出率较高。IL-6主要是一种白介素-6,是活化的T细胞以及纤维细胞的淋巴因子,其能够有效的使得患者人体中的骨髓细胞逐渐出现生长以及分化现象,从而起到增强自然杀伤细胞的裂解作用,因此,当患者受到感染时,其IL-6的指标会逐渐上升。相关资料曾经表明,PCT是一种无激素活性的降钙素前肽物质,当PCT数值逐渐升高时,表明患者出现感染性疾病,其是区别感染性疾病中比较灵敏的指标。
降钙素原(procalcitonin,PCT)是由甲状腺合成与分泌的一种蛋白质,感染时受内毒素、TNF-α等细胞因子的影响在多种组织均可产生。当严重细菌、脓毒症、多脏器功能衰竭时PCT在血浆中的水平升高,自身免疫、过敏和病毒感染时PCT不会升高。PCT反映了全身炎症反应的活跃程度,是一种用于细菌感染早期诊断、鉴别诊断及治疗监控的最新特异性指标。
优选的,所述步骤S3中,所述封闭液为BSA牛血清白蛋白或酪蛋白或无蛋白化合物。
优选的,在所述步骤S4中,采集数据包括有丝网印刷电极中工作电极和参考电极之间的阻抗或电压差。
本发明的技术方案基于丝网印刷电极的电化学检测方法,通过电化学技术检测电信号来检测试剂。通过发生(电)化学反应/相互作用并转化为一个可检测的分析信号,从而检测试剂反应。具体的:将样本固定在电极表面,然后通过生物分子间的特异性识别作用,使目标分子捕获到电极表面,基础电极将浓度信号转成电流,可测量的电信号作为相应信号,从而实现对目标分析物的定量或者定性分析。
附图说明
图1是本发明基于丝网印刷电极的电化学检测方法的流程图。
具体实施方式
为了加深对本发明的理解,下面将结合附图和实施例对本发明做进一步详细描述,该实施例仅用于解释本发明,并不对本发明的保护范围构成限定。
本实施例的基于丝网印刷电极的电化学检测方法,如图1所示,具体包括以下步骤:
S1蛋白结合基团的制备:对丝网印刷电极基片上的工作电极进行处理,粘附有蛋白结合基团;
S2基础蛋白反应:加入基础蛋白,所述基础蛋白上的活性基团与所述步骤S1中的蛋白结合基团进行反应;
S3封闭基础蛋白:采用封闭液对所述步骤S2中反应后的基础蛋白进行封闭处理,得到待检测丝网印刷电极;
所述封闭液为BSA牛血清白蛋白或酪蛋白或无蛋白化合物;
S4电化学检测:加入含有目标蛋白的检测试剂,所述基础蛋白与所述目标蛋白进行特异反应,并对反应后的工作电极采集数据进行电化学分析,得出检测结果;
采集数据包括有丝网印刷电极中工作电极和参考电极之间的阻抗或电压差。
所述步骤S2中的所述基础蛋白和所述步骤S4中的所述目标蛋白是相对应的,用于不同的检测项目,不同的检测项目,根据不同的基础蛋白对应有不同的目标蛋白;例如,(1)所述步骤S2中的所述基础蛋白为肌钙蛋白cTnI抗体;所述步骤S4中的所述目标蛋白是为肌钙蛋白cTnI,用于肌钙蛋白cTnI检测项目;(2)所述步骤S2中的所述基础蛋白为肌红蛋白MYO特异性单克隆抗体;所述步骤S4中的所述目标蛋白是为肌红蛋白MYO;用于肌红蛋白MYO检测项目;(3)所述步骤S2中的所述基础蛋白为肌酸激酶同工酶CK-MB抗体;所述步骤S4中的所述目标蛋白是为肌酸激酶同工酶CK-MB;用于肌酸激酶同工酶CK-MB检测项目;(4)所述步骤S2中的所述基础蛋白为N末端脑钠肽前体NT-proBNP抗体;所述步骤S4中的所述目标蛋白是为N末端脑钠肽前体NT-proBNP;用于N末端脑钠肽前体NT-proBNP检测项目;(5)所述步骤S2中的所述基础蛋白为血清淀粉样蛋白A抗体、C反应蛋白抗体;所述步骤S4中的所述目标蛋白是为血清淀粉样蛋白A、C反应蛋白;用于血清淀粉样蛋白A/C反应蛋白检测项目;(6)所述步骤S2中的所述基础蛋白为降钙素原PCT抗体;所述步骤S4中的所述目标蛋白是为降钙素原PCT;用于降钙素原PCT检测项目。
在所述步骤S1中,对所述工作电极的表面进行预处理,所述预处理的具体方法为:
S11清洁电极表面:机械研磨,抛光至镜面;
S12鉴定电极表面清洁度:在1×10-3mol/L的K3Fe(CN)6磷酸盐缓冲溶液中扫描,直到出现可逆的阴极和阳极峰,即表面已清洁,否则返回所述步骤S11。
在所述步骤S1中,所述蛋白结合基团为醛基或羧基或酰基;醛基可以采用在工作电极基片表面用25%(摩尔浓度)的戊二醛处理作为露出的蛋白结合基团。
对于本领域的普通技术人员而言,具体实施例只是对本发明进行了示例性描述,显然本发明具体实现并不受上述方式的限制,只要采用了本发明的方法构思和技术方案进行的各种非实质性的改进,或未经改进将本发明的构思和技术方案直接应用于其它场合的,例如更换基础蛋白和目标蛋白进行其他检测项目,均在本发明的保护范围之内。
Claims (12)
1.一种基于丝网印刷电极的电化学检测方法,其特征在于,具体包括以下步骤:
S1蛋白结合基团的制备:对丝网印刷电极基片上的工作电极进行处理,粘附有蛋白结合基团;
S2基础蛋白反应:加入基础蛋白,所述基础蛋白上的活性基团与所述步骤S1中的蛋白结合基团进行反应;
S3封闭基础蛋白:采用封闭液对所述步骤S2中反应后的基础蛋白进行封闭处理,得到待检测丝网印刷电极;
S4电化学检测:加入含有目标蛋白的检测试剂,所述基础蛋白与所述目标蛋白进行特异反应,并对反应后的工作电极采集数据进行电化学分析,得出检测结果。
2.根据权利要求1所述的基于丝网印刷电极的电化学检测方法,其特征在于,在所述步骤S1中,对所述工作电极的表面进行预处理,所述预处理的具体方法为:
S11清洁电极表面:机械研磨,抛光至镜面;
S12鉴定电极表面清洁度:在1×10-3mol/L的K3Fe(CN)6磷酸盐缓冲溶液中扫描,直到出现可逆的阴极和阳极峰,即表面已清洁,否则返回所述步骤S11。
3.根据权利要求2所述的基于丝网印刷电极的电化学检测方法,其特征在于,在所述步骤S1中,所述蛋白结合基团为醛基或羧基或酰基。
4.根据权利要求1-3任一项所述的基于丝网印刷电极的电化学检测方法,其特征在于,所述步骤S2中的所述基础蛋白和所述步骤S4中的所述目标蛋白是相对应的,用于不同的检测项目。
5.根据权利要求4所述的基于丝网印刷电极的电化学检测方法,其特征在于,所述步骤S2中的所述基础蛋白为肌钙蛋白cTnI抗体;所述步骤S4中的所述目标蛋白是为肌钙蛋白cTnI,用于肌钙蛋白cTnI检测项目。
6.根据权利要求4所述的基于丝网印刷电极的电化学检测方法,其特征在于,所述步骤S2中的所述基础蛋白为肌红蛋白MYO特异性单克隆抗体;所述步骤S4中的所述目标蛋白是为肌红蛋白MYO;用于肌红蛋白MYO检测项目。
7.根据权利要求4所述的基于丝网印刷电极的电化学检测方法,其特征在于,所述步骤S2中的所述基础蛋白为肌酸激酶同工酶CK-MB抗体;所述步骤S4中的所述目标蛋白是为肌酸激酶同工酶CK-MB;用于肌酸激酶同工酶CK-MB检测项目。
8.根据权利要求4所述的基于丝网印刷电极的电化学检测方法,其特征在于,所述步骤S2中的所述基础蛋白为N末端脑钠肽前体NT-proBNP抗体;所述步骤S4中的所述目标蛋白是为N末端脑钠肽前体NT-proBNP;用于N末端脑钠肽前体NT-proBNP检测项目。
9.根据权利要求4所述的基于丝网印刷电极的电化学检测方法,其特征在于,所述步骤S2中的所述基础蛋白为血清淀粉样蛋白A抗体、C反应蛋白抗体;所述步骤S4中的所述目标蛋白是为血清淀粉样蛋白A、C反应蛋白;用于血清淀粉样蛋白A/C反应蛋白检测项目。
10.根据权利要求4所述的基于丝网印刷电极的电化学检测方法,其特征在于,所述步骤S2中的所述基础蛋白为降钙素原PCT抗体;所述步骤S4中的所述目标蛋白是为降钙素原PCT;用于降钙素原PCT检测项目。
11.根据权利要求4所述的基于丝网印刷电极的电化学检测方法,其特征在于,所述步骤S3中,所述封闭液为BSA牛血清白蛋白或酪蛋白或无蛋白化合物。
12.根据权利要求5所述的基于丝网印刷电极的电化学检测方法,其特征在于,在所述步骤S4中,采集数据包括有丝网印刷电极中工作电极和参考电极之间的阻抗或电压差。
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