CN113740398A - 一种比率型生物传感器及用于检测muc1的方法 - Google Patents
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Abstract
一种比率型生物传感器及用于检测MUC1的方法,属生物传感器检测技术领域。构建了一种基于DNA四面体的比率型电化学适体传感器并用于真实样品中MUC1的定量测定。以电解质溶液硫堇作为参比探针(IIR),Co‑MOFs标记的适配体作为信号探针(ISP),构建比率型电化学传感器。传感器具有较强的抗干扰能力,仅需要与一种电活性物质结合。修改过程可以大大简化。具有很高的稳定性,即使在一个电极上进行十次重复扫描后,电流比(ISP/IIR)仍保持不变,极大地减少了随机误差。此外,DNA NTH有效消除了非特异性吸附,提高了检测精度。具有较大比表面积和良好电导率的黑磷被用作传感界面,进一步提高检测灵敏度,检测极限为1.34fM。
Description
技术领域
本发明属于比率型生物传感器检测技术领域,具体为一种用基于DNA四面体的比率型传感器和检测MUC1的方法。
背景技术
近年来,癌症已成为威胁世界各地人们健康的最严重疾病之一。癌症的早期诊断对于及时治疗和降低死亡率至关重要。生物标志物的存在可以指示某些疾病或感染的存在,在临床筛查癌症疾病中已变得越来越重要。MUC1是一种跨膜糖蛋白,具有超过200KD的高分子量[Florea A,Taleat Z,Cristea C,et al.Label free MUC1 aptasensors basedon electrodeposition of gold nanoparticles on screen printed electrodes[J].Electrochemistry Communications,2013,33:127-130.]。它已被用于癌症的早期诊断和生物治疗,因为它通常在恶性肿瘤组织的上皮细胞上异常表达,例如乳腺癌[Jo H H,JinH,Ban C.Dual aptamer-functionalized silica nanoparticles for the highlysensitive detection of breast cancer[J].Biosensors&Bioelectronics,2015,71:129-136.],肺[Raina D,Kosugi M,Ahmad R,et al.Dependence on the MUC1-COncoprotein in Non–Small Cell Lung Cancer Cells[J]. Molecular CancerTherapeutics,2011,10(5):806-816.],胰腺癌,卵巢癌,前列腺癌,膀胱癌,结肠癌和胃癌[Baldus S E,Zirbes T K,Engel S,et al.Correlation of the immunohistochemicalreactivity of mucin peptide cores MUC1 and MUC2 with the histopathologicalsubtype and prognosis of gastric carcinomas[J].International Journal ofCancer,1998,79(2):133–138.]。由于细胞癌变的早期阶段MUC1的含量低不易检测,因此MUC1的超灵敏检测对于癌症,尤其是乳腺癌的早期诊断非常重要。
电化学生物传感器已广泛应用于环境分析、食品加工和医学诊断等各个领域。因其仪器简单、灵敏度高等特点受到了广泛关注。近年来,比率型传感器已成为提高传感系统实际应用的一种智能选择,可用于DNA和蛋白质之间的特异性检验。比率型传感器通过内部参考探针(IR),信号探针(SP)之间的比率响应用于内置校正,可以消除DNA负载浓度不同、温度变化和非靶诱导的DNA解离所产生的影响,提供更准确的信号。目前,常见的比率电化学DNA传感器有两种工作类型。第一种类型将信号分子固定在电极上。例如谢等人设计了一种基于双信号发夹DNA的比率测定策略用于粘蛋白1的检测[Deng C Y,Pi X M,Qian P, etal.High-Performance Ratiometric Electrochemical Method Based on theCombination of Signal Probe and Inner Reference Probe in One Hairpin-Structured DNA[J].Analytical chemistry,2017,89(1):966-973.]。艾灵顿等人开发了比率式电化学DNA传感器,用于检测单核苷酸多态性(SNP)[Du Y,Lim B J,Li B L,etal.ratiometric electrochemical DNA sensors with improved robustness andreproducibility[J].Analytical chemistry,2016,86(15): 8010-8016.]。一般需要两个信号标签来标记核酸。一个用作IR,另一个用作SP。通常,二茂铁和亚甲蓝被用作比率式DNA电化学传感器的信号标签。第二种是在电解质溶液中加入 IR分子。与第一种类型相比,只需要一种电活性物质与检测探针(如核酸)偶联。因此,第二种类型更适合构建比率电化学生物传感器。
DNA作为遗传信息的载体存在于细胞核内大量存在。根据DNA特殊的空间结构,可根据碱基互补配对的原则进行自组装,与此同时空间结构具有较高的可控度和精密度,故易于组装成多种形态的DNA纳米材料。相比于传统材料,DNA纳米材料表现出易穿透带负电的细胞膜,低毒性,能抵抗核酶,具有高稳定性,能根据需要进行丰富的功能性位点修饰等优点。DNA四面体是由4条相互配对的单链DNA构成的DNA纳米结构。它通常需设计 4条单链DNA的碱基序列,根据碱基互补配对的原则,将合成的4条单链DNA等量加入到缓冲液中,通过一步退火操作,4条单链即可自动互补杂交形成具有四面体形状的三维DNA 结构[Chen XQ,Zhou G B,Song P,et al.Ultrasensitive electrochemical detection of prostate-specific antigen by using antibodies anchored on a DNA nanostructuralscaffold[J].Analytical chemistry,2014,86(15):7337-7342.]。DNA四面体可以有效消除对 DNA和蛋白质的非特异性吸附,具有很强的抗干扰能力。具有三维(3D)支架的DNA四面体也可以调节探针的空间需求,从而方便识别探针的可及性,同时提高信号标签的负载量。并且可在它单链的5'或3'端修饰特异性功能分子,实现DNA四面体功能化。
适配体是单链RNA或DNA寡核苷酸,能够特异性地有效地结合一系列蛋白质和细胞[Tuerk C,Gold L.Systematic Evolution of Ligands by Exponential Enrichment:RNALigands to Bacteriophage T4 DNA Polymerase[J].Science,1990,249(4968):505-510.]。与抗体相比,适配体更稳定,适应性更强,修饰更灵活。因此,开发一种新型的基于aptasensor 的高灵敏度和选择性定量MUC1是可取的。
发明内容
本发明的目的:为了克服现有技术的缺点,提供一种比率型生物传感器及用于检测MUC1 的方法,以提高MUC1检测的灵敏性、重现性,准确性和稳定性。
本发明产品比率型生物传感器是基于DNA四面体的传感器,其特征在于按以下步骤制得:
1、合成DNA四面体、制备Au NPs、Co-MOFs、BP无水乙醇分散液。
2、将玻碳电极GCE依次进行抛光,表面洁净化处理和干燥;最好是在麂皮绒上用Al2O3粉末进行不少于3次的抛光,然后依次用V水:V硝酸=1:1的HNO3溶液洗涤、无水乙醇洗涤,超纯水超声2.5~3.5min,晾干。
3、将9~11μL等体积混合的Au NPs@BP分散液与壳聚糖溶液滴加到GCE表面,室温下晾干。
4、将合成的四面体DNA滴于步骤3冲洗好的GCE电极上,于25~38℃下培育过夜后用PBS缓冲溶液冲洗;最好是于37℃下培育过夜。
5、将浓度为0.9~1.1mM,体积为9~11μL的MCH滴加到步骤4制备的电极表面,于36~38℃下培育8~12min,封闭非特异性结合位点;最好是将浓度为1mM的10μL MCH 滴加到步骤4制备的电极表面,于37℃下培育10min,封闭非特异性结合位点。
6、向步骤5制备的电极表面滴加9~11μL浓度为0.004pM~400pM的MUC1稀释液,于36~38℃下培育0.8~1.2h;最好是向步骤5制备的电极表面依次滴加10μL浓度为0.004pM;0.02pM;0.04pM;0.4pM;2pM;4pM;20pM;40pM;200pM;400pM的 MUC1稀释液,于37℃下培育1h。
7、向步骤6制备的电极表面滴加9~11μL标记有信号探针的Au NPs@Co-MOFs,于36~38℃培育0.8~1.2h,最后得到基于DNA四面体的比率型生物传感器。最好是向步骤6制备的电极表面滴加10μL标记有信号探针的Au NPs@Co-MOFs于37℃培育1h。
合成DNA四面体、制备Au NPs、Co-MOFs、BP无水乙醇分散液的方法为在此之前公开文献报道的已有技术。
本发明产品比率型生物传感器用于检测MUC1的方法是:将制备的传感器在硫堇电解质中通过差分脉冲伏安法DPV检测剩余在电极上的标记物中Co-MOFs的电流。
本发明构建了一种基于DNA四面体的比率型电化学适体传感器并用于真实样品中MUC1的定量测定。本发明以电解质溶液硫堇作为参比探针(IIR),Co-MOFs标记的适配体作为信号探针(ISP),构建比率型电化学传感器。比率型电化学适体传感器具有较强的抗干扰能力,可以有效地消除由于不同DNA负载密度、环境影响和仪器效率等因素造成的干扰。与常规的比率型适体传感器相比,该DNA适体传感器仅需要与一种电活性物质结合。修改过程可以大大简化。所构造的比例式适体传感器具有很高的稳定性,即使在一个电极上进行十次重复扫描后,电流比(ISP/IIR)仍保持不变。因此,极大地减少了随机误差。此外, DNANTH有效消除了非特异性吸附,提高了检测精度。具有较大比表面积和良好电导率的黑磷被用作传感界面,以进一步提高检测灵敏度。该生物传感器具有高灵敏度,检测极限为 1.34fM。由于这些优越的性能,这种比例适体传感器实现了高重现性,准确性,稳定性和灵敏性。这种通用比例传感器可用于根据适体的诱导变化检测其他目标分析物。
本发明的优点:与传统的茎环或线性DNA探针相比,本发明利用DNA四面体,可以增加信号物质的装载量,有效消除DNA和蛋白质的非特异性吸附。当引入H1/H2/DNA2三条引物扩增链,生物传感器的灵敏度显著地提高,这一特性可以用于构建新型的双信号放大型的生物传感器。实验结果表明,与传统的方法相比较,该生物传感提高了灵敏性、重现性,准确性和稳定性,并且操作简单。
附图说明
图1为实施例纳米材料的透射电镜图,其中的(A)为Co-MOFs材料,15000X;(B)为Au NPs@Co-MOFs材料,15000X;(C)为BP材料,20000X;(D)为Au NPs@BP材料, 15000X;(E)为DNA四面体。
图2为实施例的的传感体系不同修饰电极的电化学阻抗谱图,a:bare GCE;b:AuNPs@BP/GCE;c:NTH/Au NPs@BP/GCE;d:MCH/NTH/Au NPs@BP/GCE;e: MUC1/MCH/NTH/AuNPs@BP/GCE;f:DNA1-Au NPs@Co-MOFs/MUC1/MCH/NTH/ Au NPs@BP/GCE)。
图3为实施例的传感体系不同修饰电极的循环伏安法谱图,a:bare GCE;b:AuNPs@BP/GCE;c:NTH/Au NPs@BP/GCE;d:MCH/NTH/Au NPs@BP/GCE;e: MUC1/MCH/NTH/AuNPs@BP/GCE;f:DNA1-Au NPs@Co-MOFs/MUC1/MCH/NTH/Au NPs@BP/GCE)。.
图4显示实施例不同的pH值条件对传感性能的影响。
图5显示实施例MUC1的孵育时间对传感性能的影响。
图6为实施例无H1/H2/DNA2三条引物扩增链时,加入不同浓度的MUC1之后传感器的DPV响应,自下到上的8条曲线(a,b,c,d,e,f,g,h)MUC1的浓度分别为0pM;0.4pM;2pM;4pM;20pM;40pM;200pM;400pM。
图7为实施例ICo-MOFs-SP和IThi-IR分别与MUC1浓度的对数关系。
图8为实施例电流比值ICo-MOFs-SP/IThi-IR随MUC1浓度增加的响应关系。
图9为实施例电流比值ICo-MOFs-SP/IThi-IR响应和目标浓度对数的关系。
图10为实施例有H1/H2/DNA2三条引物扩增链时,加入不同浓度的MUC1之后传感器的DPV响应,自下到上的11条曲线(a,b,c,d,e,f,g,h,I,j,k)MUC1的浓度分别为0pM;0.004pM; 0.02pM;0.04pM;0.4pM;2pM;4pM;20pM;40pM;200pM;400pM。
图11为实施例ICo-MOFs-SP和IThi-IR分别与MUC1浓度的对数关系。
图12为实施例电流比值ICo-MOFs-SP/IThi-IR随MUC1浓度增加的响应关系。
图13为实施例电流比值ICo-MOFs-SP/IThi-IR响应和目标浓度对数的关系。
图14为实施例检测方法的特异性,Blank为背景实验,AFP为甲胎蛋白,CEA为癌胚抗原,PSA为前列腺特异抗原,HCG为人绒毛膜促性腺激素,CRP为C反应蛋白,Thrombin 为凝血酶,MUC1为粘蛋白。
图15为实施例检测方法的重复性。
图16为实施例检测方法的重现性,三个不同电极的电化学信号响应曲线。
图17为非比率电化学传感器的电化学信号响应比值。
图18为比率电化学传感器的电化学信号响应比值。
实施例:见图1~18,表1,表2及表3。
1.仪器和试剂
N,N-二甲基甲酰胺(DMF)均购于西陇化工有限公司(中国,广州);无水乙醇(CH3CH2OH)购于成都格雷西亚化学技术有限公司(中国,成都);6-巯基己醇(MCH, 97%),凝血酶,MUC1,癌胚抗原(CEA),C反应蛋白(CRP),前列腺特异抗原(PSA),人绒毛膜促性腺激素(HCG),甲胎蛋白(AFP),壳聚糖(CHIT),1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC),磷酸盐缓冲液(PBS,0.01mol/L,0.138mol/L NaCl, pH 7.4)和N-羟丁二酰亚胺(NHC)购于美国Sigma公司(美国);黑磷(BP)购于先丰纳米材料技术有限公司(中国,南京);氯金酸(HAuCl4·4H2O)和硝酸钴六水合物(Co (NO3)2·6H2O)购于国药集团化学试剂有限公司(中国,上海);柠檬酸三钠和4,4'-联吡啶(Bpy)购于上海阿拉丁生化技术有限公司(中国,上海);2,5-噻吩二甲酸(Tdc) 购于上海麦克林生物化学有限公司(中国,上海);硫堇购于大连美伦生物技术有限公司(中国,大连);本研究中使用的DNA样本购自上海生工生物工程股份有限公司(中国,上海)。表1给出了所有寡核苷酸的序列。所有其他试剂均为分析纯试剂,可直接使用。
TGL16离心机为长沙湘智离心机仪器有限公司产品;PHS-29A型pH计为上海精科雷磁产品;真空干燥箱为上海博迅实业有限公司产品;K-Alpha+型X-射线光电子能谱仪为美国赛默飞世尔科技公司;TEM2100透射电镜为日本电子株式会社产品;ST2200HP超声波清洗器为上海科导超声仪器有限公司产品;CHI650E电化学工作站为上海辰华仪器公司产品。
2.材料的合成
2.1四面体DNA(DNA NTH)材料的合成
将购买的Tetra-A,Tetra-B,Tetra-C和Tetra-D链分别溶于一定量的缓冲溶液(20mM tris,50mM MgCl2,pH=8.0)中。然后,将四条DNA链均匀混合,在95℃下加热2min后,在4℃下保存30min。最后将合成的DNA NTH与缓冲液以1:1的比例混合(混合后浓度为 2.5μM)[参照文献:Li J,Fan C,Pei H,et al.Smart Drug Delivery Nanocarriers withSelf-Assembled DNA Nanostructures[J].Advanced Materials,2013,25:4386-4396.]
2.2 Au NPS@Co-MOFs的合成
根据文献报道采用柠檬酸盐还原四氯金酸的方法制备金纳米颗粒[例如文献:Huang M F, Kuo Y C,Huang C C,et al.Separation of Long Double-Stranded DNA byNanoparticle-Filled Capillary Electrophoresis[J].Analytical Chemistry,2004,76(1):192-196.],量取50mL水和 500μL HAuCI4·4H2O于100mL的圆底烧瓶中,在搅拌下小火加热至混合液沸腾后,迅速加入1%的柠檬酸钠溶液1.75mL。待溶液变为酒红色后停止加热,冷却至室温就得到了金溶胶。
金属有机框架材料MOFs的合成按现有技术[例如文献:Hau H J,Bai Z Y,Wang XB, et al.Highly dispersed Co nanoparticles inlayed in S,N-doped hierarchicalcarbon nanoprisms derived from Co-MOFs as efficient electrocatalysts foroxygen reduction reaction[J].Catalysis Today,2018,318:126-131.]称取0.582g六水合硝酸钴于100mL烧杯中,溶于40mL N,N-二甲基甲酰胺(VH2O:VDMF=1:1)得到溶液A;再称取0.344g 2,5-噻吩二羧酸和0.312g 4,4’-联吡啶倒入另一烧杯中,同时缓慢加入溶液A得到溶液B。把溶液B转移至反应釜中,105℃下反应72h后,过滤得到粉红色粉末,乙醇洗3次,于45℃真空干燥 12h,即制得Co-MOFs材料。
称取50mg Co-MOFs粉末溶于25mL灭菌水中,在搅拌下缓慢滴加上述合成的金溶胶25mL,常温下搅拌24h,然后在常温下离心5min,用灭菌水清洗3次,于60℃下真空烘干24h。
2.3 Au NPs@BP复合材料的合成
称取0.0120g黑磷分散于200mL无水乙醇中,解离超声4h,离心,取上清液,即制得黑磷的无水乙醇分散液。[参照文献:Tiouitchi G,Ali M A,Benyoussef A,et al.An easyroute to synthesis high-quality black phosphorus from amorphous redphosphorus[J]. Materials Letters,2018,236:56-59.]将体积为20mL黑磷的无水乙醇分散液和Au NPs以 1:1比例混合反应搅拌24h,离心(6000rpm),用灭菌水清洗3次,分散在水中(2mg/mL)。
2.3 Au NPs@Co-MOFs标记的信号探针DNA1
将10mg Au NPs@Co-MOFs溶解在500μL H2O中。200μL NHS(100mM)和200μL EDC(400mM)转移到上述混合物中,室温下振荡2h;离心,将沉淀物分散在200μL蒸馏水中,将200μL链霉亲和素(0.5mg/L)添加到该混合溶液中于4℃下振荡4h;紧接着滴加250μL DNA1(10μM)反应2h。最后,将1mL巯基乙醇(1mM)加入上述混合物中封闭非特异性结合位点,并在4℃下摇动1h,离心(10000rpm),并用PBS冲洗。最后,将DNA1标记的Au NPs@Co-MOFs分散在500μL无菌水中,在4℃下保存备用。
2.4 Au NPs@Co-MOFs标记的信号探针H1/H2
将10mg Au NPs@Co-MOFs溶解在500μL H2O中。200μL NHS(100mM)和200μL EDC(400mM)转移到上述混合物中,室温下振荡2h;离心,将沉淀物分散在200μL蒸馏水中,将200μL链霉亲和素(0.5mg/L)添加到该混合溶液中于4℃下振荡4h;紧接着滴加250μL H1/H2(10μM)反应2h。最后,将1mL巯基乙醇(1mM)加入上述混合物中封闭非特异性结合位点,并在4℃下摇动1h,离心(10000rpm),并用PBS冲洗。最后,将DNA2标记的Au NPs@Co-MOFs分散在500μL无菌水中,在4℃下保存备用。
3.检测方法
具体操作如下:
Ⅰ.将玻碳电极(GCE)在麂皮绒上用不同粒径的Al2O3粉末进行抛光,依次用HNO3溶液(V水:V硝酸=1:1)、无水乙醇和超纯水超声3min后晾干。
Ⅱ.10μL等体积混合的Au NPs@BP分散液与壳聚糖溶液滴加到GCE表面,室温下晾干。
Ⅲ.将合成的四面体DNA滴于上述冲洗好的GCE电极上,于37℃下培育过夜后用PBS缓冲溶液冲洗。
Ⅴ.加入10μL MCH(1mM)于37℃下培育10min,封闭非特异性结合位点。
Ⅵ.加入10μL的不同浓度的MUC1稀释液,于37℃下培育1h。
Ⅶ.加入10μL Au NPs@Co-MOFs/DNA1(信号未扩大)Au NPs@ Co-MOFs/DNA2/H1/H2(信号扩大),于37℃下培育1h。
Ⅷ.最后冲洗电极后在硫堇电解质中通过差分脉冲伏安法(DPV)检测剩余在电极上的标记物中Co-MOFs的电流,硫堇电解质的电流变化不大,Co-MOFs的电流增大,从而制备了一个比率型的适体传感器。详细的原理如图1所示。
4.结果与分析
4.1.材料表征
本实施例选取BP和Co-MOFs材料作为纳米框架材料的研究对象。利用高倍透射电镜 (TEM)确定本实施例中的纳米材料是否合成成功,并观察纳米颗粒的微观形貌特征。由图1(A)可知中Co-MOFs材料为纳米棒结构。由图1(B)可知大量的Au NPs密集分布在纳米棒材料上;BP材料的结构如图1(C)所示具有清晰的晶格条纹;当金颗粒负载至BP材料上后,从图1(D)中可观察到许多小黑点在BP材料上,说明金颗粒已负载成功,利于和DNA 四面体形成稳定的Au-S键。以上结果证明纳米材料是合成成功的。
4.2修饰电极的电化学表征
电化学阻抗谱(EIS)和循环伏安法(CV)技术用于研究修饰的电极在5.0mM[Fe(CN)6] 3-/4-溶液中的固定步骤。图2显示了EIS结果,其对应于GCE表面的每个制备过程步骤。在裸电极上测出一个小的半圆(图2,曲线a,Ret=280Ω)。当Au NPs@BP沉积在GCE的表面上时,观察到较小的半圆直径(图2,曲线b,Ret=340Ω)。DNA四面体修饰修饰后,Ret (曲线c)增加到2200Ω,原因是带负电荷的寡核苷酸与带负电荷的氧化还原探针[Fe(CN)6]3-/4-之间产生静电排斥。由于MCH的存在会阻碍电子的转移,因此MCH的固定化后可能导致 Ret值增加到(图2,曲线d,Ret=2500Ω)。与MUC1连接后,Ret显着增强(图2,曲线e, Ret=3300Ω),MUC1作为生物大分子而没有优异的导电性。当标记为Au NPs@Co-MOFs 的信号探针与修饰电极特异性结合时,Ret进一步增加(图2,曲线f,Ret=4200Ω)。
图3显示了[Fe(CN)6]3-/4-在电极不同表面上的电子转移的循环伏安(CV)曲线。如图3 所示,裸露的GCE电极显示出具有优良峰形的氧化还原峰(曲线a)。当在裸露的GCE上涂覆Au NPs@BP时(图3,曲线b),氧化还原峰值电流几乎恒定,显示出这些材料的优异电导率。与DNA NTH孵育后(图3,曲线c),氧化还原峰值电流降低。然后固定了MCH,并屏蔽了活性位点后,氧化还原峰值电流被明显抑制(图3,曲线d)。当修饰的电极表面与MUC1 结合时,氧化还原峰值电流进一步降低(图3,曲线e)。将信号探针插入电极后,氧化还原峰值电流的信号再次降低(图3,曲线f)。
4.3不同pH值条件对传感性能的影响
为了达到最佳的传感性能,本实施例进行不同的pH值条件下的优化。实验结果表明随着pH值的升高,体系的信号值增大。但是当pH值超过7.5时,体系的信号值逐渐减小,当pH值为7.5条件时为传感系统提供了最大的信号与背景比值(见图4)。因此,使用 7.5的pH值进行以后的实验。
4.4.MUC1的孵育时间对传感性能的影响
为了达到最佳的传感性能,体系对MUC1的孵育时间进行了优化。实验结果表明随着 MUC1的孵育时间的增加,体系的信号值增大。但是当MUC1的孵育时间达到60min后,体系的信号值逐渐减小(见图5)。因此,使用MUC1的孵育时间为60min进行以后的实验。
4.5.分析性能
为了考察该本实施例的比率型生物传感器可以用于生物分子的定量分析,在最佳的反应条件下,传感体系检测了一系列不同浓度的MUC1。结果如图6所示当目标MUC1浓度从0.4 pM增加到400pM时,所提出的比率式适体传感器的DPV响应逐渐增加。从图7可见,当MUC1的浓度增加时,Co-MOFs-SP的电化学信号逐渐增加,但Thi-IR的DPV响应几乎相同。因此,比例响应ICo-MOFs-SP/IThi-IR随着MUC1浓度的增加而逐渐增加(图8)。图9 所示的线性回归方程为ICo-MOFs-SP/IThi-IR=1.4881lgC+1.4186,相关系数(R2)为0.997。当信噪比为3时,检测限(LOD)计算为0.134pM。
除此之外,我们引入了H1/H2/DNA2三条引物扩增链进一步降低该生物传感器的检测下限。如图10所示随着MUC1的浓度从0.004pM增加到400pM,所提出的比率式适体传感器的DPV响应逐渐增加;从图11可见,当MUC1的浓度增加时,Co-MOFs-SP的电化学信号逐渐增加,但Thi-IR的DPV响应几乎相同。因此,比例响应ICo-MOFs-SP/IThi-IR随着MUC1浓度的增加而逐渐增加(图12)。图13所示的线性回归方程为ICo-MOFs-SP/IThi-IR=1.289lgC+3.5462,相关系数(R2)为0.992。当信噪比为3时,检测限(LOD)计算为1.34fM。比未引入扩增链的放大体系的灵敏度降了2个数量级。这些结果表明引入 H1/H2/DNA2三条引物扩增链显著提高了生物传感器的灵敏度。
4.6.电化学生物传感器的选择性和重复性
特异性是一个成功的实验体系的另一个关键因素,选择了2pM的AFP,CEA,PSA,HCG, CRP和凝血酶作为干扰物质,并用于研究特异性。如图14所示,与干扰物质相比,即使当MUC1浓度低10倍时,靶物的电流响应率仍比干扰物质大得多。测量结果表明所提出的比率生物传感平台具有优异的选择性。当MUC1浓度(2pM)时,在十次重复扫描下单电极的电化学信号响应率没有明显变化,图15显示了十次重复扫描下单电极的DPV曲线,以及相对标准偏差(RSD)为2.8683%。
4.7.电化学生物传感器的重现性
为了研究该本实施例的电化学生物传感器的重现性,在相同条件下,使用十个工作电极和30个测量值来检测MUC1(2pM)。如图16所示,来自三个不同电极的电化学信号响应曲线,每个电极表面上电流信号比值几乎相同。从图17中,在30个测量值中,非比率电化学传感器的电化学信号响应变化较大,平均值为19.98μA,但在我们提出的比率电化学生物传感器中,这种变化被显著降低,30个试验的相对标准偏差(RSD)为3.18%,平均值为3.710(图 18)。上述结果表明,与非比率测量方法相比,比率电化学生物传感器更加可靠,可重现和可重复(表2)。采用比例双信号策略,可以减少背景干扰并获得出色的灵敏度。测量结果表明比率型电化学生物传感器具有优异的重现性。
4.8.电化学生物传感器的回收率
为了评价该本实施例在实际样品中的应用,采用标准加入法,将该传感器应用于人血清样品中MUC1的检测。结果见表3,三种血清样品中的MUC1(0.04μM、0.4μM和4.0μM),回收率为97.0%~104.4%,相对标准偏差为1.219%~3.569%。这些结果表明我们提出的比率生物传感器可以用于实际样品的分析。
表1为所有寡核苷酸的序列。
表2比率和非比率比率电化学传感器的重现性对比。
表3为10倍稀释人血清样品中MUC1的回收。
表1:所有寡核苷酸的序列。
表2:比率和非比率比率电化学传感器的重现性对比。
表3:10倍稀释人血清样品中MUC1的回收。
Claims (3)
1.一种比率型生物传感器,其特征在于按以下步骤制得:
(1)、合成DNA四面体、制备Au NPs、Co-MOFs、BP无水乙醇分散液;
(2)、将玻碳电极GCE依次进行抛光,表面洁净化处理和干燥;
(3)、将9~11μL等体积混合的Au NPs@BP分散液与壳聚糖溶液滴加到GCE表面,室温下晾干;
(4)、将合成的四面体DNA滴于步骤(3)冲洗好的GCE电极上,于25~38℃下培育过夜后用PBS缓冲溶液冲洗;
(5)、将浓度为0.9~1.1mM,体积为9~11μL的MCH滴加到步骤(4)制备的电极表面,于36~38℃下培育8~12min,封闭非特异性结合位点;
(6)、向步骤(5)制备的电极表面滴加9~11μL浓度为0.004pM~400pM的MUC1稀释液,于36~38℃下培育0.8~1.2h;
(7)、向步骤(6)制备的电极表面滴加9~11μL标记有信号探针的Au NPs@Co-MOFs,于36~38℃培育0.8~1.2h,最后得到基于DNA四面体的比率型生物传感器。
2.如权利要求书1所述的一种比率型生物传感器,其特征在于按以下步骤制得:
(1)、合成DNA四面体、制备Au NPs、Co-MOFs、BP无水乙醇分散液;
(2)、将玻碳电极GCE在麂皮绒上用Al2O3粉末进行不少于3次的抛光,然后依次用V水:V硝酸=1:1的HNO3溶液洗涤、无水乙醇洗涤,超纯水超声2.5~3.5min,晾干;
(3)、将9~11μL等体积混合的Au NPs@BP分散液与壳聚糖溶液滴加到GCE表面,室温下晾干;
(4)、将合成的四面体DNA滴于步骤3冲洗好的GCE电极上,于37℃下培育过夜后用PBS缓冲溶液冲洗;
(5)、将浓度为1mM的10μL MCH滴加到步骤(4)制备的电极表面,于37℃下培育10min,封闭非特异性结合位点;
(6)、向步骤(5)制备的电极表面依次滴加10μL浓度为0.004pM;0.02pM;0.04pM;0.4pM;2pM;4pM;20pM;40pM;200pM;400pM的MUC1稀释液,于37℃下培育1h;
(7)、向步骤(6)制备的电极表面滴加10μL标记有信号探针的Au NPs@Co-MOFs于37℃培育1h,最后得到基于DNA四面体的比率型生物传感器。
3.如权利要求书1所述的比率型生物传感器用于检测MUC1的方法,其特征在于:将传感器在硫堇电解质中通过差分脉冲伏安法DPV检测剩余在电极上的标记物中Co-MOFs的电流。
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