CN114034747A - 一种检测β-淀粉样蛋白寡聚物的阴极光致电化学生物传感器及其构建方法 - Google Patents
一种检测β-淀粉样蛋白寡聚物的阴极光致电化学生物传感器及其构建方法 Download PDFInfo
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Abstract
本申请公开了一种检测β‑淀粉样蛋白寡聚物的阴极光致电化学生物传感器及其构建方法,首先在ITO电极上滴加BP QDs分散液,真空过夜干燥,然后滴加PLL溶液,4℃孵育,使PLL包覆BP QDs,再取适量Apt2适体链滴加在偶联活化电极上,37℃环境下孵育过夜,使Apt2适体链中的氨基与PLL上的羧基结合,取适量6‑巯基己醇进行封板,最后将不同浓度的Aβ40寡聚体溶液与Au NRs‑Apt1探针溶液混合,滴加在电极上,37℃环境下孵育过夜。通过金纳米棒,增强大黑磷量子点在可见光和近红外区域的光电转换,提高稳定性,避免了空间位阻和表位的限制,其用于β‑淀粉样蛋白的检测,在10f mol·L‑1~100nmol·L‑1范围内具有良好线性。
Description
技术领域
本发明属于阴极光致电化学检测领域,具体涉及一种基于金纳米颗粒增强阴极光电致电化学生物传感器,传感器中采用双适体链放大技术,用于检测类淀粉蛋白。
背景技术
光致电化学检测技术以其低成本、灵敏度高、易于小型化等优点而备受关注,在临床和环境等分析领域具有广泛的应用。其中,阴极光致电化学检测技术作为一种崭新的研究方向,在生物分析领域更具有应用潜力。在阴极光致电化学过程中,工作电极一般为p型半导体。
黑磷是一种p型半导体材料,它具有柔性和机械剥离能力。近年来,黑磷量子点被成功合成,并表现出许多优异的特性,如紫外-可见吸收、荧光、近红外光热转换性能(GaoL.F.,Xu J.Y.,Zhu Z.Y.,et al.,Nanoscale,2016,8(33),15132-6.)。
发明内容
为了克服现有技术中的缺陷,本发明的目的在于提供一种阴极光致电化学检测技术、一种基于纳米金棒作为载体的双适体放大技术用于检测β-淀粉样蛋白寡聚物。
术语“Aβ”是指:β-淀粉样蛋白。术语“BP QDs”是指:黑磷量子点。术语“AβOs”是指:β-淀粉样蛋白寡聚物。术语“PLL”是指:聚赖氨酸。术语“Au NRs”是指:金纳米棒。术语“Apt1”是指:适体1。术语“Apt2”是指:适体2。术语“ITO电极”是指:氧化铟锡玻璃电极。术语“MCH”是指:6-巯基己醇。术语“bulk BP QDs”是指:裸BP。术语“BP sheets”是指:BP纳米片。术语“large BP QDs”是指:大粒径BP QDs(12nm)。术语“small BP QDs”是指:小粒径BP QDs(5nm)。
为了实现上述目的,本发明采用以下技术方案:
一种检测β-淀粉样蛋白寡聚物的阴极光致电化学生物传感器的构建方法,具体包括以下步骤:
(1)在ITO电极上滴加BP QDs分散液,真空过夜干燥,重复操作至少两次,增加BPQDs负载;
(2)将PLL溶液滴加在步骤(1)处理后的电极上,4℃孵育,使PLL包覆BP QDs;
(3)用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺溶液在室温下偶联活化步骤(2)处理的电极;
(4)取适量Apt2适体链滴加在步骤(3)处理的电极上,37℃环境下孵育过夜,使Apt2适体链中的氨基与PLL上的羧基结合;
(5)取适量6-巯基己醇滴在步骤(4)处理的电极上,室温孵育封板;
(6)将不同浓度的Aβ40寡聚体(β-淀粉样蛋白寡聚物)溶液与Au NRs-Apt1探针溶液混合,滴加在步骤(5)处理的电极上,37℃环境下孵育过夜;
所述步骤(6)中Au NRs-Apt1探针的制备方法如下:
(101)将Au NRs分散于含0.01%十二烷基硫酸钠的超纯水中;
(102)取适量TCEP溶液和巯基修饰的Apt1溶液室温下反应一段时间,加入步骤(101)所得的Au NRs溶液和氯化钠溶液室温下反应,生成Au NRs-Apt1探针;
(103)将步骤(102)所得Au NRs-Apt1溶液离心,弃上清液,上述步骤重复三遍,将所得沉淀分散于TE缓冲液中;
所述Apt1序列为:5ˊ–SH–TTTTTTTTTGCTGCCTGTGGTGTTGGGGCGGGTGCG-3ˊ,所述Apt2序列为:5ˊ–NH2–GGTGGCTGGAGGGGGCGCGAACG-3ˊ。
具体地,所述BP QDs粒径范围为5-12nm,更优选地,所述BP QDs粒径为8nm。
进一步地,步骤(6)中Aβ40寡聚体溶液的制备方法,具体包括以下步骤:
(201)取一定量Aβ单体(β-淀粉样蛋白)溶液,在恒温摇床箱中孵育,形成Aβ40寡聚体(β-淀粉样蛋白寡聚物)溶液。
优选地,所述步骤(4)中,Apt2浓度为0.01~0.1μM,可以选择0.01μM、0.1μM;最优选地,Apt2最佳浓度为0.1μM。
优选地,所述步骤(102)中,所述Au NRs溶液中金纳米棒浓度为0~1.0M,可以选择0.1M、0.2M、0.3M、0.4M、0.5M、0.6M、0.7M、0.8M、0.9M、1.0M;最优选地,金纳米棒最佳浓度为0.2M。
优选地,所述步骤(102)中,所述Apt1溶液中Apt1浓度为0.01~100μM;最优选地,Apt1最佳浓度为5.0μM。
本发明的有益效果:
(1)本发明建立的阴极光致电化学检测技术可以用于β-淀粉样蛋白的检测,在10fmol·L-1~100nmol·L-1范围内具有良好线性。
(2)金纳米棒具有表面等离子体共振特性,可增强大黑磷量子点在可见光和近红外区域的光电转换,提高稳定性。
(3)基于双适体Au NRs传感器的优势,避免了空间位阻和表位的限制;操作简单,成本低廉;可用于检测Aβ40寡聚物。
附图说明
图1A为实施例1制备的AuNRs的透射电镜图。
图1B为实施例1中涉及的Apt1、Au NRs和Au NRs-Apt1的紫外吸收光谱图;其中,曲线a为Apt1的紫外吸收光谱,曲线b为Au NRs的紫外吸收光谱,c为Au NRs-Apt1的紫外吸收光谱。
图1C为实施例2中制备的Aβ寡聚物的粒径分布图。
图2为实施例3中PEC检测Aβ40寡聚体过程中不同步骤的光电响应图(A)和电化学阻抗谱表征图(B);其中,曲线a对应ITO电极,曲线b对应BP/ITO电极,曲线c对应PLL/BP/ITO电极,曲线d对应Apt2/PLL/BP/ITO电极,曲线e对应MCH/Apt2/PLL/BP/ITO,曲线f对应Au NRs-Apt1/AβOs/MCH/Apt2/PLL/BP/ITO电极。
图3为实施例2构建的阴极光致电化学生物传感器对不同浓度的Aβ寡聚体的定量分析结果图(A)及相应的标准曲线图(B)。
图4为实施例2构建的阴极光致电化学生物传感器检测Aβ寡聚体选择性实验结果图(A)和电化学检测Aβ寡聚体稳定性实验结果图(B)。
图5为不同形貌的黑磷和不同粒径的黑磷量子点的光电流响应图。
具体实施方式
下面结合附图并通过具体实施方式来进一步说明本发明的技术方案。
本发明试验中所使用到的材料以及试验方法进行一般性的描述。虽然为实现本发明目的所使用的许多材料和操作方法是公知的,但是本发明仍然在此作尽可能的详细描述。
除非特别指明,以下实施例中所用的β-淀粉样蛋白购自上海生工生物股份有限公司。Aβ40序列为:DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAILGLMVGGVV。
除非特别指明,实验所用化学试剂都是分析纯。实验所用溶液均用超纯水配制(Milli-Q水净化系统,18.2MΩ/cm)。
以下实施例中使用的试剂和仪器如下:
试剂:
NaCl购自博迪试剂有限公司;6-巯基己醇购自阿拉丁试剂有限公司。
仪器:
超声仪,型号SB-3200DT,购自新芝生物科技有限公司。
磁力加热搅拌器,型号CJJ78-1,购自山东鄄城华鲁电热仪器有限公司。
离心机,型号TG18KR,购自东旺仪器。
真空干燥箱,购自天津泰斯特仪器有限公司。
电化学工作站,型号CHI832B,购自上海辰华仪器有限公司。
实施例1
本实施例涉及的金纳米棒(Au NRs)及Au NRs-Apt1探针的制备方法,具体包括以下步骤:
(1)将0.1mL HAuCl4溶液(5mM)与1mL CTAB溶液(0.20M)混合并保持温度为28.0℃,得深橙色溶液。加入新鲜配制的0.12mL NaBH4溶液(0.01M),搅拌2min,溶液变成浅棕色,为Au NRs种子溶液。
(2)取5mL HAuCl4溶液(5mM)与5mL CTAB溶液(0.2M)和4mL水混合,加入0.1M抗坏血酸溶液65μL,0.01M AgNO3溶液0.125mL,搅拌约2min,得无色溶液。加入0.05mL Au NR种子液,轻轻搅拌约20s,室温下老化4h,生成Au NRs。
(3)将5mL Au NRs以10000rpm离心15min,再分散于2.5mL含0.01%十二烷基硫酸钠(SDS)的超纯水中。将巯基修饰的Apt1用10mM的TE缓冲液(含1mM EDTA,pH=8)溶解,95℃加热5分钟,冰箱冷却至室温备用。
(4)取50μL,10mM TCEP(摩尔比为1:100)和50μL巯基修饰的Apt1(10μM)室温下反应1h,断开二硫键,加入1mL Au NRs溶液和300μL,0.1M氯化钠在摇床中室温下反应12h。再以12000rpm/min的转速离心10min去除未结合的Au NRs,弃上清液,上述步骤重复三遍,所得沉淀即为Au NRs-Apt1探针,将其分散于200μL TE缓冲液中备用。
所述Apt1序列为:5ˊ–SH–TTTTTTTTTGCTGCCTGTGGTGTTGGGGCGGGTGCG-3ˊ。
实施例2
Aβ40寡聚体的制备:
Aβ40单体储备液用0.01mM PBS缓冲溶液稀释,在恒温摇床箱中孵育2h(37℃,300rpm)。
图1A是Au NRs的透射电镜图像。从图中可以看出Au NRs的平均直径为20nm,长度为70±5nm(长宽比约为3.5)。图1B为制备的Au NRs-Apt1探针和Au NRs的紫外-可见吸收光谱。可以看出Apt1的特征吸收峰位于260nm(曲线a)。Au NRs的特征吸收峰位于分别位于510nm和865nm(曲线b),Au NRs-Apt1探针的吸收峰在523nm处出现了红移(曲线c),这是由于Apt1与Au NRs发生了共价结合增强了金纳米棒的聚集。紫外-可见光谱证实成功制备了Au NRs-Apt1探针。图1C为Aβ寡聚物的粒径分布图,统计分析结果显示Aβ40寡聚物的大小约为26.8±0.65nm(图1C)。
实施例3
Aβ的光致电化学检测:
(1)在ITO电极上滴加20μL BP QDs分散液,真空过夜干燥,重复两次,得到BP/ITO电极。所述BP QDs的粒径优选为8nm。
(2)将20μL,2mg/mL的PLL(二次水配制)溶液滴加在BP/ITO电极上,4℃孵育12h,得到PLL/BP/ITO电极。
(3)用0.1M 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和0.025M N-羟基琥珀酰亚胺溶液在室温下偶联活化PLL/BP/ITO电极1h。
(4)移取20μL,0.1μM的Apt2适体链滴加在偶联活化后的PLL/BP/ITO电极上,37℃环境下孵育过夜,随后使用PBS缓冲液冲洗未结合的Apt2,得到Apt2/PLL/BP/ITO电极。
(5)取10μL,10mM MCH滴在电极上室温孵育1h封板,得到MCH/Apt2/PLL/BP/ITO电极。
(6)移取10μL,不同浓度的Aβ40寡聚体溶液与10μL实施例1制备的Au NRs-Apt1探针溶液混合,滴加在MCH/Apt/PLL/BP/ITO电极上,37℃环境下孵育过夜,得到PEC传感器。
所述Apt2序列为:5ˊ–NH2–GGTGGCTGGAGGGGGCGCGAACG-3ˊ。
图2为实施例3不同步骤制备电极的光电响应和电化学阻抗谱表征,说明了方法的可行性。
图3为实施例3制备的PEC传感器对不同浓度的Aβ40寡聚物进行定量测定的结果。光电流强度随样品中目标Aβ40寡聚物浓度的增加而增加(图3A),在10.0fM~100.0nM的范围内,光电信号强度与β-淀粉样蛋白浓度的对数呈良好的线性关系图(3B)。
图4为基于双适体放大阴极光致电化学检测Aβ寡聚体选择性和稳定性实验,说明此方法具有良好的选择性和稳定性。
为了研究PEC传感器的特异性和选择性,将实施例3步骤(6)中的Au NRs-Apt1与AuNRs-Apt1的混合溶液替换为人血清中一些可能存在的癌症标志物,如甲胎蛋白(AFP)、癌胚抗原(CEA)、Aβm、Aβf。如图4A所示,对非目标分析物产生的光电流与新制备的光电阴极传感器几乎相同(即在没有目标Aβ40寡聚体的情况下)。相反,靶标Aβ40寡聚体的存在导致PEC传感器的光电流强度大幅增加。更重要的是,非检测物与检测物Aβ40寡聚体共存时,光电流没有显著的变化,这表明基于PEC传感器能够以良好的特异性和选择性适用于复杂体系中靶标Aβ40寡聚体的检测。
此外,研究了PEC传感器在450W氙灯连续“开-关”状态下的再现性。从图4B中可以看出,PEC传感器在多次光照射下具有较高的稳定性和重现性。
实施例3中所述BP QDs粒径为8nm,如图5所示BP QDs粒径范围为5、12nm具有光电信号很微弱,粒径为8nm的BP QDs显示出较高的光电流信号。
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<110> 青岛科技大学
<120> 一种检测β-淀粉样蛋白寡聚物的阴极光致电化学生物传感器及其构建方法
<130> 1
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tttttttttg ctgcctgtgg tgttggggcg ggtgcg 36
<210> 2
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ggtggctgga gggggcgcga acg 23
Claims (10)
1.一种检测β-淀粉样蛋白寡聚物的阴极光致电化学生物传感器的构建方法,其特征在于,具体包括以下步骤:
(1)在ITO电极上滴加BP QDs分散液,真空过夜干燥,重复操作至少两次,增加BP QDs负载;
(2)将PLL溶液滴加在步骤(1)处理后的电极上,4℃孵育,使PLL包覆BP QDs;
(3)用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺溶液在室温下偶联活化步骤(2)处理的电极;
(4)取适量Apt2适体链滴加在步骤(3)处理的电极上,37℃环境下孵育过夜,使Apt2适体链中的氨基与PLL上的羧基结合;
(5)取适量6-巯基己醇滴在步骤(4)处理的电极上,室温孵育封板;
(6)将不同浓度的Aβ40寡聚体溶液与Au NRs-Apt1探针溶液混合,滴加在步骤(5)处理的电极上,37℃环境下孵育过夜;
所述步骤(6)中Au NRs-Apt1探针的制备方法如下:
(101)将Au NRs分散于含0.01%十二烷基硫酸钠的超纯水中;
(102)取适量TCEP溶液和巯基修饰的Apt1溶液室温下反应一段时间,加入步骤(101)所得的Au NRs溶液和氯化钠溶液室温下反应,生成Au NRs-Apt1探针;
(103)将步骤(102)所得Au NRs-Apt1溶液离心,弃上清液,上述步骤重复三遍,将所得沉淀分散于TE缓冲液中;
所述Apt1序列为:5ˊ–SH–TTTTTTTTTGCTGCCTGTGGTGTTGGGGCGGGTGCG-3ˊ,所述Apt2序列为:5ˊ–NH2–GGTGGCTGGAGGGGGCGCGAACG-3ˊ。
2.根据权利要求1所述的检测β-淀粉样蛋白寡聚物的阴极光致电化学生物传感器的构建方法,其特征在于,所述BP QDs粒径为8nm。
3.根据权利要求1所述的检测β-淀粉样蛋白寡聚物的阴极光致电化学生物传感器的构建方法,其特征在于,步骤(6)中Aβ40寡聚体溶液的制备方法,具体为:取一定量Aβ单体溶液,在恒温摇床箱中孵育,形成Aβ40寡聚体溶液。
4.根据权利要求1所述的检测β-淀粉样蛋白寡聚物的阴极光致电化学生物传感器的构建方法,其特征在于,步骤(4)Apt2浓度为0.01~0.1μM。
5.根据权利要求1所述的检测β-淀粉样蛋白寡聚物的阴极光致电化学生物传感器的构建方法,其特征在于,所述步骤(4)中Apt2浓度为0.1μM。
6.根据权利要求1所述的检测β-淀粉样蛋白寡聚物的阴极光致电化学生物传感器的构建方法,其特征在于,步骤(102)所述Au NRs溶液浓度为0~1.0M。
7.根据权利要求1所述的检测β-淀粉样蛋白寡聚物的阴极光致电化学生物传感器的构建方法,其特征在于,步骤(102)所述Au NRs溶液浓度为0.2M。
8.根据权利要求1所述的检测β-淀粉样蛋白寡聚物的阴极光致电化学生物传感器的构建方法,其特征在于,步骤(102)所述Apt1溶液浓度为0.01~0.1μM。
9.根据权利要求1所述的检测β-淀粉样蛋白寡聚物的阴极光致电化学生物传感器的构建方法,其特征在于,步骤(102)所述Apt1溶液浓度为5.0μM。
10.根据权利要求1-9任一项所述的构建方法构建的检测β-淀粉样蛋白寡聚物的阴极光致电化学生物传感器。
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