CN110082403A - 基于金钯纳米花/石墨烯复合材料的组蛋白乙酰转移酶计时-电流传感器及其应用 - Google Patents
基于金钯纳米花/石墨烯复合材料的组蛋白乙酰转移酶计时-电流传感器及其应用 Download PDFInfo
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Abstract
本发明公开了基于金钯纳米花/石墨烯复合材料的组蛋白乙酰转移酶计时‑电流传感器及其应用,首先将底物多肽利用Au‑S作用固定于金电极表面,通过乙酰化反应将乙酰辅酶A上的乙酰基转移至底物多肽的特定赖氨酸残基上,利用生成的乙酰化多肽将具有较强催化能力的乙酰基抗体‑金钯纳米花/石墨烯复合材料特异性吸附,在含有双氧水的电解质溶液中产生明显的电化学信号。在乙酰化反应中,改变p300浓度及其小分子抑制剂浓度,通过乙酰基和乙酰基抗体的特异性结合作用,探究对所制备的一系列传感器电化学信号的影响。优点是特异性好、灵敏度高、检测速度快、结果准确可靠、成本低。
Description
技术领域
本发明涉及一种计时-电流传感器及其应用,尤其是涉及基于金钯纳米花/石墨烯复合材料的组蛋白乙酰转移酶电化学检测方法,属于功能生物材料和生物传感技术领域。
背景技术
组蛋白的翻译后修饰,如乙酰化、磷酸化、甲基化、ADP-核糖基化、泛素化等在真核生物体的生理和病理过程中起着很重要的作用。其中,组蛋白的乙酰化主要由组蛋白乙酰化转移酶(HAT)催化进行,以乙酰辅酶A为反应底物,进行酶促反应,将乙酰辅酶A上的乙酰基水解,并转移到组蛋白尾部特定的赖氨酸残基上,这是一个可逆的过程。p300是一种典型组蛋白乙酰化转移酶,具有广泛的生物学功能,参与细胞的周期调控、基因转录的调控,维持某些蛋白质的功能及稳定性。在病毒癌蛋白的转录翻译、胚胎的发育等方面也具有重要作用。近年来,许多研究表明组蛋白的修饰改变在很多不同的肿瘤中都有报道。除肿瘤以外,赖氨酸的乙酰化酶功能异常会导致很多其它疾病,如炎症反应、心脏病,糖尿病等。因此,实现组蛋白乙酰化转移酶活性的超灵敏检测具有十分重要的意义。
早期检测组蛋白乙酰化转移酶的方法主要是依赖于放射自显影技术或者放射性同位素标记技术,但这两种方法需要标记放射性元素,存在耗时费力、成本较高且对环境污染较大等缺点。近年来,研究者们试图寻找一些不需要标记放射性同位素的方法对组蛋白乙酰化转移酶进行检测,但组蛋白乙酰化转移酶研究只是“冰山一角”,还处于研究的初级阶段,因此,亟待开发一种灵敏、准确、快速、简便的组蛋白乙酰转移酶检测方法。电化学传感器由于其高的灵敏度、简单的制备和操作以及快速的响应时间受到了广泛的关注。为了提高电化学传感器的灵敏度,多种不同的信号放大策略被提出。其中,酶催化受到了大量的关注由于酶的高特异性和和有效性。通常情况下,酶被标记在生物探针或纳米材料上。然而,酶标记的过程复杂且耗时。而且,在极酸或极碱的条件下,酶容易失活,这限制了酶的应用。纳米材料,作为21世纪极具发展前景的材料,不仅具备良好的导电性,优良的稳定性和高的机械强度,而且拥有可观的电化学催化能力。因此,这种无酶的电化学传感器为组蛋白乙酰化转移酶的灵敏检测提供了一个新的机遇。
本发明基于金钯纳米花/石墨烯复合材料,利用计时-电流法构建了一种新型的无酶电化学传感器,用于检测组蛋白乙酰转移酶(以HATp300为例)活性。金钯纳米花/石墨烯复合材料有两个作用:通过大的比表面积以提高乙酰基抗体的固载量;利用纳米材料本身的催化性质,构建模拟酶实现信号的放大。电化学输出信号的大小可以通过控制HATp300的量来调节,因此,可以实现HATp300活性及其抑制剂的检测。目前国内外未见基于金钯纳米花/石墨烯复合材料的组蛋白乙酰转移酶计时-电流传感器的制备及其应用的相关报道。
发明内容
本发明所要解决的技术问题是提供一种特异性好、灵敏度高、检测速度快、结果准确可靠、成本低的基于金钯纳米花/石墨烯复合材料的组蛋白乙酰转移酶计时-电流传感器及其应用。
本发明解决上述技术问题所采用的技术方案为:基于金钯纳米花/石墨烯复合材料的组蛋白乙酰转移酶计时-电流传感器及其应用,具体步骤如下:
(1)乙酰基抗体-金钯纳米花/石墨烯复合材料(AbAc-AuPd@GO)的制备
将0.1~1g石墨烯(GO)分散在10~30mL去离子水超声0.1~1h,再加入1~5mL 0.1~3g/L的氯金酸(HAuCl4)溶液和1~5mL 0.1~3g/L的氯钯酸(H2PdCl4)溶液,磁力均匀搅拌0.1~1h,置于氙弧灯下照射0.1~1h,再转入干燥箱中,30~100℃反应10~36h。最后,将样本置于氢气管式炉中100~500℃反应0.5~5h。将制备的AuPd@GO分散在去离子水中得到0.1~5mg/mL的AuPd@GO分散液,备用。
取1~300μL 0.1~5g/L乙酰基抗体(AbAc)置于氦氖激光治疗仪中照射5~60s,再添加到0.1~3mL上述AuPd@GO分散液中,30~42℃反应1~8h。
(2)组蛋白乙酰转移酶计时-电流传感器的制备
a.Au
金电极(Au)使用前分别用直径为0.1~0.5μm和0.01~0.1μm的Al2O3粉末进行打磨,利用超纯水通过超声清洗1~5次,之后置于氮气流中干燥,再将其浸泡于0.01~0.5MH2SO4溶液中,在(-0.1~0.5)V~(+0.5~1.5)V范围内进行循环伏安扫描5~30min,最后再经超纯水清洗后氮气吹干备用。
b.peptide/Au
将5~20μL 0.01~1mM底物多肽(peptide)滴涂于处理好的Au表面反应1~8h,通过Au-S键固定的底物多肽自组装单分子层,随后蒸馏水缓缓冲洗电极,再滴涂5~20μL组蛋白乙酰转移酶反应液【HATp300(200~1000nM,1~5μL)与乙酰辅酶A(1~5mM,1~5μL)在磷酸缓冲溶液(PBS)中充分混合】,将电极置于25~37℃恒温箱中孵育30~120min。
c.AbAc-AuPd@GO/peptide/Au
取(1)中制备的AbAc-AuPd@GO溶液2~12μL滴涂于peptide/Au电极上,30~42℃下静置10~60min,随后蒸馏水缓缓冲洗电极,之后用于计时-电流检测。
在电极制备过程的乙酰化反应中,改变p300浓度,探究其对电化学信号的影响。所述的电化学参数条件如下:计时-电流法,电压:-0.3V;时间:100s。实验中用到的磷酸缓冲溶液(PBS)(O.1M,pH 7.0),通过0.1M的磷酸二氢钠和0.1M的磷酸氢二钠进行配置,调节体积比来控制pH,作为电解质溶液时,含有5mM的双氧水溶液。
发明原理:利用上述基于金钯纳米花/石墨烯复合材料的组蛋白乙酰转移酶计时-电流传感器及其应用,基于乙酰基抗体-金钯纳米花/石墨烯复合材料,利用计时-电流法构建了一种新型的无酶电化学传感器,用于检测组蛋白乙酰转移酶活性。首先将底物多肽利用Au-S作用固定于金电极表面,再将乙酰化反应液滴涂于电极表面进行乙酰化反应,将乙酰辅酶A上的乙酰基转移至底物多肽的特定赖氨酸残基上,生成乙酰化多肽,再在电极表面滴入具有较强的催化能力乙酰基抗体-金钯纳米花/石墨烯复合材料,将制备好的电极置于含有双氧水的电解质溶液中,出现明显的电化学催化信号。乙酰化反应过程中,随着p300浓度变大,被乙酰化的底物多肽变多,可以通过乙酰基和乙酰基抗体的特异性结合作用,增加乙酰基抗体-金钯纳米花/石墨烯复合材料的负载量。显然,在浓度一定范围内,目标物浓度越大,电流响应越明显。实验结果表明,电流的大小与目标物的浓度在一定范围内呈线性关系,实现对目标物的检测。其优点在于:
(1)高灵敏度。实验得出传感器的电化学响应对p300浓度对数值的线性相关方程为y=10.084lgCp300+40.988,R2=0.9889,线性范围为0.001~1000nM,检测限为0.37pM,由此说明传感器对p300可实现高灵敏检测。
(2)高特异性。常见其他相关酶对本检测体系均无干扰。原因在于:本发明是基于p300催化乙酰化反应将乙酰辅酶A上的乙酰基转移到底物多肽的赖氨酸残基上,乙酰基的生成影响到乙酰基抗体-金钯纳米花/石墨烯复合材料的负载量,从而影响电化学信号的生成,其他酶无法催化乙酰化反应,故对本检测体系无干扰。
(3)结果准确。回收率均在90%~110%之间。
(4)抑制剂。利用该计时-电流传感器对双氧水的电化学响应,实现对HATp300抑制剂(如C646)的检测,可以得出传感器的电化学响应与HATp300抑制剂的相关关系。
(5)制备与检测方法试剂用量少、检测速度快、成本低。
综上所述,本发明制备基于金钯纳米花/石墨烯复合材料的组蛋白乙酰转移酶计时-电流传感器及其应用,具有灵敏度高、选择性好、操作简单、分析快速、易于操作等优点,可以实现低浓度HATp300的检测及其小分子抑制剂的筛选,具有良好的应用前景。
附图说明
图1为本发明传感器的可行性实验图;
图2为本发明传感器对有无p300的电化学响应;
图3为本发明传感器对不同浓度p300的电化学响应对p300浓度的对数校准曲线图;
图4为本发明传感器的选择性和干扰性实验图;
图5为不同浓度C646对p300活性的抑制作用。
具体实施方式
以下结合附图实施例对本发明作进一步详细描述。
实施例1 传感器的制备
(1)乙酰基抗体-金钯纳米花/石墨烯复合材料(AbAc-AuPd@GO)的制备
将0.5g石墨烯(GO)分散在20mL去离子水超声0.5h,再加入2.5mL 1g/L的氯金酸(HAuCl4)溶液和2.5mL 1g/L的氯钯酸(H2PdCl4)溶液,磁力均匀搅拌0.5h,置于氙弧灯下照射0.5h,再转入干燥箱中,60℃反应24h。最后,将样本置于氢气管式炉中300℃反应2h。将制备的AuPd@GO分散在去离子水中得到1mg/mL的AuPd@GO分散液,备用。
取100μL 1g/L乙酰基抗体(AbAc)置于氦氖激光治疗仪中照射30s,再添加到1mL上述AuPd@GO分散液中,37℃反应4h。
(2)组蛋白乙酰转移酶计时-电流传感器的制备
a.Au
金电极(Au)使用前分别用直径为0.3μm和0.05μm的Al2O3粉末进行打磨,利用超纯水通过超声清洗3次,之后置于氮气流中干燥,再将其浸泡于0.1M H2SO4溶液中,在-0.3V~+1.2V范围内进行循环伏安扫描5min,最后再经超纯水清洗后氮气吹干备用。
b.peptide/Au
将10μL 0.2mM底物多肽(peptide)滴涂于处理好的Au表面反应4h,通过Au-S键固定的底物多肽自组装单分子层,随后蒸馏水缓缓冲洗电极,再滴涂10μL组蛋白乙酰转移酶反应液【HATp300(500nM,2μL)与乙酰辅酶A(2.5mM,2μL)在磷酸缓冲溶液(PBS)中充分混合】,将电极置于30℃恒温箱中孵育80min。
c.AbAc-AuPd@GO/peptide/Au
取(1)中制备的AbAc-AuPd@GO溶液6μL滴涂于peptide/Au电极上,37℃下静置30min,随后蒸馏水缓缓冲洗电极,之后用于计时-电流检测。
检测制备的三种电极在PBS(0.1M,pH 7.0)电解质溶液中的电化学响应,见图1。可看出制备的AbAc-AuPd@GO/peptide/Au相比较于其他两种电极,电化学响应很明显,证明所希望的电极成功组装,也说明了AbAc-AuPd@GO的电催化能力。
实施例2 有无p300的电化学响应
基于金钯纳米花/石墨烯复合材料的组蛋白乙酰转移酶计时-电流传感器及其应用,利用实施例1制备我们的生物传感器探讨组蛋白乙酰转移酶的检测。见图2,无p300时,传感器在PBS(0.1M,pH 7.0)中基本无电化学响应,而在p300存在时,存在明显的电化学响应,证明该传感器可用于p300活性检测。
实施例3 p300活性检测
基于金钯纳米花/石墨烯复合材料的组蛋白乙酰转移酶计时-电流传感器及其应用,传感器的制备步骤同具体实施例1,在乙酰化反应过程中,依次改变p300的浓度,p300的浓度为:0,0.001,0.005,0.01,0.02,0.05,0.1,0.2,0.5,1,2,5,10,20,50,100,150,200,300,500,700,1000nM,随后用于制备传感器。记录传感器在PBS(0.1M,pH 7.0)中的电化学响应,根据实验结果,获得一系列不同浓度p300对应的电化学响应曲线,建立电化学响应电流大小与p300浓度之间的定量关系,根据两者之间的定量关系,确定待测样品中p300的浓度。实验结果如图3所示,说明随着p300浓度增大,传感器的电化学响应越明显,线性相关方程为y=10.084lgCp300+40.988,R2=0.9889,线性范围为0.001~1000nM,检测限为0.37pM,说明传感器对p300活性可实现高灵敏检测。
实施例4 特异性及抗干扰性检测
选择性实验中p300及其他酶的浓度均为100nM,所用到的其他酶的缩写如下:蛋白激酶(PKA)、乙酰胆碱酯酶(AChE)、末端转移酶(TdT)、碱性磷酸酶(ALP)。按上述实施例1的传感器制备步骤,乙酰化反应中,用其他相同浓度的酶代替p300,制备得到传感器。结果如图4所示,与p300对比,传感器对其他酶的电化学响应非常小,基本接近空白信号,说明传感器对于p300的检测有很好的选择性。同时,将这四种酶分别添加到p300反应体系中(与p300共存),发现对电化学信号影响可以忽略不计,说明了该传感器的强抗干扰能力。
实施例5 p300抑制剂C646的检测
基于金钯纳米花/石墨烯复合材料的组蛋白乙酰转移酶计时-电流传感器及其应用,传感器的制备步骤同具体实施例1,在乙酰化反应过程中,p300的浓度为100nM,依次加入不同浓度的抑制剂C646,C646浓度为0,0.1,0.5,1,5,10,20,30,50,70,100,150,200,300,400,500,600,700,800,1000μM,随后用于制备传感器。记录传感器在PBS(0.1M,pH7.0)中的电化学响应。根据实验结果得知(如图5),随着抑制剂C646浓度的增大,相对应的电流响应越弱,说明C646对p300活性的抑制作用越强,半抑制浓度为29.5μM。
当然,上述说明并非对本发明的限制,本发明也并不限于上述举例。本技术领域的普通技术人员在本发明的实质范围内做出的变化、改型、添加或替换,也应属于本发明保护范围。
Claims (5)
1.基于金钯纳米花/石墨烯复合材料的组蛋白乙酰转移酶计时-电流传感器及其应用,其特征在于,机理如下:利用乙酰基抗体-金钯纳米花/石墨烯复合材料,通过计时-电流法构建了一种新型的无酶电化学传感器,用于检测组蛋白乙酰转移酶活性。金钯纳米花/石墨烯复合材料有两个作用:通过大的比表面积以提高乙酰基抗体的固载量;利用纳米材料本身的催化性质,构建模拟酶实现信号的放大。
2.基于金钯纳米花/石墨烯复合材料的组蛋白乙酰转移酶计时-电流传感器及其应用,具体步骤如下:
(1)乙酰基抗体-金钯纳米花/石墨烯复合材料(AbAc-AuPd@GO)的制备
将0.1~1g石墨烯(GO)分散在10~30mL去离子水超声0.1~1h,再加入1~5mL0.1~3g/L的氯金酸(HAuCl4)溶液和1~5mL 0.1~3g/L的氯钯酸(H2PdCl4)溶液,磁力均匀搅拌0.1~1h,置于氙弧灯下照射0.1~1h,再转入干燥箱中,30~100℃反应10~36h。最后,将样本置于氢气管式炉中100~500℃反应0.5~5h。将制备的AuPd@GO分散在去离子水中得到0.1~5mg/mL的AuPd@GO分散液,备用。
取1~300μL 0.1~5g/L乙酰基抗体(AbAc)置于氦氖激光治疗仪中照射5~60s,再添加到0.1~3mL上述AuPd@GO分散液中,30~42℃反应1~8h。
(2)组蛋白乙酰转移酶计时-电流传感器的制备
a.Au
金电极(Au)使用前分别用直径为0.1~0.5μm和0.01~0.1μm的Al2O3粉末进行打磨,利用超纯水通过超声清洗1~5次,之后置于氮气流中干燥,再将其浸泡于0.01~0.5M H2SO4溶液中,在(-0.1~0.5)V~(+0.5~1.5)V范围内进行循环伏安扫描5~30min,最后再经超纯水清洗后氮气吹干备用。
b.peptide/Au
将5~20μL 0.01~1mM底物多肽(peptide)滴涂于处理好的Au表面反应1~8h,通过Au-S键固定的底物多肽自组装单分子层,随后蒸馏水缓缓冲洗电极,再滴涂5~20μL组蛋白乙酰转移酶反应液【HAT p300(200~1000nM,1~5μL)与乙酰辅酶A(1~5mM,1~5μL)在磷酸缓冲溶液(PBS)中充分混合】,将电极置于25~37℃恒温箱中孵育30~120min。
c.AbAc-AuPd@GO/peptide/Au
取(1)中制备的AbAc-AuPd@GO溶液2~12μL滴涂于peptide/Au电极上,30~42℃下静置10~60min,随后蒸馏水缓缓冲洗电极,之后用于计时-电流检测。
3.根据权利要求1~2所述的电化学传感器的制备,其特征在于联合新型金钯纳米花/石墨烯复合材料和计时-电流法来实现p300的电化学检测。
4.根据权利要求1~3所述的计时-电流传感器的制备,可以实现不同浓度p300的检测,检测限低至0.37pM。
5.根据权利要求1~4所述的电化学传感器的制备,对p300小分子抑制剂C646进行检测,半抑制浓度为29.5μM。
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