CN108828030A - 基于AuNPs增强Na2S2O8/O2的ECL效应的蛋白激酶检测方法 - Google Patents
基于AuNPs增强Na2S2O8/O2的ECL效应的蛋白激酶检测方法 Download PDFInfo
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Abstract
本发明公开了一种基于AuNPs增强Na2S2O8/O2的ECL效应的蛋白激酶检测方法,属于电致化学发光技术领域。将多肽修饰于玻碳电极表面,然后在蛋白激酶和巯基三磷酸腺苷的作用下,使得多肽发生磷酸化反应,之后加入AuNPs,通过Au‑S键作用将AuNPs连接到多肽的巯基磷酸化位点上,从而拉近了AuNPs与电极表面的距离,AuNPs良好的电子传导性使得Na2S2O8/O2体系的ECL信号大大增强,且ECL增强的强度与蛋白激酶浓度呈正相关,据此可实现蛋白激酶活性的高灵敏和选择性检测,并用于对蛋白激酶抑制剂的筛选。
Description
技术领域
本发明涉及一种基于AuNPs增强Na2S2O8/O2的ECL效应的蛋白激酶检测方法,属于电致化学发光技术领域。
背景技术
蛋白激酶,又称蛋白质磷酸化酶,在蛋白质或多肽磷酸化的过程中发挥催化调节的作用。蛋白质或多肽的磷酸化反应主要发生在苏氨酸、丝氨酸和酪氨酸残基侧链的羟基位点上,不同类型的蛋白激酶能够识别并修饰的蛋白质位点也有所不同。蛋白激酶A(PKA),是目前所发现的具有最简单的生物结构以及最清楚生化特性的一种激酶,且活化的PKA可以识别蛋白质或多肽的丝氨酸残基侧链的羟基并催化其发生磷酸化,从而改变蛋白质或多肽的特性,最终影响有关基因的表达,而许多疾病以及癌症的发生都与PKA的异常表达有着紧密的联系。目前,蛋白激酶活性的测定方法主要有比色法、放射法、生物素/巯基标记法以及表面等离子共振法等,这些方法虽然能有效检测蛋白激酶的活性,但是存在偶联操作繁琐、标记步骤复杂且耗时等问题。因此,发展简单易行的蛋白激酶活性分析新方法具有重要意义。
电致化学发光(ECL)是在反应体系中施加一定的电压,发光试剂在电极表面发生氧化或还原反应形成激发态,激发态返回到基态过程中伴随着ECL信号的产生。ECL方法具有节约试剂、背景信号低、良好的时间和空间可控性以及易于分离目标物等优点,是一种有效的检测工具。发明人成功制备了生物素-DNA标记的葡萄糖氧化酶/AuNPs(GOx/AuNPs/DNA-biotin)纳米探针,利用生物素-亲和素作用,将GOx/AuNPs/DNA-biotin组装到经PKA和三磷酸腺苷(ATP)作用的磷酸化多肽修饰电极上,放大了鲁米诺的ECL信号并用于检测PKA活性(Liang R-P,Xiang C-Y,Zhao H-F,Qiu J-D.Highly sensitive electrogeneratedchemiluminescence biosensor in profiling protein kinase activity andinhibition using amultifunctional nanoprobe.Analytica ChimicaActa,2014,812,33-40)。金属纳米粒子的性质介于孤立的原子与大块材料之间,具有良好的水溶性、生物相容性、低毒性以及表面等离子共振等特性。AuNPs具有独特的结构以及量子尺寸效应、表面效应、体积效应等独特性能,成为材料学研究的热点,在生物分析领域有着广泛的应用。AuNPs可以提供大的电极界面面积,加速ECL发光体与电极之间的电子转移,放大ECL信号。Guo等人将两种不同的癌胚抗原(CEA)适配体分别修饰在二氧化硅掺杂的Ru(bpy)3 2+(Ru@SiO2)与AuNPs表面,存在CEA时,可形成多分子层Ru@SiO2-AuNPs结构,以Ru@SiO2作为ECL发光体,AuNPs作为局域等离子共振源用来增强ECL信号,通过ECL信号的增强效果检测CEA的浓度(Wang D,Li Y,Lin Z,Qiu B,Guo L-H.Surface-enhancedelectrochemiluminescence of Ru@SiO2for ultrasensitive detection ofcarcinoembryonic antigen.Analytical Chemistry,2015,87(12),5966-5972)。然而,基于AuNPs增强Na2S2O8/O2体系的ECL效应在蛋白激酶检测中尚未见报道。
发明内容
本发明旨在提供一种基于AuNPs增强Na2S2O8/O2的ECL效应的蛋白激酶检测方法,该方法具有极高的灵敏度和选择性,首次将AuNPs对Na2S2O8/O2体系的ECL放大效应应用于蛋白激酶活性的检测及其抑制剂分析筛选中,具有良好的应用前景。
本发明的原理是:
基于AuNPs增强Na2S2O8/O2的ECL效应的蛋白激酶检测方法,先将多肽偶联到壳聚糖修饰的玻碳电极表面制成多肽修饰电极,将该电极浸入含有蛋白激酶和巯基三磷酸腺苷(ATP-s)的三羟甲基氨基甲烷盐酸盐缓冲溶液中,使多肽发生巯基磷酸化,将巯基磷酸化多肽修饰电极浸入AuNPs溶液中,通过Au-S键作用将AuNPs连接到多肽的巯基磷酸化位点上,制成AuNPs/巯基磷酸化多肽修饰电极,以该电极为工作电极、铂丝为对电极和Ag/AgCl电极作为参比电极来构建三电极系统,将三电极系统置于Na2S2O8/O2溶液中,通过MPI-B型多参数化学分析测试系统测试光电倍增管高压为800V时AuNPs/巯基磷酸化多肽修饰电极在在-1.8~0V电位范围的ECL信号;在工作电极上施加的负电位下,O2和S2O8 2-在电极表面同时得到电子,生成相应的自由基O2 ·和SO4·-,进而O2 ·和SO4·-反应生成激发态的O2 *,激发态O2 *在回到基态的过程中产生ECL信号,AuNPs具有良好的导电性,能够催化电极表面的电子转移反应,促进O2和S2O8 2-在电极表面得到电子的进程,产生更多的O2 ·和SO4·-,进一步生成更多的激发态O2 *,从而增强了Na2S2O8/O2体系的ECL信号;随着蛋白激酶浓度的增加,组装到电极表面的AuNPs增多,AuNPs对Na2S2O8/O2体系的ECL信号增强效应越强,蛋白激酶浓度的对数与Na2S2O8/O2的ECL信号增强程度呈正相关,据此实现对蛋白激酶活性的高灵敏检测;此外,Na2S2O8/O2的ECL强度随着蛋白激酶抑制剂浓度的增加而降低,据此关系计算出蛋白激酶抑制剂的半抑制浓度,用于评价抑制剂对蛋白激酶活性的抑制效果。
本发明的技术方案如下:
基于AuNPs增强Na2S2O8/O2的ECL效应的蛋白激酶检测方法,包括如下步骤:
以AuNPs/巯基磷酸化多肽修饰电极为工作电极、铂丝为对电极和Ag/AgCl电极作为参比电极来构建三电极系统,将三电极系统置于Na2S2O8/O2溶液中,通过MPI-B型多参数化学分析测试系统测试光电倍增管高压为800V时AuNPs/巯基磷酸化多肽修饰电极在-1.8~0V电位范围的ECL信号,并根据加入的蛋白激酶浓度的对数与Na2S2O8/O2体系的ECL信号的强度之间的线性关系来实现对蛋白激酶活性的高灵敏检测;此外,还可根据加入的蛋白激酶抑制剂浓度与Na2S2O8/O2的ECL信号强度之间的线性关系来计算出蛋白激酶抑制剂的半抑制浓度,用于评价抑制剂对蛋白激酶活性的抑制效果。
本发明的蛋白激酶检测方法中所述的Na2S2O8/O2溶液为含有0.1M Na2S2O8和0.1MKCl的0.1M pH 7.4的磷酸盐缓冲溶液;
本发明中,所述AuNPs/巯基磷酸化多肽修饰电极的制备方法包括如下步骤:
(1)AuNPs的制备:在50mL烧瓶中加入0.5mL质量比为2%的HAuCl4·4H2O溶液和24.5mL超纯水,磁力搅拌并加热溶液,待溶液沸腾后迅速加入1mL质量比为5%的柠檬酸钠溶液,继续搅拌并保持沸腾,溶液在3min内逐渐变成深红色,继续保持溶液沸腾5min后,将溶液在搅拌下自然冷却至室温,制成AuNPs溶液,保存在4℃冰箱中;
(2)制备AuNPs/巯基磷酸化多肽修饰电极:将7μL壳聚糖溶液滴涂到清洗干净的玻碳电极表面,晾干后,将电极浸入含5mM N-乙基-N’-1-(3-二甲氨基丙基)碳二亚胺盐酸盐、8mM N-羟基琥珀酰亚胺和120μM多肽的HEPES缓冲溶液中反应过夜,用超纯水清洗电极表面后,将电极浸入含有100μM巯基三磷酸腺苷和不同浓度蛋白激酶的三羟甲基氨基甲烷盐酸盐缓冲溶液中,在37℃水浴中反应100min,用超纯水清洗电极表面后,将电极置于AuNPs溶液中孵育1h,制成AuNPs/巯基磷酸化多肽修饰电极。
本发明的AuNPs/巯基磷酸化多肽修饰电极制备方法中,所述的壳聚糖溶液的质量百分浓度为0.2%,配制方法为将壳聚糖加入到质量百分浓度为1%的醋酸溶液中超声溶解;所述的HEPES缓冲溶液的浓度为10mM,pH为7.4;所述的三羟甲基氨基甲烷盐酸盐缓冲溶液的浓度为20mM,pH为7.4,含20mM的MgCl2。
本发明方法应用于检测蛋白激酶活性时,Na2S2O8/O2体系的ECL信号强度与PKA浓度的对数在0.0005-0.5U/mL范围内呈良好的线性关系,检测限为0.0002U/mL。
本发明相较于现有技术,其有益效果是:
(1)本发明利用AuNPs对Na2S2O8/O2体系ECL信号的增强效应,构建了一种新型ECL方法用于蛋白激酶活性及其抑制剂分析。
(2)本发明建立的基于AuNPs增强Na2S2O8/O2的ECL效应的蛋白激酶检测方法具有灵敏度高和选择性好的特点。
附图说明
图1是(A)AuNPs的TEM图和(B)UV-Vis光谱图。
图2是ECL传感器用于检测PKA活性的原理图。
图3是电极组装过程的(A)CV和(B)EIS曲线。(a)裸电极,(b)CS/GCE,(c)多肽/CS/GCE,(d)磷酸化多肽/CS/GCE,(e)组装AuNPs后的修饰电极。电解液是含有5mM[Fe(CN)6]3-/4-和0.1M KCl的磷酸盐缓冲溶液。
图4是(a)裸电极,(b)CS/GCE,(c)多肽/CS/GCE,(d)磷酸化多肽/CS/GCE,(e)未磷酸化多肽/CS组装Au NPs后的修饰电极,(f)磷酸化多肽/CS组装AuNPs后的修饰电极的ECL图。测试溶液组成为含0.1M Na2S2O8和0.1M KCl的磷酸盐(0.1M,pH 7.4)缓冲溶液,电位范围为-1.8~0V,扫描速率为100mV/s,光电倍增管电压为800V。
图5是(A)ECL传感器对不同浓度PKA的ECL强度-时间图,(B)ECL强度与PKA浓度之间的关系曲线,内插图为ECL-lgCPKA标准曲线。
图6是(A)不同浓度鞣花酸对PKA抑制后的ECL强度-时间图,(B)ECL强度与鞣花酸浓度之间的关系曲线。
具体实施方式
下面结合附图和具体实施例对本发明作进一步阐述,本发明并不限于此。
实施例1
AuNPs的制备及表征
在50mL烧瓶中加入0.5mL质量比为2%的HAuCl4·4H2O溶液和24.5mL超纯水,磁力搅拌并加热溶液,待溶液沸腾后迅速加入1mL质量比为5%的柠檬酸钠溶液,继续搅拌并保持沸腾,溶液在3min内逐渐变成深红色,继续保持溶液沸腾5min后,将溶液在搅拌下自然冷却至室温,制成AuNPs溶液,保存在4℃冰箱中。
利用透射电子显微镜(TEM)对AuNPs的形貌进行表征,结果如图1A所示。由图1A可见,本发明方法制备的AuNPs的平均粒径约为13nm,并有良好的均一性和分散性。
采用紫外-可见吸收(UV-Vis)光谱法对AuNPs的光谱性质进行表征,结果如图1B所示。由图1B可见,AuNPs在520nm处出现了AuNPs的表面等离子共振峰,表明采用本发明方法成功制备了球形的AuNPs。
实施例2
AuNPs/巯基磷酸化多肽修饰电极的制备及表征
玻碳电极(GCE)先在粒径为1.0、0.3、0.05μm的α-Al2O3糊在麂皮上抛光,再用硝酸溶液、乙醇和超纯水分别清洗1min,用N2吹干表面;将7μL质量百分浓度为0.2%的壳聚糖(CS)溶液滴涂到玻碳电极表面,晾干后,将电极浸入含5mM N-乙基-N’-1-(3-二甲氨基丙基)碳二亚胺盐酸盐、8mM N-羟基琥珀酰亚胺和120μM多肽(序列为H2N-LRRASLGGGGR-COOH)的10mM pH 7.4的HEPES缓冲溶液中反应过夜,用超纯水清洗电极表面后,将电极浸入含有蛋白激酶和100μM巯基三磷酸腺苷(ATP-s)的三羟甲基氨基甲烷盐酸盐缓冲溶液(20mM,pH7.4,含20mM的MgCl2)中,在37℃水浴中反应100min,用超纯水清洗电极表面后,将电极置于AuNPs溶液中孵育1h,制成AuNPs/巯基磷酸化多肽修饰电极。电极的制备过程如图2所示。
以AuNPs/巯基磷酸化多肽修饰电极作为工作电极、铂丝为对电极和Ag/AgCl电极作为参比电极来构成三电极系统,采用电化学工作站进行循环伏安和电化学阻抗谱测试。
采用循环伏安(CV)法和电化学阻抗谱(EIS)对ECL传感器的构建过程进行表征,结果如图3所示。由图3A可见,氧化还原探针[Fe(CN)6]3-/4-在GCE上有一对可逆的氧化还原峰(曲线a);[Fe(CN)6]3-/4-在CS/GCE上的氧化还原峰电流稍有降低(曲线b);当把多肽修饰到CS/GCE上,峰电流进一步减小(曲线c),这是由于修饰到电极表面的多肽阻碍了Fe(CN)6 3-/4-在电极表面的电子传递和传质过程;在PKA和ATP-s的作用下,多肽发生磷酸化反应,[Fe(CN)6]3-/4-在巯基磷酸化多肽/CS/GCE上的氧化还原峰电流进一步降低(曲线d),表明在PKA的催化作用下,ATP-s上的巯基磷酸根成功转移到了多肽上;当AuNPs通过Au-S键连接到电极上之后,[Fe(CN)6]3-/4-在AuNPs/巯基磷酸化多肽/CS/GCE上的峰电流值显著降低(曲线e),表明AuNPs成功修饰到电极上,由于AuNPs表面带负电荷,同种电荷有静电排斥作用,阻碍了[Fe(CN)6]3-/4-向电极表面的靠近。
此外,还采用EIS法对ECL传感器的构建过程进行表征,结果如图3B所示。由图3B可见,在含[Fe(CN)6]3-/4-的磷酸盐缓冲溶液中,GCE的EIS的半圆直径很小(曲线a),随着CS(曲线b)、多肽(曲线c)、多肽磷酸化(曲线d)以及AuNPs(曲线e)的逐步组装,电极的电子传递阻抗逐渐增加。
电化学阻抗谱的结果与循环伏安法的结果相一致,表明采用本发明方法成功制备了ECL传感器,并可用于对蛋白激酶的检测。
实施例3
ECL响应机理探讨
将以不同物质修饰的玻碳电极作为工作电极、铂丝为对电极和Ag/AgCl电极作为参比电极构成的三电极系统置于含有0.1M Na2S2O8和0.1M KCl的0.1M pH 7.4的磷酸盐缓冲溶液中,通过MPI-B型多参数化学分析测试系统测试光电倍增管高压为800V时工作电极在-1.8~0V电位范围内的ECL信号。
对不同修饰电极分别进行ECL测量,得到ECL强度-时间曲线,结果如图4所示。由图4可见,在Na2S2O8/O2反应体系中,GCE的ECL信号强度约为400a.u.(曲线a);当把CS(曲线b)、多肽(曲线c)和磷酸化多肽(曲线d)修饰到电极表面后,Na2S2O8/O2体系的ECL信号都很低;将多肽/CS/GCE在0U/mLPKA和100μMATP-s中反应100min,再浸入AuNPs溶液中,所得电极的ECL信号(曲线e)只比磷酸化多肽/CS/GCE(曲线d)的ECL信号略有增强,因此可排除AuNPs在电极表面的非特异性吸附的影响;当将磷酸化多肽/CS/GCE浸入AuNPs溶液中,AuNPs通过Au-S键结合到多肽磷酸化位点上,AuNPs/巯基磷酸化多肽/CS/GCE在Na2S2O8/O2体系中的ECL信号显著增强(曲线f)。这是由于:Na2S2O8/O2体系中,O2和S2O8 2-在电极表面同时得到电子,生成相应的自由基O2 ·和SO4·-,进而O2 ·和SO4·-反应生成激发态的O2 *,激发态O2 *在回到基态的过程中发射ECL信号。AuNPs具有良好的导电性,能够催化电极表面的电子转移反应,促进O2和S2O8 2-在电极表面得到电子的进程,产生更多的自由基O2 ·和SO4·-,进一步生成更多的激发态O2 *,产生增强的ECL信号。
实施例4
检测蛋白激酶活性
将多肽/CS/GCE浸入含有100μM的ATP-s和不同活度PKA的三羟甲基氨基甲烷盐酸盐缓冲溶液(20mM,pH 7.4,含20mM的MgCl2)中,在37℃水浴中反应100min,用超纯水清洗电极表面后,将电极置于AuNPs溶液中孵育1h,制成AuNPs/巯基磷酸化多肽CS/GCE;将以AuNPs/巯基磷酸化多肽CS/GCE作为工作电极、铂丝为对电极和Ag/AgCl电极作为参比电极构成的三电极系统置于含有0.1M Na2S2O8和0.1M KCl的0.1M pH 7.4的磷酸盐缓冲溶液中,通过MPI-B型多参数化学分析测试系统测试光电倍增管高压为800V时工作电极在-1.8~0V电位范围内的ECL信号。根据Na2S2O8/O2体系的ECL信号强度与PKA浓度的对数的线性关系来实现对PKA的检测。
图5A为ECL传感器对不同浓度PKA的ECL强度-时间曲线。由图5A可见,随着PKA浓度的增大(a-h:0,0.0005,0.001,0.005,0.01,0.05,0.1,0.5U/mL),Na2S2O8/O2体系的ECL信号逐渐增强。由图5B可见,,Na2S2O8/O2体系的ECL信号强度与PKA浓度的对数在0.0005-0.5U/mL范围内呈良好的线性关系,检测限为0.0002U/mL。本方法的灵敏度非常高,是采用电化学方法(0.001-10U/mL)的5倍(Cui,L.;Li,Y.;Lu,M.;Tang,B.;Zhang,C.-Y.Anultrasensitive electrochemical biosensor for polynucleotide kinase assaybased on gold nanoparticle-mediated lambda exonuclease cleavage-inducedsignal amplification.Biosensors and Bioelectronics,2017,99,1-7),是荧光法(0.005-0.02U/mL)的10倍(Wang,M.;Lin,Z.;Liu,Q.;Jiang,S.;Liu,H.;Su,X.DNA-hostedcopper nanoclusters/graphene oxide based fluorescent biosensor forproteinkinase activity detection.Analytica ChimicaActa,2018,1012,66-73)。可见,本发明构建的基于AuNPs增强Na2S2O8/O2的ECL效应的蛋白激酶检测方法的灵敏度高、检测限低且线性范围宽,可用于对PKA活性的高灵敏检测。
实施例5
以鞣花酸为例对PKA的抑制剂进行筛选研究。由图6A可见,Na2S2O8/O2体系的ECL信号随鞣花酸浓度的增加而降低,当鞣花酸浓度超过6μM时,ECL信号非常小。由图6B可见,鞣花酸对PKA的半抑制浓度为3.58μM(曲线a),而另外两种非PKA特异性抑制剂如槲皮素(曲线b)和5,6-二氯-l-β-D-呋喃核糖基苯丙咪唑(曲线c)则对Na2S2O8/O2体系的ECL信号几乎没有影响,以上结果表明,鞣花酸对PKA的抑制效果最强。
Claims (9)
1.基于AuNPs增强Na2S2O8/O2的ECL效应的蛋白激酶检测方法,其特征在于,包括如下步骤:
以AuNPs/巯基磷酸化多肽修饰电极为工作电极而构建三电极系统,将三电极系统置于Na2S2O8/O2溶液中,检测AuNPs/巯基磷酸化多肽修饰电极的ECL信号,并根据加入的蛋白激酶浓度的对数与Na2S2O8/O2体系的ECL信号的强度之间的线性关系来实现对蛋白激酶活性的高灵敏检测;此外,还可根据加入的蛋白激酶抑制剂浓度与Na2S2O8/O2的ECL信号的强度之间的线性关系来计算出蛋白激酶抑制剂的半抑制浓度,用于评价抑制剂对蛋白激酶活性的抑制效果。
2.如权利要求1所述的基于AuNPs增强Na2S2O8/O2的ECL效应的蛋白激酶检测方法,其特征在于,所述三电极系统是以AuNPs/巯基磷酸化多肽修饰玻碳电极作为工作电极、铂丝为对电极和Ag/AgCl电极作为参比电极而构建的三电极系统。
3.如权利要求1所述的基于AuNPs增强Na2S2O8/O2的ECL效应的蛋白激酶检测方法,其特征在于,所述检测AuNPs/巯基磷酸化多肽修饰电极的ECL信号是通过MPI-B型多参数化学分析测试系统测试光电倍增管高压为800V时AuNPs/巯基磷酸化多肽修饰玻碳电极在-1.8~0V电位范围的ECL信号。
4.如权利要求1所的述基于AuNPs增强Na2S2O8/O2的ECL效应的蛋白激酶检测方法,其特征在于,所述的Na2S2O8/O2溶液为含有0.1M Na2S2O8和0.1M KCl的0.1M pH 7.4的磷酸盐缓冲溶液。
5.如权利要求1所述的基于AuNPs增强Na2S2O8/O2的ECL效应的蛋白激酶检测方法,其特征在于,AuNPs/巯基磷酸化多肽修饰电极的制备方法包括如下步骤:
将7μL壳聚糖溶液滴涂到清洗干净的玻碳电极表面,晾干后,将电极浸入含5mM N-乙基-N’-1-(3-二甲氨基丙基)碳二亚胺盐酸盐、8mM N-羟基琥珀酰亚胺和120μM多肽的HEPES缓冲溶液中反应过夜,用超纯水清洗电极表面后,将电极浸入含有100μM巯基三磷酸腺苷和不同浓度蛋白激酶的三羟甲基氨基甲烷盐酸盐缓冲溶液中,在37℃水浴中反应100min,用超纯水清洗电极表面后,将电极置于AuNPs溶液中孵育1h,制成AuNPs/巯基磷酸化多肽修饰电极。
6.如权利要求5所述基于AuNPs增强Na2S2O8/O2的ECL效应的蛋白激酶检测方法,其特征在于,所述的壳聚糖溶液的质量百分浓度为0.2%,配制方法为将壳聚糖加入到质量百分浓度为1%的醋酸溶液中超声溶解。
7.如权利要求5所述基于AuNPs增强Na2S2O8/O2的ECL效应的蛋白激酶检测方法,其特征在于,所述的三羟甲基氨基甲烷盐酸盐缓冲溶液的浓度为20mM,pH为7.4,含20mM的MgCl2。
8.如权利要求5所述基于AuNPs增强Na2S2O8/O2的ECL效应的蛋白激酶检测方法,其特征在于,所述的HEPES缓冲溶液的浓度为10mM,pH为7.4。
9.如权利要求5所述基于AuNPs增强Na2S2O8/O2的ECL效应的蛋白激酶检测方法,其特征在于,所述AuNPs溶液的制备方法包括如下步骤:
在50mL烧瓶中加入0.5mL质量比为2%的HAuCl4·4H2O溶液和24.5mL超纯水,磁力搅拌并加热溶液,待溶液沸腾后迅速加入1mL质量比为5%的柠檬酸钠溶液,继续搅拌并保持沸腾,溶液在3min内逐渐变成深红色,继续保持溶液沸腾5min后,将溶液在搅拌下自然冷却至室温,制成AuNPs溶液,保存在4℃冰箱中。
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