CN110804435B - 基于木瓜蛋白酶为模板的荧光铂纳米簇、制备方法及其应用 - Google Patents
基于木瓜蛋白酶为模板的荧光铂纳米簇、制备方法及其应用 Download PDFInfo
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Abstract
本发明提供了一种基于木瓜蛋白酶为模板的铂纳米簇及其制备方法和应用。其具体包括如下步骤:将木瓜蛋白酶溶液与高价铂配合物的水溶液进行混合,然后加NaOH溶液调节pH值为8~13,避光水浴反应完全后即得。本发明制备的铂纳米簇对溶菌酶响应灵敏,检测体系操作简便、可控、耗时短,可应用于尿液中溶菌酶含量的检测。本发明制备的木瓜蛋白酶铂纳米簇极大地增强了木瓜蛋白酶的活性,故该合成物质除木瓜蛋白酶原有的应用如在医药、食品、饲料、纺织等方面外,还可基于其荧光特性用于追踪木瓜蛋白酶的应用效果及检测样品中溶菌酶的含量。
Description
技术领域
本发明属于荧光探针技术领域,具体涉及一种以木瓜蛋白酶为模板的铂纳米簇及其制备方法和应用。
背景技术
木瓜蛋白酶(Papain)是一种主要来源于番木瓜青果乳汁中的巯基蛋白酶,具有水解酰胺键和酯键的特性,有很强的分解蛋白质能力,同时木瓜蛋白酶具有热稳定性好、酶活力高等特点,在医药、食品、纺织、制革等领域具有广泛的应用。木瓜蛋白酶由212个氨基酸组成,Cys25、His159和Asp158三个氨基酸构成了它的催化三联体,另外六个半胱氨酸残基组成了三对二硫键,且都不在活性位点上。我国广东、广西、海南、云南、福建、四川、贵州及台湾等省具有丰富的番木瓜资源,但尚未充分加以利用。因此,为了充分利用我国丰富的番木瓜资源,拓展木瓜蛋白酶的应用具有一定的意义。
探针技术与一般的检测方法相比具有专一性强、灵敏度高、快速准确等特点,这些特点使得探针技术成为现代科学技术不可或缺的重要检测手段。作为新一代荧光标记材料,金属纳米簇以其易于合成、超小尺寸、良好的生物相容性以及生物相容好等优点备受研究者关注。其中铂纳米簇作为近年来发展起来的荧光探针中的一种,在食品、环境、医学等检测领域发挥了重要作用。
溶菌酶是一种结构清楚、化学性质稳定、来源广泛的酶,已成为一种模式蛋白应用于研究生理条件的变化对于蛋白质结构功能的影响,并逐渐应用于人类疾病的研究上。例如通过监测尿液中溶菌酶的含量,可以初步鉴别肾小管疾病、急性单核细胞白血病等病症。但是怎样高效、简便地检测尿液中的溶菌酶含量是目前需要解决的一大难题。
发明内容
为了解决上述技术问题,本发明提供了一种生物相容性好、光稳定性强的木瓜蛋白酶-铂纳米簇,本发明还提供了纳米簇的制备方法和应用。本发明的木瓜蛋白酶-铂纳米簇荧光信号强且稳定,制备方法简便易操作。其既可以追踪木瓜蛋白酶的应用效果,又可以应用于溶菌酶的定量检测,检测方法简便、快捷、高效、易实现。
本发明的一种荧光铂纳米簇,所述纳米簇以木瓜蛋白酶作为模板和还原剂。
优选纳米簇的粒径小于5nm,其荧光最大激发波长为380nm,最大发射波长为490nm。
本发明还提供了所述的荧光铂纳米簇的制备方法,所述方法为将木瓜蛋白酶的水溶液与高价铂配合物的水溶液进行混合,然后加NaOH溶液调节pH值为8~13,避光水浴反应完全后即得。
本发明所述高价铂配合物为六氯铂酸(H2PtCl6)、六氯铂酸盐、四氯铂酸或四氯铂酸盐中的任意一种或多种组合。进一步优选所述高价铂配合物为H2PtCl6。
更具体的,本发明所述铂纳米簇的制备方法包括如下步骤:
(1)制备25-50mg/mL的木瓜蛋白酶水溶液;
(2)制备浓度为25-100mM的H2PtCl6的水溶液;
(3)将H2PtCl6溶液加入木瓜蛋白酶水溶液中,使其中的木瓜蛋白酶与H2PtCl6两者的摩尔比为1.5:1-10.5:1,充分混匀;
(4)向步骤(3)得到的混合溶液中添加NaOH调节溶液的pH值至8-13,涡旋器充分混匀5-10min;
(5)将步骤(4)得到的混合溶液在25℃~65℃条件下水浴6~48h后即得。
优选所述步骤(1)中木瓜蛋白酶水溶液的浓度为50mg/mL;步骤(2)中H2PtCl6溶液浓度为25mM;步骤(3)中木瓜蛋白酶与H2PtCl6两者的摩尔为3.5:1;步骤(4)中NaOH的浓度为1M,溶液pH值为12;所述步骤(5)的水浴温度为37℃,水浴时间为36h。
本发明还提供了所述的荧光铂纳米簇在溶菌酶检测中的应用。
优选其检测溶菌酶的方法包括如下步骤:
(1)将荧光铂纳米簇溶液和不同浓度的溶菌酶溶液混合,采用荧光分光光度计分别检测各混合溶液的荧光强度值,绘制各溶液荧光强度值和与之对应的不同浓度的溶菌酶溶液之间的标准曲线;
(2)将荧光铂纳米簇溶液和待测样品进行混合,检测加入待测样品后的溶液的荧光强度值,将数值带入上述标准曲线计算即可得到待测样品中的溶菌酶浓度值。
优选其检测溶菌酶的线性范围为10μg/mL-200μg/mL,检测限为0.55μg/mL。
本发明所述的荧光铂纳米簇在溶菌酶检测中的应用,优选其应用范围是可以用在人体尿液中溶菌酶的检测。
本发明还提供了所述的荧光铂纳米簇在追踪木瓜蛋白酶的应用效果中的应用。
经过本发明实验发现,本发明以木瓜蛋白酶为模板制备的荧光铂纳米簇比木瓜蛋白酶的活性有极大的增强,故该合成物质除木瓜蛋白酶原有的应用如在医药、食品、饲料、纺织等方面外,还可基于其荧光特性用于追踪木瓜蛋白酶的应用效果。
有益效果
本发明利用简单的一步合成法成功制备了一种发绿色荧光的铂纳米簇。本发明制备的木瓜蛋白酶-铂纳米簇(Papain-Pt NCs)合成简便,荧光信号强且稳定。本发明极大地增强了木瓜蛋白酶的活性,可以追踪木瓜蛋白酶的应用效果。另外本发明经过研究发现溶菌酶可定量影响木瓜蛋白酶铂纳米簇荧光强度,在此基础上建立了一种以木瓜蛋白酶铂纳米簇为荧光探针定量检测溶菌酶的方法,可应用于人体尿液中检测等。本发明对溶菌酶的检测简便、快捷、高效、易实现。
附图说明
图1为实施例1所制的Papain-Pt NCs的透射电镜图。
图2为实施例1所制的Papain-Pt NCs的激发-发射荧光光谱图。
图3为实施例1所制的Papain-Pt NCs的紫外-可见吸收光谱图。
图4为实施例1所制的Papain-Pt NCs的红外-可见吸收光谱图。
图5为实施例1所制的Papain-Pt NCs及Papain酶活检测图。
图6为实施例1所制的Papain-Pt NCs应用于酶检测的柱状图。
图7为实施例1所制的Papain-Pt NCs应用于检测溶菌酶的荧光光谱图。
图8为实施例1所制的Papain-Pt NCs应用于检测溶菌酶的线性关系图。
具体实施方式
下面结合说明书附图介绍本发明的较佳实施例,举例证明本发明可以实施,通过向本领域中的技术人员完整介绍本发明,使其技术内容更加清楚和便于理解。本发明可以通过许多不同形式的实施例来得以体现,其保护范围并非仅限于文中提到的实施例,本文的附图和说明本质上是举例说明而不是限制本发明。
下述实施例中的实验方法,如无特殊说明,均为常规方法。
下述实施例中所用的原材料、试剂等,如无特殊说明,均可从商业途径得到或已公开。
实施例1:
木瓜蛋白酶包裹的铂纳米簇的制备
具体步骤如下:
(1)取500mg木瓜蛋白酶加入10mL纯水中制备成50mg/mL的木瓜蛋白酶溶液;
(2)将H2PtCl6加水配置成浓度为25mM的H2PtCl6溶液;
(3)将160μL的H2PtCl6溶液加入到5.6mL的木瓜蛋白酶溶液中,使得混合溶液中木瓜蛋白酶和H2PtCl6的摩尔比为3.5:1,涡旋器充分混匀5min;
(4)加入1M NaOH于步骤(3)得到的溶液中调节pH值为12,涡旋器再次混匀5min;
(5)将所制备的样品在37℃下避光水浴36h,得到铂纳米簇的聚合物,5,000rpm/min离心10min后,取上清液并置于4℃冰箱避光保存备用。
本实施例铂纳米簇的透射电镜图(TED图)如图1所示,从图1可知,铂纳米簇大小均一呈分散状态,粒径小于5nm;
本实施例铂纳米簇的荧光激发发射光谱图如图2所示,从图2中可知,绿色荧光的铂纳米簇的最大激发波长在380nm处,最大发射波长在490nm处;
本实施例铂纳米簇的UV-vis吸收光谱图如图3所示,从图3中可知,实施例1所制的Papain-Pt NCs在300-350nm范围具有较宽的吸收图谱,而Papain在此处没有最大吸收,证明实施例1所制的Papain-Pt NCs和Papain本身不是同一种物质。
本实施例铂纳米簇在自然光下为浅棕色,在365nm的紫外灯下为绿色;
本实施例铂纳米簇的红外光谱图如图4所示,Papain和实施例1所制的Papain-PtNCs在1000-1250cm-1处有明显不同,证明实施例1所制的Papain-Pt NCs和Papain本身不是同一种物质。
实施例2:木瓜蛋白酶包裹的铂纳米簇的制备
具体步骤如下:
(1)取木瓜蛋白酶加入纯水中制备成50mg/mL的木瓜蛋白酶溶液;
(2)将H2PtCl6加水配置成浓度为25mM的H2PtCl6溶液;
(3)将H2PtCl6溶液加入到木瓜蛋白酶溶液中,使其中的木瓜蛋白酶与H2PtCl6两者的摩尔比为2.5:1,涡旋器充分混匀10min;
(4)加入1M NaOH于步骤(3)得到的溶液中调节pH值为8,涡旋器再次混匀10min;
(5)将所制备的样品在25℃下避光水浴14h,得到铂纳米簇的聚合物,5,000rpm/min离心10min后,取上清液并置于4℃冰箱避光保存备用。
实施例3:木瓜蛋白酶包裹的铂纳米簇的制备
具体步骤如下:
(1)取木瓜蛋白酶加入纯水中制备成50mg/mL的木瓜蛋白酶溶液;
(2)将H2PtCl6加水配置成浓度为25mM的H2PtCl6溶液;
(3)将H2PtCl6溶液加入到木瓜蛋白酶溶液中,使其中木瓜蛋白酶与H2PtCl6两者的摩尔比为4:1,涡旋器充分混匀6min;
(4)加入1M NaOH于步骤(3)得到的溶液中调节pH值为9,涡旋器再次混匀8min;
(5)将所制备的样品在65℃下避光水浴48h,得到铂纳米簇的聚合物,5,000rpm/min离心10min后,取上清液并置于4℃冰箱避光保存备用。
实施例4:Papain-Pt NCs及Papain酶活检测
采用紫外分光光度法对Papain及实施例1所制的Papain-Pt NCs进行酶活性测定。检测标准体系为1mL:940μL的pH值为6.8的PBS缓冲液,10μL 2mg/mL酪蛋白,50μL不同浓度的Papain或Papain-Pt NCs,25℃温育5min,使用TU-1810紫外-可见分光光度计在275nm下检测样品的吸光值,实验独立重复3次,结果见图5。
图5结果表明,制备的Papain-Pt NCs对Papain的活性有极大的增强,故该合成物质除木瓜蛋白酶原有的应用如在医药、食品、饲料、纺织等方面外,还可基于其荧光特性用于追踪木瓜蛋白酶的应用效果。
实施例5:Papain-Pt NCs对不同酶的选择性实验
具体步骤如下:
对实施例1所制备的Papain-Pt NCs进行酶选择性实验。使用RF-5301荧光分光光度计检测不同的酶对Papain-Pt NCs纳米簇的荧光强度的影响。所用的9种酶分别为胃蛋白酶,果胶酶,淀粉酶,菠萝蛋白酶,脂肪酶,溶菌酶,糖化酶,蜗牛酶,胰蛋白酶。
检测标准体系为1mL,其中浓度均为1mg/mL的不同酶100μL,Papain-Pt NCs 50μL,加去离子水补足至1mL,25℃水浴2h后,在激发光波长为380nm的条件下,检测溶液在490nm的发射光的峰高。结果显示胃蛋白酶,果胶酶,淀粉酶,菠萝蛋白酶,脂肪酶,糖化酶,蜗牛酶,胰蛋白酶均对体系的荧光强度没有明显的影响,而加入溶菌酶可以使体系的荧光强度显著降低,说明铂纳米簇对溶菌酶具有良好的选择性(图6)。
实施例6:一种以Papain-Pt NCs为荧光探针检测溶菌酶的方法,
具体步骤如下:
向铂纳米簇体系中加入一系列不同浓度的溶菌酶溶液,检测标准体系为1mL,其中不同浓度溶菌酶溶液100μL,Papain-Pt NCs 50μL,加去离子水补足至1mL,25℃水浴2h后,使用RF-5301荧光分光光度计,在激发光波长为380nm的条件下,检测溶液在490nm的发射光的峰高。结果显示,Papain-Pt NCs荧光淬灭程度随溶菌酶浓度的增大而增强(图7),其相对荧光强度线性检测线为y=0.00297x+0.9918(R2=0.99568),所构建的检测溶菌酶的线性范围为10μg/mL-200μg/mL,其检测限为0.55μg/mL(图8)。结果表明,所制备的铂纳米簇对溶菌酶具有高度选择性。
实施例7:实施例1所制的Papain-Pt NCs对实际人体尿液样品的检测
为了验证该方法在实际应用中的可行性,将其应用于实际尿液样品中溶菌酶的测定。选取5位志愿者尿液样本,稀释100倍后转移至新的EP管中储存在4℃冰箱备用。
(1)实际尿液样品的检测:
检测体系为1mL,其中50μL 25mg/mL的Papain-Pt NCs溶液,950μL稀释100倍待测的尿液样品;将此体系于25℃水浴2h,在激发光波长为380nm的条件下,检测溶液在490nm的发射光的峰高。将五种尿液样品分别按照上述方法进行检测,实验独立重复3次。将测试结果带入实施例6绘制的标准曲线中计算,五种样品的检测结果均为0μM。
(2)检测加标回收率:
检测标准体系为1mL,其中50μL 25mg/mL的Papain-Pt NCs溶液,900μL稀释100倍待测的尿液样品,50μL不同浓度的溶菌酶,25℃水浴2h,在激发光波长为380nm的条件下,检测溶液在490nm的发射光的峰高,实验独立重复3次,同时对尿液样品的回收率进行计算。从表1可以看出,回收率在95%以上的可接受范围内。说明该方法是有效的,可应用于实际尿液样品中溶菌酶的检测。
表1 Papain-Pt NCs对实际尿液样品中溶菌酶的检测
Claims (5)
1.一种荧光铂纳米簇在溶菌酶检测中的应用,其特征在于所述纳米簇以木瓜蛋白酶作为模板和还原剂。
2.根据权利要求1所述的一种荧光纳米簇在溶菌酶检测中的应用,其特征在于所述纳米簇的制备方法为将木瓜蛋白酶的水溶液与高价铂配合物的水溶液进行混合,然后加NaOH溶液调节pH值为8~13,避光水浴反应完全后即得。
3.根据权利要求2所述的一种荧光纳米簇在溶菌酶检测中的应用,其特征在于所述纳米簇的制备方法包括如下步骤:
(1)制备25-50mg/mL的木瓜蛋白酶水溶液;
(2)制备浓度为25-100mM的H2PtCl6的水溶液;
(3)将H2PtCl6溶液加入木瓜蛋白酶水溶液中,使其中的木瓜蛋白酶与H2PtCl6两者的摩尔比为1.5:1-10.5:1,充分混匀;
(4)向步骤(3)得到的混合溶液中添加NaOH调节溶液的pH值至8-13,涡旋器充分混匀5-10min;
(5)将步骤(4)得到的混合溶液在25℃~65℃条件下水浴6~48h后即得。
4.根据权利要求1所述的荧光铂纳米簇在溶菌酶检测中的应用,其特征在于其检测溶
菌酶的方法为包括如下步骤:
(1)将荧光铂纳米簇溶液和不同浓度的溶菌酶溶液混合,采用荧光分光光度计分别检测各混合溶液的荧光强度值,绘制各溶液荧光强度值和与之对应的不同浓度的溶菌酶溶液之间的标准曲线;
(2)将荧光铂纳米簇溶液和待测样品进行混合,检测加入待测样品后的溶液的荧光强度值,将数值带入上述标准曲线计算即可得到待测样品中的溶菌酶浓度值。
5.根据权利要求1所述的荧光铂纳米簇在溶菌酶检测中的应用,其特征在于其检测溶
菌酶的线性范围为10 μg/mL-200 μg/mL,检测限为0.55 μg/mL。
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