CN113640235B - 纳米酶催化辅助的比率型比色检测肝素的方法 - Google Patents
纳米酶催化辅助的比率型比色检测肝素的方法 Download PDFInfo
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Abstract
本发明属于分析化学技术领域,涉及纳米酶催化辅助的比率型比色检测肝素的方法,包括:向若干1.5mL离心管中加入900μL 0.1M的磷酸盐缓冲液,摇匀静置;分别加入50μL 1 mg/mL的FeMoO4纳米棒水溶液、25μL 5mM TMB溶液和不同浓度的肝素溶液,保证溶液总体积为1mL;用紫外可见光谱仪获取652nm和565nm处的吸光度值,以肝素浓度为横坐标,以652nm和565nm处的吸光度比值(A652/A565)为纵坐标,绘制标准工作曲线;将待测样品重复操作,并与标准工作曲线对比,即得待测样品中肝素的浓度。基于待测物与纳米酶催化产生的带正电荷物种之间的特定静电相互作用,本发明所述方法实验步骤简单、快捷,可实现对肝素的超灵敏和特异性定量检测,为临床血样中肝素的测定提供可靠的结果。
Description
技术领域
本发明属于分析化学技术领域,涉及肝素的检测方法,尤其涉及纳米酶催化辅助的比率型比色检测肝素的方法。
背景技术
作为一种广泛应用于临床的抗凝剂,肝素可预防在心肺手术中形成血栓。此外,它能加速多种凝血因子的失活速率,也常被用于治疗紧急的静脉血栓形成。然而,过量使用肝素会引发一系列严重的并发症,包括肝素引起的血小板减少和出血。在术后护理中肝素的指导治疗剂量为0.2-1.2U/mL,在心血管手术中为2-8U/mL。此外,肝素还参与调节一些生理和病理过程,如抗炎、抗过敏等。因此,监测临床样品中的肝素含量水平非常重要。
至今,已有多种分析和定量肝素的方法。传统上,采用活化凝血时间(ACT)、活化部分凝血酶活时间(APTT)和抗Xa试剂盒用于间接检测肝素。然而由于多种因素的干扰,这些传统方法不够精准。目前,色谱法、比色法、荧光法和电化学法已被开发用于肝素的直接高性能监测,例如:
中国专利CN201810413400.3《一种快速检测肝素和/或低分子肝素中肝素二糖含量的方法》公开了一种检测肝素的色谱法,首先将肝素与肝素酶混合反应,得到酶解产物;然后将酶解产物利用反相离子对色谱进行检测,进而获得肝素的含量信息。
中国专利CN201710413862.0《基于变性牛血清蛋白为模板的铜纳米簇的肝素检测方法》公开了一种检测肝素的荧光法,采用一锅法合成以变性的牛血清蛋白为模板的铜纳米簇,利用分析物对铜纳米簇荧光的猝灭效应,以磷酸缓冲溶液、铜纳米簇分散体系和待测样品组成肝素检测体系,通过检测加入待测样品前后的荧光强度变化,得到待测样品中肝素的含量。
中国专利CN201710925956.6《基于碳量子点淬灭的荧光标记DNA的比例性荧光方法检测肝素钠》公开了一种基于碳量子点淬灭的荧光标记DNA比率型荧光检测肝素钠的方法,该方法利用碳量子点和FAM标记的DNA作为两种荧光来源;碳量子点会猝灭FAM标记的DNA荧光;肝素的加入可以抑制上述猝灭行为,使得碳量子点的荧光始终保持稳定,而标记的DNA荧光得以增强;据此建立比率型荧光检测肝素的方法。
中国专利CN201110302864.5《一种肝素的检测方法》公开了一种电化学检测肝素的方法,该方法利用聚合物敏感膜相中鱼精蛋白与水相中肝素的特异性结合,通过电位测定仪测定加入不同浓度肝素后的电位变化,根据初始电位变化速率,绘制标准工作曲线,对照标准工作曲线即得未知样品中肝素的浓度。
上述已公开的肝素的检测方法虽然具有一定的检测效果,但仍具有以下缺点或不足:
(1)色谱检测方法需要较昂贵的仪器设备,检测操作步骤相对复杂;
(2)大多数比色和荧光方法都是基于目标诱导的单信号变化,在实际应用中容易受到外界因素的干扰,导致检测方法的可重复性和鲁棒性不佳;
(3)大多数荧光和比色方法涉及显色底物的制备,增加了检测成本和操作难度;
(4)部分比率型检测方法涉及多种显色底物的使用,使得检测过程复杂化。
发明内容
为解决上述现有技术中存在的问题与不足,本发明旨在提供一种方便、快捷、高性能的比率型比色检测肝素的方法。
为实现上述目的,本发明所使用的技术方案是:
一种纳米酶催化辅助的比率型比色检测肝素的方法,包括如下步骤:
(1)向若干1.5mL离心管中加入900μL 0.1M的磷酸盐缓冲液,摇匀静置;
(2)向上述缓冲溶液中分别加入50μL 1mg/mL的FeMoO4纳米棒水溶液、25μL 5mM用乙醇配制的TMB溶液和不同浓度的肝素溶液,保证溶液总体积为1mL,反应1~10min,优选5min;其中,所述肝素在体系内的最终浓度在0.1~4.0μg/mL范围;
(3)利用紫外可见光谱仪测量上述溶液的吸光信号,获取在652nm和565nm处的吸光度值,并以肝素浓度为横坐标,以652nm和565nm处的吸光度比值(A652/A565)为纵坐标,绘制标准工作曲线;
(4)将待测样品重复步骤(1)~(3),用紫外可见光谱仪测定待测样品在652nm和565nm处的吸光度值,通过将吸光度比值(A652/A565)与标准工作曲线对比,即可得到待测样品中肝素的浓度。
本发明较优公开例中,步骤(1)中所述磷酸盐缓冲液的pH值为7.0。
本发明较优公开例中,步骤(4)中待测样品的可测浓度范围为0.1~3.0μg/mL(0.017~0.51U/mL或0.0162~0.486μM),检测极限为53ng/mL(0.009U/mL或8.59nM)。
本发明所用的FeMoO4纳米棒为自制。
以下公开一种FeMoO4纳米棒制备方法:将含有3.976g FeCl2·4H2O的水溶液缓慢加入到含有4.839g Na2MoO4·H2O的水溶液中,连续搅拌5min后,将棕色的混合物转移至不锈钢高压反应釜中,120℃水热反应12h,冷却至室温后,将形成的固体沉淀离心收集,用去离子水洗涤多次,干燥后备用。
本发明所制备的FeMoO4纳米棒在生理介质中展现出良好的类氧化酶活性,可催化无色的3,3',5,5'-四甲基联苯胺(TMB)氧化为蓝色的TMBox物种,在652nm处提供一个显著的可见光信号。若存在肝素,由于强烈的静电相互作用,它会在短时间内触发带正电荷的TMBox物种的组装,产生一种紫色的Heparin-TMBox复合物,在565nm处呈现另一个可见光信号。随着肝素浓度的增加,溶液颜色由蓝色变为靛蓝色,再变为紫色。
本发明所用其他试剂均为市售。
在本说明书中,术语“TMB”是化合物“3,3',5,5'-四甲基联苯胺”的缩写名称,二者可互换使用。
在本说明书中,术语“heparin”是指肝素,二者可互换使用。
有益效果
本发明公开了一种比率型比色快速检测肝素的方法,是基于待测物与纳米酶催化产生的带正电荷物种之间的特定静电相互作用。本发明所述方法实验步骤简单、快捷,可实现对肝素的超灵敏和特异性定量检测,为临床血样中肝素的测定提供可靠的结果。
附图说明
图1.FeMoO4纳米棒展现出类氧化酶活性,可催化无色TMB氧化生成蓝色TMBox;
图2.肝素诱导TMBox发生团聚,影响溶液显色;
图3.肝素的添加顺序对检测体系的影响;
图4.利用FeMoO4纳米棒+TMB体系定量检测肝素的浓度;
图5.利用FeMoO4纳米棒+TMB体系检测肝素的选择性效果图。
具体实施方式
为了使本发明的目的、技术方案以及优点更加清晰明了,下面将结合实施例对本发明进行详细说明,以便本领域技术人员更好地理解本发明,但并不局限于以下实施例。
实施例1
FeMoO4纳米棒展现类氧化酶活性,催化无色TMB氧化生成蓝色TMBox
(1)向1.5mL离心管中加入900μL 0.1M的磷酸盐缓冲液(pH 7.0),摇匀;
(2)向上述缓冲溶液中分别加入50μL 1mg/mL的FeMoO4纳米棒水溶液和50μL 5mMTMB溶液(用乙醇配制),保证溶液总体积为1.0mL,反应1-10min;
(3)利用紫外可见光谱仪测量上述溶液的吸光信号,获取在652nm处的吸光度值。
结果如图1所示,图1中TMB底物不能被溶解氧直接氧化,低浓度的FeMoO4水溶液(50μg/mL)也没有背景底色。然而,在含有溶解氧的TMB溶液中加入FeMoO4纳米棒后,快速发生明显的显色反应,在652nm处出现一个明显的可见吸收信号,原无色溶液在短时间内变成深蓝色。这种颜色变化归因于无色底物TMB被氧化成其氧化物TMBox,表明FeMoO4纳米棒可诱导溶解氧和TMB发生氧化还原。为证明FeMoO4确实作为纳米酶而不是简单的氧化剂来诱导TMB的氧化,比较了在空气或氮气饱和缓冲液中发生的反应。与空气饱和溶液相比,在氮气饱和缓冲液中TMB的氧化反应明显受到抑制。这些结果证实了FeMoO4纳米棒作为类氧化酶催化溶解氧与TMB之间的氧化还原反应。
实施例2
肝素诱导TMBox发生团聚,影响溶液显色
(1)向1.5mL离心管中加入900μL 0.1M的磷酸盐缓冲液(pH 7.0),摇匀;
(2)向上述缓冲溶液中分别加入50μL 1mg/mL的FeMoO4纳米棒水溶液和25μL 5mMTMB溶液(用乙醇配制),反应5min;
(3)向上述溶液中加入3μg/mL的肝素溶液,反应2min;
(4)利用紫外可见光谱仪测量上述溶液的吸光信号,获取在652nm和565nm处的吸光度值。
结果如图2所示,由于在FeMoO4纳米棒的催化下,TMB被氧化成TMBox,使得FeMoO4+TMB溶液呈现蓝色。当加入肝素后(3.0μg/mL,相当于0.51U/mL或0.486μM)时,溶液变成紫色,在565nm左右出现一个新的明显信号,而652nm处的吸光度降低。此外,发现只有肝素能触发565nm处信号的产生。上述现象归因于TMBox与肝素的特殊静电相互作用:由纳米酶催化产生的蓝色TMBox物种带有强正电性,可与带有强负电荷的肝素发生静电相互作用,后者引发前者的静电组装,产生一种紫色的Heparin-TMBox絮状复合物,从而引发565nm处信号的产生;因蓝色TMBox物种被消耗,导致652nm处的吸光度降低。
实施例3
肝素的添加顺序对检测体系的影响
在本检测方法中,分析物肝素有两种添加方法(图3):一种是待TMB完全被催化氧化成蓝色TMBox物种后再添加,形成两步法检测方法;另一种是与TMB等同时添加,形成一锅法检测方法。为查验两种操作方法是否会造成检测结果差异,比较了不同添加方法对同一肝素浓度(1.0μg/mL)的检测结果,结果如图3所示。发现上述两种添加方法对肝素的检测无明显差异,为节约检测时间,优化地,采用一锅法检测肝素。
实施例4
利用FeMoO4纳米棒+TMB体系定量检测肝素的浓度
(1)向1.5mL离心管中加入900μL 0.1M的磷酸盐缓冲液(pH 7.0),摇匀;
(2)向上述缓冲溶液中分别加入50μL 1mg/mL的FeMoO4纳米棒水溶液、25μL 5mMTMB溶液(用乙醇配制)和不同浓度的肝素溶液,保证溶液总体积为1mL,反应1-10min;其中,所述肝素在体系内的最终浓度在0.1-4.0μg/mL范围;
(3)利用紫外可见光谱仪测量上述溶液的吸光信号,获取在652nm和565nm处的吸光度值,并以肝素浓度为横坐标,以652nm和565nm处的吸光度比值(A652/A565)为纵坐标,绘制标准工作曲线。
结果如图4所示,当逐渐增加肝素浓度时,蓝色TMBox物种的消耗导致652nm处信号逐渐降低,而紫色Heparin-TMBox复合物的生成导致565nm处的信号逐渐增加。因此,随着肝素浓度的增加,652nm和565nm处的吸光度比值(A652/A565)逐渐降低。A652/A565与肝素浓度在0.1-3.0μg/mL(0.017-0.51U/mL或0.0162-0.486μM)范围内呈现良好的线性关系,线性方程为A652/A565=-0.582[肝素浓度]+2.064(R2=0.992)。根据三倍信噪比规则计算出检测限(LOD)为53ng/mL(0.009U/mL或8.59nM)。
实施例5
利用FeMoO4纳米棒+TMB体系检测肝素的选择性评价
(1)向1.5mL离心管中加入900μL 0.1M的磷酸盐缓冲液(pH 7.0),摇匀;
(2)向上述缓冲溶液中分别加入50μL 1mg/mL的FeMoO4纳米棒水溶液、25μL 5mMTMB溶液(用乙醇配制)和不同物种的溶液,保证溶液总体积为1mL,反应1-10min;
(3)利用紫外可见光谱仪测量上述溶液的吸光信号,获取在652nm和565nm处的吸光度值,绘制对比响应图;
结果如图5所示,图5是用FeMoO4纳米棒+TMB体系检测肝素的选择性的柱状图,柱状图标号从1到32依次为Fe3+、Cu2+、Zn2+、CO3 2-、SO4 2-、NO3 -、Cl-、甘氨酸、丙氨酸、亮氨酸、异亮氨酸、缬氨酸、脯氨酸、苯丙氨酸、蛋氨酸、色氨酸、丝氨酸、谷氨酰胺、苏氨酸、半胱氨酸、天冬酰胺、酪氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸、组氨酸、三磷酸腺苷、谷胱甘肽、多巴胺、葡萄糖、肝素。从图中可以看出,只有肝素可以显著降低652nm和565nm处的吸光度比值(A652/A565),而其他物种共存时不会对该比值产生明显影响。
实施例6
利用FeMoO4纳米棒+TMB体系检测临床血清中的肝素含量
(1)临床血清样品由合作医院提供,并利用磷酸盐缓冲液稀释至5%;
(2)向上述样品中分别添加0.8和2.0μg/mL的肝素储存液,待用;
(3)向1.5mL离心管中加入900μL 0.1M的磷酸盐缓冲液(pH 7.0),摇匀;
(4)向上述缓冲溶液中分别加入50μL 1mg/mL的FeMoO4纳米棒水溶液、25μL 5mMTMB溶液(用乙醇配制)和25μL样品,保证溶液总体积为1mL,反应1-10min;
(4)利用紫外可见光谱仪测量上述溶液的吸光信号,获取在652nm和565nm处的吸光度值,与标准工作曲线对比,获得样品中肝素的含量信息。
测定结果如表1所示:
表1本方法对临床血清样品的检测结果
由上表可知,FeMoO4纳米棒+TMB体系对临床样品中肝素含量的变化响应准确可靠,可实现目标物的高灵敏、高选择性、快速、低成本检测。
以上所述,仅为本发明较佳的具体实施方式,并非因此限制本发明的专利范围。凡是利用本发明说明书所作的等效结构或等效流程变换,或直接或间接运用在其他相关的技术领域,均同理包括在本发明的专利保护范围内。
Claims (5)
1.一种纳米酶催化辅助的比率型比色检测肝素的方法,其特征在于,包括如下步骤:
(1)向若干1.5mL离心管中加入900μL 0.1 M 的磷酸盐缓冲液,摇匀静置;
(2) 向上述缓冲液中分别加入50μL 1mg/mL的FeMoO4纳米棒水溶液、25μL 5mM 用乙醇配制的含有溶解氧的TMB溶液和不同浓度的肝素溶液,保证溶液总体积为1mL,反应1~10min;其中,所述肝素在体系内的最终浓度在0.1~4.0μg/mL范围;
(3)利用紫外可见光谱仪测量上述溶液的吸光信号,获取在652nm和565 nm处的吸光度值,并以肝素浓度为横坐标,以652nm和565nm处的吸光度比值(A652/A565)为纵坐标,绘制标准工作曲线;
(4)将待测样品重复步骤(1)~(3),用紫外可见光谱仪测定待测样品在652nm和565nm处的吸光度值,通过将吸光度比值(A652/A565)与标准工作曲线对比,即可得到待测样品中肝素的浓度。
2.根据权利要求1所述纳米酶催化辅助的比率型比色检测肝素的方法,其特征在于:步骤(1)中所述磷酸盐缓冲液的pH值为7.0。
3.根据权利要求1所述纳米酶催化辅助的比率型比色检测肝素的方法,其特征在于:步骤(2)中所述反应时间为5min。
4.根据权利要求1所述纳米酶催化辅助的比率型比色检测肝素的方法,其特征在于:步骤(4)中待测样品的可测浓度范围为0.1~3.0μg/mL。
5.根据权利要求1所述纳米酶催化辅助的比率型比色检测肝素的方法,其特征在于:步骤(4)中待测样品的检测限为53ng/mL。
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