CN105067612A - 纳米金催化铜沉积介导的浓度依赖多色转变比色分析方法和应用 - Google Patents
纳米金催化铜沉积介导的浓度依赖多色转变比色分析方法和应用 Download PDFInfo
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Abstract
本发明公开了纳米金催化铜沉积介导的浓度依赖多色转变比色分析方法,具体将捕获样品中待检测物的特异性识别分子包被在固相载体上,然后将待检测物与包被在固相载体上的特异性识别分子进行特异性反应,再在反应后的待检测物表面引入纳米金标记的检测探针,接着在纳米金表面沉积铜,再加入含Fe3+和[Fe(CN)6]3-的显色剂显色,最后通过颜色半定量判断待测检物含量或利用紫外可见分光光度计定量检测待检测物,本发明的方法操作简单、成本较低,且能通过肉眼半定量检测,很适合于快速的初期检测,有望在疾病标志物早期筛查、食品安全控制等方面得到广泛应用。
Description
技术领域
本发明属于检测领域,具体涉及纳米金催化铜沉积介导的浓度依赖多色转变比色分析方法及应用。
背景技术
定量或半定量比色分析作为生化检测的主要手段,在环保评价、食品安全、疾病诊断、肿瘤标志物检测方面有着广泛应用。比色分析因其方法简便直观、分析时间短、所用仪器少脱颖而出,成为生化分析检测的重要手段。对于已有的比色分析法,大都是从深到浅或从浅到深的一种颜色变化,这样的比色法难以用肉眼判断待测物的大致含量,必须借助精密仪器来测定,这大大限制了比色法在实际应用中的发展。因此,急需一种能够多色转变的分析方法,能够通过肉眼进行半定量分析,借助仪器实现定量检测。
发明内容
有鉴于此,本发明的目的之一在于提供纳米金催化铜沉积介导的浓度依赖多色转变比色分析方法;本发明的目的之二在于提供该分析方法在定量或半定量检测生化分子含量中的应用。
为实现上述发明目的,本发明提供如下技术方案:
1、纳米金催化铜沉积介导的浓度依赖多色转变比色分析方法,将捕获样品中待检测物的特异性识别分子包被在固相载体上,然后将待检测物与包被在固相载体上的特异性识别分子进行特异性反应,再在反应后的待检测物表面引入纳米金标记的检测探针,接着在纳米金表面沉积铜,再加入含Fe3+和[Fe(CN)6]3-的显色剂显色,最后通过颜色半定量判断待测检物含量或利用紫外可见分光光度计定量检测待检测物。
优选的,沉积铜的方法如下:向引入纳米金标记检测探针的固相载体上加入含12~500mM硫酸铜和12~500mM抗坏血酸的沉积铜试剂,孵育,清洗即可。
优选的,所述显色剂为含1~5mM三氯化铁和1~5mM铁氰化钾的溶液。
优选的,特异性识别分子包被固相载体的方法如下:将终浓度1~100μgmL-1的抗待检测物特异性单克隆抗体加入固相载体上,4~8℃过夜或37℃孵育1~3h,然后封闭,再加入待检测物,37℃孵育0.5~2h,随后加入纳米金标记的检测探针孵育。
更优选的,所述待检测物为生物分子或化学分子,具体为蛋白质、糖类、脂类、核酸、细胞因子、生物毒素、农药、兽药等。
更优选的,所述检测探针为特异识别待检测物的生物分子。
最优选的,所述特异识别待检测物的生物分子为抗体、多肽、核酸适配体、凝集素或核酸探针。
2、所述分析方法在定量或半定量检测蛋白质、糖类、脂类、核酸、细胞因子、生物毒素、农药或兽药等生物或化学分子含量中的应用。
优选的,所述分析方法在定量或半定量生化分析中的应用。
本发明的有益效果在于:本发明公开了纳米金催化铜沉积介导的浓度依赖多色转变比色分析方法,采用纳米金标记的检测探针,避免了传统的酶标探针不易储存,反应条件苛刻等缺点,提供了更有利的反应条件;而且基于纳米金催化铜沉积的信号放大策略,通过调整沉积试剂的浓度和沉积时间长短,大大放大了检测信号,取得了更高的灵敏度和更低的检出限;使用三氯化铁和铁氰化钾显色,构造了一种浓度依赖的多色转变比色分析法,使得借助肉眼半定量判断待测物含量或浓度成为现实。使用常规的紫外可见分光光度计也可定进行量检测。
本发明基于纳米金催化铜沉积介导的浓度依赖的多色比色分析法,拥有操作简单、成本较低,且能通过肉眼半定量检测。很适合于快速的初期检测,有望在疾病标志物早期筛查、食品安全控制等方面得到广泛应用。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚,本发明提供如下附图:
图1为纳米金催化铜沉积介导的浓度依赖的多色比色分析法检测原理图。
图2为不同浓度氯化亚铁和铁氰化钾混合溶液的显色图和紫外可见吸收光谱图。
图3为验证本发明是否可行实验显色图和紫外可见吸收光谱图(a和b分别没有包被纳米金,没有沉积铜显色之后的照片;c为在包被纳米金、沉积铜、显色之后的照片)。
图4为纳米金沉积前(a)后(b)及金铜复合物溶解后(c)的FE-SEM图。a图中红色箭头所指即为纳米金。
图5为对纳米金催化铜沉积不同沉积时间和沉积试剂浓度显色结果图。
图6为用纳米金催化铜沉积介导的浓度依赖的多色比色分析法检测结果图。
图7为用纳米金催化铜沉积介导的浓度依赖的多色比色分析法检测兔抗原时的紫外可见吸收光谱及标准曲线。图中的照片是俯拍和侧拍的显色结果。
图8为用纳米金催化铜沉积介导的浓度依赖的多色比色分析法对肿瘤标志物PSA检测得到的标准曲线。
具体实施方式
下面将结合附图,对本发明的优选实施例进行详细的描述。实施例中未注明具体条件的实验方法,通常按照常规条件或按照制造厂商所建议的条件。
纳米金催化铜沉积介导的浓度依赖多色转变比色检测方法,具体步骤将捕获样品中待检测物的特异性识别分子包被在固相载体上,然后将待检测物与包被在固相载体上的特异性识别分子进行特异性反应,再在反应后的待检测物表面引入纳米金标记的检测探针,接着在纳米金表面沉积铜,再加入含Fe3+和[Fe(CN)6]3-(如含氯化铁和铁氰化钾)的显色剂显色,最后通过颜色半定量判断待测检物含量或利用紫外可见分光光度计定量检测待检测物,其检测原理如图1所示。为了解氯亚化铁和铁氰化钾的显色颜色及吸收特征,分析浓度分别为0mM,0.05mM,0.1mM,0.25mM,0.5mM,0.75mM,1mM的氯化亚铁和铁氰化钾混合溶液的颜色和紫外可见吸收光谱,结果如图2所示。结果显示,当氯化亚铁的浓度由低到高逐渐增加时,显色剂的颜色出现黄色、绿色、蓝色等多色转变,并且OD值也呈现不同吸收谱,表明可以利用氯化亚铁和铁氰化钾进行多色转变显色实现半定量,利用紫外吸收光谱进行定量检测。
为验证本发明是否可行,分别检测是未包被纳米金,未沉积铜,包被纳米金、沉积铜之后显色的吸收光谱和对应照片,结果如图3所示。
图4为纳米金沉积前后及金铜复合物溶解后的FE-SEM图(a:纳米金沉积铜前,图中箭头所指即为纳米金;b纳米金沉积铜后;c纳米金铜复合物溶解后)。结果显示,纳米金沉积铜后在纳米金表面覆盖一层厚厚的铜,溶解后体积变小。
图5为纳米金催化铜沉积时的沉积时间和显色剂浓度的优化结果。结果显示,最优沉积时间为6min,最优显色剂浓度为3mM氯化铁+3mM铁氰化钾。
图6为纳米金催化铜沉积介导的浓度依赖的多色比色分析法检测结果。结果显示,紫外可见光谱吸收值随时间的变化。
按上述检测方法,检测待检测物兔抗原rabbitIgG和肿瘤标志物PSA的含量。
实施例1
纳米金催化铜沉积介导的浓度依赖多色转变比色免疫分析法检测兔抗原rabbitIgG,具体步骤如下:
(1)向孔板中加入50μL10μgmL-1鼠抗兔单克隆抗体(溶于pH=9.6,碳酸盐(CBS)缓冲溶液),4℃过夜,然后用300μL质量分数为15%的脱脂奶粉(溶于pH=9.6,CBS缓冲溶液)在37℃下封闭孔板1h,再将50μL分别含0pg·mL-1,1pg·mL-1,10pg·mL-1,100pg·mL-1,1ng·mL-1,10ng·mL-1,100ng·mL-1,1μg·mL-1兔抗原(溶于PBS)加入孔板,37℃孵育1h,最后加入50μL纳米金标记的山羊抗兔多克隆抗体37℃孵育0.5h;
(2)将步骤(1)处理好的孔板用二次去离子水洗三次,拍干,然后加入100μL沉积铜试剂在37℃反应6min,然后倒掉孔板中的液体,再用二次去离子水彻底清洗三次,其中沉积铜试剂为硫酸铜和抗坏血酸终浓度均为100mM的混合液;
(3)向步骤(2)处理后的孔板加入80μLpH=5醋酸缓冲液,10μL30mM三氯化铁溶解形成的纳米铜,再加入10μL30mM铁氰化钾溶液形成普鲁士蓝,通过肉眼半定量检测兔抗原rabbitIgG,结果如图7所示。同时通过紫外可见分光光度计定量检测,然后以吸收值为纵坐标,rabbitIgG浓度的对数为横坐标,绘制标准曲线,结果如图7所示。结果表明,利用本发明的方法检测到不同的颜色,不同的颜色表示不同的含量,可通过肉眼实现半定量检测;同时也可以通过紫外可见分光光度计检测吸光值实现定量检测。并且定量检测时,在优化条件下,rabbitIgG的最低检测限可低至0.28pg·mL-1检测的线性范围为1pg·mL-1至100ng·mL-1。
实施例2
纳米金催化铜沉积介导的浓度依赖多色转变比色免疫分析法检测肿瘤标志物PSA,具体步骤如下:
(1)向孔板中加入50μL10μgmL-1抗前列腺特异性抗原单克隆抗体(溶于pH=9.6CBS缓冲溶液),4℃过夜,然后用300μL质量分数为15%脱脂奶粉(溶于pH=9.6CBS缓冲溶液)在37℃下封闭孔板1h,再将50μL分别含0pg·mL-1,1pg·mL-1,10pg·mL-1,100pg·mL-1,1ng·mL-1,10ng·mL-1,100ng·mL-1,1μg·mL-1肿瘤标志物PSA的溶液(溶于质量分数10%的人血清)加入孔板,37℃孵育1h;随后加入50μL1μg·mL-1生物素标记的抗前列腺特异性抗体,37℃孵育1h,最后加入50μL纳米金标记的链霉菌链亲和素(也可以在孵育肿瘤标志物PSA后直接使用纳米金抗前列腺特异性抗体),37℃孵育0.5h,每步后均用300μLTBS缓冲液(含质量分数为0.05%吐温-20)清洗三次,拍干;
(2)将步骤(1)处理好的孔板用二次去离子水洗三次,拍干,每孔加入100μL沉积铜试剂,在37℃反应6min,然后倒掉孔板中的液体,用二次去离子水彻底清洗三次,其中沉积铜试剂为硫酸铜和抗坏血酸终浓度均为100mM的混合液;
(3)将步骤(2)处理后的孔板加入80μLpH=5醋酸缓冲液,10μL30mM三氯化铁溶解形成的纳米铜,再加入10μL30mM铁氰化钾溶液形成普鲁士蓝显色,然后通过肉眼或紫外可见分光光度计检测肿瘤标志物PSA含量,紫外可见分光光度计检测结果以吸收值为纵坐标,PSA浓度的对数为横坐标,绘制标准曲线,结果如图8所示。结果表明,利用本发明的方法可以通过紫外可见分光光度计检测吸光值实现定量检测。并且定量检测时,在优化条件下,PSA的最低检测限可低至0.33pg·mL–1检测的线性范围为1pg·mL–1至100ng·mL–1。
由上述实施例,本领域技术人员可预见到,待检测物与检测探针能够发生特异结合的生物分子均可上述方法检测。因此,本发明的方法可用于定量或半定量检测蛋白质、糖类、脂类、核酸、细胞因子、生物毒素、农药、兽药等生物或化学分子。
最后说明的是,以上优选实施例仅用以说明本发明的技术方案而非限制,尽管通过上述优选实施例已经对本发明进行了详细的描述,但本领域技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离本发明权利要求书所限定的范围。
Claims (10)
1.纳米金催化铜沉积介导的浓度依赖多色转变比色分析方法,其特征在于:将捕获样品中待检测物的特异性识别分子包被在固相载体上,然后将待检测物与包被在固相载体上的特异性识别分子进行特异性反应,再在反应后的待检测物表面引入纳米金标记的检测探针,接着在纳米金表面沉积铜,再加入含Fe3+和[Fe(CN)6]3-的显色剂显色,最后通过颜色半定量判断待测检物含量或利用紫外可见分光光度计定量检测待检测物。
2.根据权利要求1所述纳米金催化铜沉积介导的浓度依赖多色转变比色分析方法,其特征在于,沉积铜的方法如下:向引入纳米金标记检测探针的固相载体上加入含12~500mM硫酸铜和12~500mM抗坏血酸的沉积铜试剂,孵育,清洗即可。
3.根据权利要求1所述纳米金催化铜沉积介导的浓度依赖多色转变比色分析方法,其特征在于:所述显色剂为含1~5mM三氯化铁和1~5mM铁氰化钾的溶液。
4.根据权利要求1所述纳米金催化铜沉积介导的浓度依赖多色转变比色分析方法,其特征在于,特异性识别分子包被固相载体的方法如下:将终浓度1~100μgmL-1的待检测物特异性识别分子加入固相载体上,4~8℃过夜或37℃孵育1~3h,然后封闭,再加入待检测物,37℃孵育0.5~2h,随后加入纳米金标记的检测探针孵育。
5.根据权利要求1~4任一项所述纳米金催化铜沉积介导的浓度依赖多色转变比色分析方法,其特征在于:所述待检测物为生物分子或化学分子。
6.根据权利要求5所述纳米金催化铜沉积介导的浓度依赖多色转变比色分析方法,其特征在于:所述待检测物为蛋白质、糖类、脂类、核酸、细胞因子、生物毒素、农药或兽药等生物或化学分子。
7.根据权利要求1~4任一项所述纳米金催化铜沉积介导的浓度依赖多色转变比色分析方法,其特征在于:所述检测探针为特异识别待检测物的生物分子。
8.根据权利要求7所述纳米金催化铜沉积介导的浓度依赖多色转变比色分析方法,其特征在于:所述特异识别待检测物的生物分子为抗体、多肽、核酸适配体、凝集素或核酸探针。
9.权利要求1~7任一项所述分析方法在定量或半定量检测蛋白质、糖类、脂类、核酸、细胞因子、生物毒素、农药或兽药含量中的应用。
10.根据权利要求9所述的应用,其特征在于:所述分析方法在定量或半定量生化分析中的应用。
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