CN112326613A - 凝血酶含量的检测方法 - Google Patents
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Abstract
本发明公开了凝血酶含量的检测方法,包括:(1)绘制凝血酶的荧光标准曲线;(2)将待测样品液与含适配体的DNA在缓冲液中混匀孵育后加入AgNO3和Cu(NO3)2混匀孵育加入NaBH4混匀孵育;加入氧化石墨烯分散液混匀孵育测定荧光值;(3)根据标准曲线计算待测样品液中凝血酶的含量。本发明先将凝血酶与含适配体的DNA特异性结合再合成DNA‑Cu/Ag NCs最后加入氧化石墨烯,既消除了背景荧光又避免了氧化石墨烯对凝血酶非特异性吸附,减少了对氧化石墨烯的修饰步骤。本发明线性范围为2‑10nM,最低检测限为0.25nM,具有成本低廉、操作简单、灵敏度高、选择性好、毒性小、生物相容性好、免标记等优点。
Description
技术领域
本发明涉及凝血酶含量的检测方法,尤其涉及基于氧化石墨烯和适配体DNA-Cu/Ag NCs结合的凝血酶检测方法,属于凝血酶含量的检测领域。
背景技术
凝血酶作为一种特殊的丝氨酸内蛋白酶,不仅在血管生成、血栓形成、凝血级联和肿瘤生长止血等许多病理生理过程中发挥重要作用,而且还调节血小板聚集、内皮细胞活化以及在血管生物学中作为激素的重要响应。机体内的凝血酶必须保持在一定的浓度范围,浓度过高会导致血栓形成,过低会导致出血。因此准确测定凝血酶的浓度对确定给定病人恰当的治疗至关重要。
适配体是通过称为“通过指数富集(SELEX)配体的系统进化”的特定过程分离的单链寡核苷酸。由于具有良好的稳定性、特异性、成本较低以及相对容易分离和修饰,因此在大分子或低分子量基质的生物传感方面具有很大的应用前景。各种基于适配体用于凝血酶检测的分析技术(包括比色法、荧光、电化学和表面增强拉曼散射)已经得到开发。
金属纳米簇是含有几个到约一百个原子的纳米材料,由于其独特的物理、光学和电学性质,被广泛地用于催化、生物医学、成像、传感以及光电设备等领域。其中,以DNA模板稳定的铜银纳米簇(简称DNA-Cu/Ag NCs)具有可优化的链长和序列、强的荧光发射、较低的毒性、较好的生物相容性等优点,倍受到研究者的重视。基于以上所述,可以将适配体与DNA-Cu/Ag NCs相结合来实现凝血酶的检测。
由于适配体与DNA-Cu/Ag NCs相结合检测凝血酶的探针有一定的背景荧光,直接影响检测的灵敏性,有待改进。
发明内容
本发明的主要目的是提供一种基于氧化石墨烯和适配体DNA-Cu/Ag NCs结合的凝血酶检测方法,该方法消除了DNA-Cu/Ag NCs的背景荧光,实现了对凝血酶简单、快速、低成本、高灵敏的检测。
为实现上述目的,本发明采取的技术方案是:
一种凝血酶含量的检测方法,包括:
(1)绘制凝血酶的荧光标准曲线,包括
(a)氧化石墨烯的预处理:将氧化石墨烯在水溶液中用超声处理得到氧化石墨烯分散液,备用;
(b)凝血酶与适配体结合的DNA-Cu/Ag NCs溶液的制备:将含适配体的DNA与不同浓度的凝血酶分别在Tris-HAc缓冲液中混匀并孵育后加入AgNO3和Cu(NO3)2避光孵育;再加入NaBH4混匀并孵育使Ag+和Cu2+还原为Ag和Cu,得到凝血酶与适配体结合的DNA-Cu/Ag NCs溶液;
(c)将步骤(a)所制得的氧化石墨烯分散液加入到步骤(b)所制得的凝血酶与适配体结合的DNA-Cu/Ag NCs溶液中孵育后,分别测定各混合溶液所对应的荧光值,根据最大荧光发射值绘制标准曲线;
(2)将待测的样品液与含适配体的DNA在Tris-HAc缓冲液中混匀孵育后加入AgNO3和Cu(NO3)2混合均匀避光孵育;再加入NaBH4混匀并孵育;最后加入氧化石墨烯分散液混合均匀孵育后测定该混合溶液的荧光值;
(3)根据标准曲线,计算得到待测样品液中凝血酶的含量。
作为本发明的一种具体的实施方式,步骤(a)所述的氧化石墨烯分散液中氧化石墨烯浓度优选为3mg/mL;所述的超声处理优选采用1500W 的功率超声处理40min;
步骤(b)所述的适配体DNA序列为: (SEQ ID NO.1);其中间斜体字部分为凝血酶的适配体,两端黑体字部分为Cu/AgNCs的成核序列,划线部分为连接碱基;所述含适配体的DNA的浓度优选为1.5μM。
作为本发明的一种具体的实施方式,步骤(b)中Cu(NO3)2、AgNO3和 NaBH4的浓度分别优选为4.5μM、12μM和30μM;所述的不同浓度的凝血酶可以为0-14nM的凝血酶,所述Tris-HAc缓冲液的浓度优选为10mM, 其pH值为7.0;步骤(b)中所述的凝血酶与适配体结合的DNA-Cu/Ag NCs 的粒径小于2nm。
作为本发明的一种具体的实施方式,步骤(b)中将含适配体的DNA与不同浓度的凝血酶分别在Tris-HAc缓冲液中混匀并孵育0.5h后加入 AgNO3和Cu(NO3)2混合均匀在4℃下避光孵育20-40min后再加入NaBH4混匀并孵育1-2h。
步骤(b)中凝血酶与适配体的结合避免了下一步骤加入的氧化石墨烯对凝血酶非特异性吸附。
作为本发明的一种具体的实施方式,步骤(c)中将步骤(a)所制得的氧化石墨烯分散液加入到步骤(b)所制得的凝血酶与适配体结合的 DNA-Cu/Ag NCs溶液中孵育20min然后在室温下分别测定各混合溶液所对应的荧光值,根据最大荧光发射值绘制标准曲线。
作为本发明的一种具体的实施方式,步骤(2)中将待测的样品液与含适配体的DNA在Tris-HAc缓冲液中混匀孵育0.5h然后将AgNO3和 Cu(NO3)2加入到该混合溶液中,在4℃下避光孵育20-40min;
作为本发明的一种具体的实施方式,步骤(2)中最后所加入的氧化石墨烯分散液至其在混合溶液中的终浓度为45μg/mL。
氧化石墨烯(简称GO)属于有一个原子层厚的二维纳米材料,具有优异的电子、机械和热学性能,发现在生物分子识别事件中具有关键的作用。它能够结合和猝灭标记在单链DNA(ssDNA)探针上的荧光团,而当互补链DNA(cDNA)或靶标出现时,形成双螺旋结构或结合靶标的DNA 二级结构使得DNA释放,从而使荧光得到恢复。本发明利用氧化石墨烯消除背景荧光,以凝血酶特异性识别的适配体为识别元件,DNA-Cu/Ag NCs为信号报告分子,通过氧化石墨烯的使用,氧化石墨烯与未结合凝血酶的DNA-Cu/Ag NCs作用,猝灭了DNA-Cu/AgNCs荧光,消除了 DNA-Cu/Ag NCs的背景荧光,从而实现了对凝血酶简单、快速、低成本、高灵敏的检测。
本发明以凝血酶特异性识别的适配体为识别元件,以适配体 DNA-Cu/Ag NCs为信号报告分子,利用氧化石墨烯消除背景荧光,可实现凝血酶高灵敏的检测,属于荧光增强型探针。其线性范围为2-10nM,凝血酶的最低检测限为0.25nM。
本发明采取先将凝血酶与DNA适配体特异性结合再合成 DNA-Cu/Ag NCs,最后加入氧化石墨烯,既消除了背景荧光,又避免了氧化石墨烯对凝血酶非特异性吸附,从而减少了对氧化石墨烯的修饰步骤。
本发明具有成本低廉、操作简单、灵敏度高、选择性好、毒性小、生物相容性好、免标记等优点。另外,本发明所述的检测方法对临床诊断和生物化学研究具有潜在的应用价值。
附图说明
图1为本发明所述的凝血酶检测方法原理图。
图2为在最大发射波长为560nm下DNA-Cu/Ag NCs的荧光强度随凝血酶浓度的变化关系图,插入图为DNA-Cu/Ag NCs的荧光强度在凝血酶浓度为2-10nM的线性关系图。
图3为加入浓度为10nM的溶菌酶、脂肪酶、胰蛋白酶、乙酰胆碱酯酶、腺苷脱氨酶和凝血酶的相对荧光强度图;F0和F分别为未加和加入上述物质的DNA-Cu/Ag NCs最大荧光发射强度。
图4为DNA-Cu/Ag NCs的TEM图,插入图为DNA-Cu/Ag NCs大小分布柱状图。
具体实施方式
下面结合具体实施例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但是应理解所述实施例仅是范例性的,不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改或替换均落入本发明的保护范围。
试验例1本发明凝血酶检测方法的选择性试验
为了评估基于氧化石墨烯与铜银纳米簇结合的凝血酶检测方法的选择性,按照如下步骤进行实验:
(1)氧化石墨烯的处理:使用前,将购置的氧化石墨烯在水溶液中 1500W超声40min,制得分散液A备用;所述分散液的浓度为3mg/mL。
(2)将含适配体的DNA(1.5μM)与浓度为10nM的溶菌酶、脂肪酶、胰蛋白酶、乙酰胆碱酯酶、腺苷脱氨酶和凝血酶分别在Tris-HAc缓冲液(10mM,pH 7.0)中混合,孵育0.5h,然后将12μM AgNO3和4.5μM Cu(NO3)2加入到上述溶液混匀,在4℃下避光孵育20-40min。最后将新制的30μM NaBH4加入到上述溶液中在4℃下混匀并避光孵育1-2h,得到上述含酶的DNA-Cu/Ag NCs溶液B。
(3)将步骤(1)所得的氧化石墨烯分散液A分别加入到步骤(2) 所制得的B溶液中至氧化石墨烯分散液的最终浓度为45μg/mL;将混合溶液孵育20min,在室温下测定各样品溶液所对应的荧光值。测定结果见图3。
实施例1采用本发明检测方法检测牛血清蛋白中凝血酶的含量
(1)氧化石墨烯的处理:使用前,将购置的氧化石墨烯在水溶液中 1500W超声40min制得氧化石墨烯分散液A备用;所述氧化石墨烯分散液A的浓度为3mg/mL。
(2)将0-14nM的凝血酶分别加入到含1.5μM的含适配体的DNA 的Tris-HAc缓冲液(10mM,pH 7.0)中混合均匀孵育0.5h,然后将12μM AgNO3和4.5μM Cu(NO3)2加入到上述溶液中混匀,在4℃下避光孵育20-40min,最后将新制的30μMNaBH4加入到上述溶液中混匀,在4℃下避光孵育1-2h,得到不同浓度凝血酶与适配体结合的系列混合溶液(DNA-Cu/Ag NCs溶液B)。
(3)将步骤(1)所得的氧化石墨烯分散液分别加入到步骤2所制得的系列混合溶液中混合均匀控制氧化石墨烯分散液在混合溶液中的终浓度为45μg/mL;将混合溶液孵育20min。
(4)在室温下测定各混合溶液所对应的荧光值,绘制标准曲线。
(5)将待测样品液(溶解在稀释了100倍的牛血清蛋白的凝血酶溶液,浓度分别为2nM、4nM、6nM和8nM)按照(1)、(2)和(3) 的步骤制备含待测凝血酶的DNA-Cu/Ag NCs并测荧光值,计算凝血酶的浓度。
表1为利用本发明方法检测加入到稀释了100倍的牛血清蛋白中的凝血酶的测定值、回收率以及相对标准偏差。表1中的数据表明本发明方法能准确的检测样品中的凝血酶的含量,可以用来检测实际样品中的凝血酶。
表1用本发明方法检测加入到牛血清蛋白中凝血酶的浓度(N=3).
a The mean of three determinations.
bSD=standard deviation.
RSD=relative standard 。
SEQUENCE LISTING
<110> 北京林业大学
<120> 凝血酶含量的检测方法
<130> BJ-1006-200804A
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<170> PatentIn version 3.5
<210> 1
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<212> DNA
<213> Artifical sequence
<400> 1
ctcttaatct cctttttggt tggtgtggtt ggtttttctc ttaatctcc 49
Claims (10)
1.一种凝血酶含量的检测方法,其特征在于,包括:
(1)绘制凝血酶的荧光标准曲线,包括
(a)氧化石墨烯的预处理:将氧化石墨烯在水溶液中用超声处理得到氧化石墨烯分散液,备用;
(b)凝血酶与适配体结合的DNA-Cu/Ag NCs溶液的制备:将含适配体的DNA与不同浓度的凝血酶分别在Tris-HAc缓冲液中混匀并孵育后加入AgNO3和Cu(NO3)2避光孵育;再加入NaBH4混匀并孵育使Ag+和Cu2+还原为Ag和Cu,得到凝血酶与适配体结合的DNA-Cu/Ag NCs溶液;
(c)将步骤(a)所制得的氧化石墨烯分散液加入到步骤(b)所制得的凝血酶与适配体结合的DNA-Cu/Ag NCs溶液中孵育后,分别测定各混合溶液所对应的荧光值,根据最大荧光发射值绘制标准曲线;
(2)将待测的样品液与含适配体的DNA在Tris-HAc缓冲液中混匀孵育后加入AgNO3和Cu(NO3)2混合均匀避光孵育;再加入NaBH4混匀并孵育;最后加入氧化石墨烯分散液混合均匀孵育后测定该混合溶液的荧光值;
(3)根据标准曲线,计算得到待测样品液中凝血酶的含量。
2.按照权利要求1所述的检测方法,其特征在于,步骤(a)所述的氧化石墨烯分散液中氧化石墨烯浓度为3mg/mL;所述的超声处理是采用1500W的功率超声处理40min。
3.按照权利要求1所述的检测方法,其特征在于,步骤(b)所述的含适配体的DNA序列为SEQ ID NO.1所示;所述含适配体的DNA浓度优选为1.5μM。
4.按照权利要求1所述的检测方法,其特征在于,步骤(b)中Cu(NO3)2、AgNO3和NaBH4的浓度分别为4.5μM、12μM和30μM。
5.按照权利要求1所述的检测方法,其特征在于,步骤(b)中所述的不同浓度的凝血酶为0-14nM的凝血酶,所述Tris-HAc缓冲液的浓度为10mM,其pH值为7.0。
6.按照权利要求1所述的检测方法,其特征在于,步骤(b)中所得到的凝血酶与适配体结合的DNA-Cu/Ag NCs的粒径小于2nm。
7.按照权利要求1所述的检测方法,其特征在于,步骤(b)中将含适配体的DNA与不同浓度的凝血酶分别在Tris-HAc缓冲液中混匀并孵育0.5h后加入AgNO3和Cu(NO3)2混合均匀在4℃下避光孵育20-40min后再加入NaBH4混匀并孵育1-2h。
8.按照权利要求1所述的检测方法,其特征在于,步骤(c)中将步骤(a)所制得的氧化石墨烯分散液加入到步骤(b)所制得的凝血酶与适配体结合的DNA-Cu/Ag NCs溶液中孵育20min然后在室温下分别测定各混合溶液所对应的荧光值,根据最大荧光发射值绘制标准曲线。
9.按照权利要求1所述的检测方法,其特征在于,步骤(2)中将待测的样品液与含适配体的DNA在Tris-HAc缓冲液中混匀孵育0.5h然后将AgNO3和Cu(NO3)2加入到该混合溶液中,在4℃下避光孵育20-40min。
10.按照权利要求1所述的检测方法,其特征在于,步骤(2)中最后所加入的氧化石墨烯分散液至其在混合溶液中的终浓度为45μg/mL。
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