CN103698320A - Construction method of chiral sensor for detecting DNA enzymatic assembly of lead ion - Google Patents

Construction method of chiral sensor for detecting DNA enzymatic assembly of lead ion Download PDF

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CN103698320A
CN103698320A CN201310691536.8A CN201310691536A CN103698320A CN 103698320 A CN103698320 A CN 103698320A CN 201310691536 A CN201310691536 A CN 201310691536A CN 103698320 A CN103698320 A CN 103698320A
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silver nanoparticles
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silver
nanoparticles
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徐丽广
胥传来
尹红红
匡华
刘丽强
马伟
宋珊珊
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Jiangnan University
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Abstract

一种测定铅离子的DNA酶组装的手性传感器的构建方法,属于纳米生物技术检测领域。本发明包括:10nm和20nm的银纳米粒子分别用一对DNA探针进行修饰,修饰的银纳米粒子在DNA酶的作用下组装成不对称手性二聚体,银纳米粒子组装体作为检测传感器用于Pb2+的检测,应用圆二色光谱(CD)进行检测。本发明应用了一种银纳米粒子探针,借助于Pb2+依赖的DNA酶的作用下,大小银纳米粒子组装成不对称的手性二聚体,通过CD信号对Pb2+进行检测。本发明方法能够实现对Pb2+的超灵敏检测,检测灵敏度高、特异性好,同时可以检测水溶液中的痕量目标物。

Figure 201310691536

The invention discloses a method for constructing a chiral sensor assembled by DNA enzyme for measuring lead ions, which belongs to the field of nanobiological technology detection. The invention includes: silver nanoparticles of 10nm and 20nm are respectively modified with a pair of DNA probes, the modified silver nanoparticles are assembled into an asymmetric chiral dimer under the action of DNA enzyme, and the silver nanoparticle assembly is used as a detection sensor For the detection of Pb 2+ , circular dichroism (CD) is used for detection. The invention uses a silver nanoparticle probe, and under the action of Pb2 + -dependent DNase, large and small silver nanoparticles are assembled into an asymmetric chiral dimer, and the Pb2 + is detected through a CD signal. The method of the invention can realize ultrasensitive detection of Pb 2+ , has high detection sensitivity and good specificity, and can detect trace target objects in aqueous solution at the same time.

Figure 201310691536

Description

一种测定铅离子的DNA酶组装的手性传感器的构建方法A method for constructing a chiral sensor assembled by DNase for measuring lead ions

技术领域 technical field

一种测定Pb2+的DNA酶组装的手性传感器的构建方法,属于纳米生物技术检测领域。 The invention relates to a method for constructing a chiral sensor assembled by DNase for measuring Pb 2+ , belonging to the field of nanobiological technology detection.

背景技术 Background technique

Pb2+是最具毒性的重金属离子之一,铅的污染来自矿山开采、冶炼、橡胶生产、染料、印刷、陶瓷、铅玻璃、焊锡、电缆及铅管等生产废水和废弃物。另外,汽车尾气中的四乙基铅是剧毒物质。随着我国工农业的迅速发展,环境中重金属的污染越来越严重。重金属污染引起的毒害持久存在,会随土壤、水体再次循环而进入食物链,对食品安全构成威胁,危害人类生命和健康。目前为止,铅在生物体内没有已知的生理作用,任何人体内的铅均被视为污染,铅中毒会引发肠胃紊乱,肝、肾以及神经损伤。美国环保署规定饮用水中Pb2+的最高含量不得超过72nM。国家环保部的调查显示,我国水体重金属污染问题十分突出,江河湖库底质的污染率高达80.1%。针对重金属污染的特点,对其发现和检测是至关重要的。 Pb 2+ is one of the most toxic heavy metal ions. Lead pollution comes from production wastewater and wastes such as mining, smelting, rubber production, dyes, printing, ceramics, lead glass, solder, cables and lead pipes. In addition, tetraethyl lead in automobile exhaust is a highly toxic substance. With the rapid development of industry and agriculture in our country, the pollution of heavy metals in the environment is becoming more and more serious. The poison caused by heavy metal pollution persists and will enter the food chain with the recirculation of soil and water, posing a threat to food safety and endangering human life and health. So far, lead has no known physiological role in living organisms. Any lead in human body is regarded as pollution. Lead poisoning can cause gastrointestinal disorders, liver, kidney and nerve damage. The United States Environmental Protection Agency stipulates that the maximum content of Pb 2+ in drinking water shall not exceed 72nM. According to the survey of the Ministry of Environmental Protection, the problem of heavy metal pollution in my country's water is very prominent, and the pollution rate of the bottom of rivers, lakes and reservoirs is as high as 80.1%. According to the characteristics of heavy metal pollution, its discovery and detection is very important.

传统的Pb2+检测方法包括电感偶合等离子体法(ICP)和原子吸收光谱法,这些方法具有灵敏度高、定量准确等优点,但是所需仪器昂贵、样品预处理复杂、耗时,且要求检测人员具备一定的专业知识,分析成本高。化学、生物传感器由于具有灵敏度高、选择性好、体积小、造价低等特点,受到了人们的青睐。近年来,随着纳米技术的发展,基于寡核苷酸功能化的纳米粒子在食品危害因子检测方面进行了大量的研究和应用。由于纳米粒子具有特殊的性质,如:光学特性、电化学特性、荧光特性、顺磁性等,可以利用这些性质所特有的信号进行放大来达到检测目标物的目的。等离子纳米粒子组装成的手性纳米结构具有CD信号,这一发现对纳米材料在检测领域的应用成为一个新的进展,可以应用手性纳米材料组装体的CD信号作为检测信号对有害物进行检测。 Traditional Pb 2+ detection methods include Inductively Coupled Plasma (ICP) and Atomic Absorption Spectrometry. The personnel have certain professional knowledge, and the analysis cost is high. Chemical and biological sensors have been favored by people because of their high sensitivity, good selectivity, small size, and low cost. In recent years, with the development of nanotechnology, a lot of research and application have been carried out in the detection of food hazard factors based on oligonucleotide functionalized nanoparticles. Since nanoparticles have special properties, such as: optical properties, electrochemical properties, fluorescence properties, paramagnetism, etc., the signals unique to these properties can be used to amplify to achieve the purpose of detecting the target. The chiral nanostructure assembled by plasmonic nanoparticles has a CD signal. This discovery has become a new development in the application of nanomaterials in the detection field. The CD signal of the chiral nanomaterial assembly can be used as a detection signal to detect harmful substances. .

DNA酶是经体外筛选得到的一种功能化的DNA分子,这种酶不同于蛋白酶,可以通过化学合成得到,具有良好的热稳定性和反应活性,同时DNA分子可以修饰多种功能基团,并且可以固定到固相支持物上,目前,大量的目标物具有能够识别的DNA酶。在重金属离子存在的条件下,重金属依赖的DNA酶的底物链在RNA碱基切割位点处被切开,Pb2+依赖的DNA酶被称作“17 E”DNA酶,由一条酶链和一条底物链组成,应用DNA酶建立的Pb2+检测传感器引起了人们广泛的研究兴趣。 DNase is a functionalized DNA molecule obtained by in vitro screening. This enzyme is different from protease and can be obtained by chemical synthesis. It has good thermal stability and reactivity. At the same time, DNA molecules can modify various functional groups. And it can be immobilized on a solid support. At present, a large number of target objects have DNA enzymes that can be recognized. In the presence of heavy metal ions, the substrate chain of the heavy metal-dependent DNase is cut at the RNA base cleavage site, and the Pb 2+ -dependent DNase is called "17 E" DNase, which consists of an enzyme chain Composed with a substrate chain, the Pb 2+ detection sensor established by using DNase has aroused widespread research interest.

本发明是借助于DNA酶的作用将DNA探针修饰的大小银纳米粒子组装成不对称的银纳米粒子二聚体,在不同浓度Pb2+存在的条件下,二聚体发生不同程度的解聚,随着Pb2+浓度的增加,二聚体的解聚程度越大,从而CD信号随之降低,根据CD信号的强度与Pb2+浓度之间的建立的对应关系,从而对Pb2+含量进行检测。 The present invention assembles silver nanoparticles of large and small sizes modified by DNA probes into asymmetric silver nanoparticle dimers by virtue of the action of DNA enzymes, and the dimers are dissociated in different degrees under the conditions of the presence of different concentrations of Pb 2+ Polymerization, as the concentration of Pb 2+ increases, the degree of disaggregation of the dimer increases, and the CD signal decreases accordingly. According to the corresponding relationship between the intensity of the CD signal and the concentration of Pb 2+ , the Pb 2 + content is tested.

发明内容 Contents of the invention

本发明的目的在于提供一种对Pb2+进行检测与定量的手性传感器方法,借助于DNA酶的作用将DNA探针修饰的大小银纳米粒子组装成不对称的银纳米粒子二聚体,在不同浓度Pb2+存在的条件下,二聚体发生不同程度的解聚,最后通过CD光谱对银纳米粒子组装体进行测定,从而间接检测目标Pb2+的含量。 The object of the present invention is to provide a kind of chiral sensor method that Pb 2+ is detected and quantified, by means of the effect of DNA enzyme, the size silver nanoparticles modified by DNA probes are assembled into asymmetric silver nanoparticle dimers, In the presence of different concentrations of Pb 2+ , the dimer depolymerized to different degrees, and finally the silver nanoparticle assembly was measured by CD spectroscopy, so as to indirectly detect the content of the target Pb 2+ .

本发明的技术方案:一种测定Pb2+的DNA酶组装的手性传感器的构建方法,包括:10nm和20nm的银纳米粒子分别用一对DNA探针进行修饰,银纳米粒子在DNA酶的作用下组装成不对称手性二聚体,该银纳米粒子组装体作为检测传感器用于Pb2+的检测,应用CD光谱进行检测;具体步骤为: Technical scheme of the present invention: a method for constructing a chiral sensor assembled by DNase for measuring Pb 2+ , comprising: silver nanoparticles of 10nm and 20nm are modified with a pair of DNA probes respectively, Under the action, it is assembled into an asymmetric chiral dimer, and the silver nanoparticle assembly is used as a detection sensor for the detection of Pb 2+ , and CD spectroscopy is used for detection; the specific steps are:

(1)10nm和20nm的银纳米粒子分别用一对DNA探针进行修饰 (1) Silver nanoparticles of 10nm and 20nm were modified with a pair of DNA probes respectively

将新合成的10nm、20nm的银纳米粒子分别在10000r/min、8000r/min的转速条件下通过离心浓缩十倍,然后将银纳米粒子分别重悬到包含有50mM NaCl的10mM Tris-HCl缓冲液中,使得10nm、20nm的银纳米粒子的终浓度分别为50 nM、20 nM;然后将DNA探针和银纳米粒子按照10:1的摩尔比例进行偶联,具体如下:1μL的20 μM S1核酸片段加入到100 μL 20 nM 的20 nm银纳米粒子中,1 μL的50 μM S2核酸片段加入到100 μL 50 nM 的10 nm银纳米粒子中,孵育12h后,成功偶联的银纳米粒子离心三次,将未偶联的DNA去除,最后纳米粒子均重悬到50 μL 25 mM Tris-醋酸缓冲液中 (pH 8.2,包含100 mM NaCl),最终得到偶联好的银纳米粒子; Concentrate the newly synthesized 10nm and 20nm silver nanoparticles by centrifugation at 10000r/min and 8000r/min respectively, and then resuspend the silver nanoparticles into 10mM Tris-HCl buffer containing 50mM NaCl In the method, the final concentrations of 10nm and 20nm silver nanoparticles were 50 nM and 20 nM respectively; then DNA probes and silver nanoparticles were coupled at a molar ratio of 10:1, specifically as follows: 1 μL of 20 μM S1 nucleic acid The fragments were added to 100 μL of 20 nM 20 nm silver nanoparticles, and 1 μL of 50 μM S2 nucleic acid fragments were added to 100 μL of 50 nM 10 nm silver nanoparticles. After incubation for 12 hours, the successfully coupled silver nanoparticles were centrifuged three times. , the uncoupled DNA was removed, and finally the nanoparticles were resuspended in 50 μL of 25 mM Tris-acetate buffer (pH 8.2, containing 100 mM NaCl), and finally the coupled silver nanoparticles were obtained;

S1:5'-TCACAGATGA GT-SH-3'; S1: 5'-TCACAGATGA GT-SH-3';

S2:5'-SH-CACGAGTTGA CA-3'; S2: 5'-SH-CACGAGTTGA CA-3';

(2)银纳米粒子在DNA酶的作用下组装成不对称手性二聚体 (2) Silver nanoparticles are assembled into asymmetric chiral dimers under the action of DNase

整个组装体系包括:70 μL S1修饰的20 nm的银纳米粒子,30 μL S2修饰的10 nm的银纳米粒子,1 μL 100 μM 底物链(Sub),2 μL 100 μM 酶链(17E);首先将整个反应体系加热到70℃,然后将其自然冷却至室温以促进银纳米粒子的组装;杂交8h后,将组装产物离心三次,获得纯化好的不对称银纳米粒子二聚体; The whole assembly system includes: 70 μL S1 modified 20 nm silver nanoparticles, 30 μL S2 modified 10 nm silver nanoparticles, 1 μL 100 μM substrate chain (Sub), 2 μL 100 μM enzyme chain (17E); Firstly, the entire reaction system was heated to 70°C, and then naturally cooled to room temperature to promote the assembly of silver nanoparticles; after hybridization for 8 hours, the assembled product was centrifuged three times to obtain a purified asymmetric silver nanoparticle dimer;

Sub:5'-ACTCATCTGT GAACTCACTA T(rA)GGAAGAGAT GTGTCAACTC GTG-3'; Sub: 5'-ACTCATCTGT GAACTCACTA T(rA)GGAAGAGAT GTGTCAACTC GTG-3';

17E:5'-CATCTCTTCT CCGAGCCGGT CGAAATAGTG AGT-3'; 17E: 5'-CATCTCTTCT CCGAGCCGGT CGAAATAGTG AGT-3';

(3)银纳米粒子组装体作为检测传感器用于Pb2+的检测,应用CD光谱进行检测 (3) The silver nanoparticle assembly is used as a detection sensor for the detection of Pb 2+ , and the CD spectrum is used for detection

在不对称银纳米粒子二聚体中加入一系列不同浓度的Pb2+,在Pb2+的作用下,底物链断裂,从而导致二聚体被分裂成单个的粒子,CD信号的强度降低,将不同目标物浓度下的反应体系用CD光谱进行检测,得到200nm-800nm的CD光谱图,随着Pb2+浓度的增加,不对称银纳米粒子二聚体的数量逐渐减少,CD信号逐渐降低,根据Pb2+浓度与CD信号强度之间的对应关系,绘制Pb2+浓度与CD信号强度的标准曲线,从而应用CD信号对Pb2+的浓度进行定量。 Adding a series of different concentrations of Pb 2+ to the asymmetric silver nanoparticle dimer, under the action of Pb 2+ , the substrate chain is broken, which causes the dimer to be split into individual particles, and the intensity of the CD signal decreases , the reaction system under different target concentrations was detected by CD spectrum, and the CD spectrum of 200nm-800nm was obtained. With the increase of Pb 2+ concentration, the number of asymmetric silver nanoparticle dimers gradually decreased, and the CD signal gradually decreased. According to the corresponding relationship between Pb 2+ concentration and CD signal intensity, draw the standard curve of Pb 2+ concentration and CD signal intensity, so as to use CD signal to quantify the concentration of Pb 2+ .

所述的10nm、20nm的银纳米粒子通过硼氢化钠还原硝酸银的方法进行合成,合成步骤:0.6 mL 0.1 M硼氢化钠溶解到20 mL冰水中,再加入5 mL 1%的聚乙烯吡咯烷酮(PVP)作为保护剂,另加入600 μL 1%的柠檬酸三钠以得到20nm银纳米粒子,以上混合物在冰浴中处于持续搅拌状态。然后用两个恒流泵以30 mL/h的速率,同时分别加入5 mL 1%的聚乙烯吡咯烷酮(PVP)和5 mL 10 mM的硝酸银。最后将反应溶液在80℃条件下孵育以去除过量的硼氢化钠,3 h后得到合成好的10nm的银纳米粒子,并放入4℃待用。 The silver nanoparticles of 10nm and 20nm were synthesized by reducing silver nitrate with sodium borohydride. Synthesis steps: 0.6 mL of 0.1 M sodium borohydride was dissolved in 20 mL of ice water, and then 5 mL of 1% polyvinylpyrrolidone ( PVP) was used as a protective agent, and 600 μL of 1% trisodium citrate was added to obtain 20nm silver nanoparticles, and the above mixture was kept stirring in an ice bath. Then, 5 mL of 1% polyvinylpyrrolidone (PVP) and 5 mL of 10 mM silver nitrate were added simultaneously at a rate of 30 mL/h using two constant-flow pumps. Finally, the reaction solution was incubated at 80°C to remove excess sodium borohydride. After 3 h, the synthesized 10nm silver nanoparticles were obtained, and placed at 4°C for use.

本发明的有益效果:本发明提供一种对Pb2+进行检测与定量的手性传感器方法,借助于DNA酶的作用将DNA探针修饰的大小银纳米粒子组装成不对称的银纳米粒子二聚体,在不同浓度Pb2+存在的条件下,二聚体发生不同程度的解聚,最后通过CD光谱对银纳米粒子组装体进行测定,从而间接检测目标Pb2+的含量。 Beneficial effects of the present invention: the present invention provides a chiral sensor method for detecting and quantifying Pb 2+ , by means of the action of DNase, silver nanoparticles of large and small sizes modified by DNA probes are assembled into asymmetric silver nanoparticles. In the presence of different concentrations of Pb 2+ , the dimer depolymerized to different degrees, and finally the silver nanoparticle assembly was measured by CD spectroscopy, so as to indirectly detect the content of the target Pb 2+ .

附图说明 Description of drawings

图1  Pb2+检测的CD光谱; Figure 1 CD spectrum of Pb 2+ detection;

图2  Pb2+检测的标准曲线。 Figure 2 Standard curve for Pb 2+ detection.

具体实施方式 Detailed ways

实施例1 Example 1

测定Pb2+的DNA酶组装的手性传感器的构建方法。包括:10nm、20nm的银纳米粒子分别用一对DNA探针进行修饰,银纳米粒子在DNA酶的作用下组装成不对称手性二聚体,该银纳米粒子组装体作为检测传感器用于Pb2+的检测,应用CD光谱进行检测;具体步骤为: Construction of a DNase-assembled chiral sensor for the measurement of Pb 2+ . Including: 10nm and 20nm silver nanoparticles are respectively modified with a pair of DNA probes, and the silver nanoparticles are assembled into an asymmetric chiral dimer under the action of DNase, and the silver nanoparticle assembly is used as a detection sensor for Pb For the detection of 2+ , CD spectrum is used for detection; the specific steps are:

(1)10nm、20nm的银纳米粒子分别用一对DNA探针进行修饰 (1) Silver nanoparticles of 10nm and 20nm were modified with a pair of DNA probes respectively

将新合成的10nm、20nm的银纳米粒子分别在10000r/min、8000r/min的转速条件下通过离心浓缩十倍,然后将银纳米粒子分别重悬到包含有50mM NaCl的10mM Tris-HCl缓冲液中,使得10nm、20nm的银纳米粒子的终浓度分别为50 nM、20 nM;然后将DNA探针和银纳米粒子按照10:1的摩尔比例进行偶联,具体如下:1μL的20 μM S1核酸片段加入到100 μL 20 nM 的20 nm银纳米粒子中,1 μL的50 μM S2核酸片段加入到100 μL 50 nM 的10 nm银纳米粒子中,孵育12h后,成功偶联的银纳米粒子离心三次,将未偶联的DNA去除,最后纳米粒子均重悬到50 μL 25 mM Tris-醋酸缓冲液中 (pH 8.2,包含100 mM NaCl),最终得到偶联好的银纳米粒子; Concentrate the newly synthesized 10nm and 20nm silver nanoparticles by centrifugation at 10000r/min and 8000r/min respectively, and then resuspend the silver nanoparticles into 10mM Tris-HCl buffer containing 50mM NaCl In the method, the final concentrations of 10nm and 20nm silver nanoparticles were 50 nM and 20 nM respectively; then DNA probes and silver nanoparticles were coupled at a molar ratio of 10:1, specifically as follows: 1 μL of 20 μM S1 nucleic acid The fragments were added to 100 μL of 20 nM 20 nm silver nanoparticles, and 1 μL of 50 μM S2 nucleic acid fragments were added to 100 μL of 50 nM 10 nm silver nanoparticles. After incubation for 12 hours, the successfully coupled silver nanoparticles were centrifuged three times. , the uncoupled DNA was removed, and finally the nanoparticles were resuspended in 50 μL of 25 mM Tris-acetate buffer (pH 8.2, containing 100 mM NaCl), and finally the coupled silver nanoparticles were obtained;

S1:5'-TCACAGATGA GT-SH-3'; S1: 5'-TCACAGATGA GT-SH-3';

S2:5'-SH-CACGAGTTGA CA-3'; S2: 5'-SH-CACGAGTTGA CA-3';

(2)银纳米粒子在DNA酶的作用下组装成不对称手性二聚体 (2) Silver nanoparticles are assembled into asymmetric chiral dimers under the action of DNase

整个组装体系包括:70 μL S1修饰的20 nm的银纳米粒子,30 μL S2修饰的10 nm的银纳米粒子,1 μL 100 μM 底物链(Sub),2 μL 100 μM 酶链(17E);首先将整个反应体系加热到70℃,然后将其自然冷却至室温以促进银纳米粒子的组装;杂交8h后,将组装产物离心三次,获得纯化好的不对称银纳米粒子二聚体;  The whole assembly system includes: 70 μL S1 modified 20 nm silver nanoparticles, 30 μL S2 modified 10 nm silver nanoparticles, 1 μL 100 μM substrate chain (Sub), 2 μL 100 μM enzyme chain (17E); Firstly, the whole reaction system was heated to 70°C, and then naturally cooled to room temperature to promote the assembly of silver nanoparticles; after hybridization for 8 hours, the assembled product was centrifuged three times to obtain a purified asymmetric silver nanoparticle dimer;

Sub:5'-ACTCATCTGT GAACTCACTA T(rA)GGAAGAGAT GTGTCAACTC GTG-3'; Sub: 5'-ACTCATCTGT GAACTCACTA T(rA)GGAAGAGAT GTGTCAACTC GTG-3';

17E:5'-CATCTCTTCT CCGAGCCGGT CGAAATAGTG AGT-3'; 17E: 5'-CATCTCTTCT CCGAGCCGGT CGAAATAGTG AGT-3';

(3)银纳米粒子组装体作为检测传感器用于Pb2+的检测,应用CD光谱进行检测 (3) The silver nanoparticle assembly is used as a detection sensor for the detection of Pb 2+ , and the CD spectrum is used for detection

在不对称银纳米粒子二聚体中加入一系列不同浓度的Pb2+,在Pb2+的作用下,底物链断裂,从而导致二聚体被分裂成单个的粒子,CD信号的强度降低。将不同目标物浓度下的反应体系用CD光谱进行检测,得到200nm-800nm的CD光谱图,随着Pb2+浓度的增加,不对称银纳米粒子二聚体的数量逐渐减少,CD信号逐渐降低,根据Pb2+浓度与CD信号强度之间的对应关系,绘制Pb2+浓度与CD信号强度的标准曲线,从而应用CD信号对Pb2+的浓度进行定量。 Adding a series of different concentrations of Pb 2+ to the asymmetric silver nanoparticle dimer, under the action of Pb 2+ , the substrate chain is broken, which causes the dimer to be split into individual particles, and the intensity of the CD signal decreases . The reaction system under different target concentrations was detected by CD spectrum, and the CD spectrum of 200nm-800nm was obtained. With the increase of Pb 2+ concentration, the number of asymmetric silver nanoparticle dimers gradually decreased, and the CD signal gradually decreased. , according to the corresponding relationship between Pb 2+ concentration and CD signal intensity, draw the standard curve of Pb 2+ concentration and CD signal intensity, so as to use CD signal to quantify the concentration of Pb 2+ .

(4)检测灵敏度研究 (4) Research on detection sensitivity

根据每个目标Pb2+浓度下对应的CD信号强度,以DNA浓度为横坐标,CD信号强度为纵坐标做出一条标准曲线,根据标准曲线计算出Pb2+的检测限为0.02 ng mL-1According to the CD signal intensity corresponding to each target Pb 2+ concentration, a standard curve was drawn with the DNA concentration as the abscissa and the CD signal intensity as the ordinate, and the detection limit of Pb 2+ was calculated as 0.02 ng mL - 1 .

(5)特异性研究 (5) Specificity research

以Mn2+、Zn2+、Mg2+、Fe2+、Ca2+、Hg2+、Cu2+为检测对象,进行特异性分析,加入浓度均为5 ng mL-1,操作方法与Pb2+检测的操作方法一致,反应体系的CD信号与阴性空白样品即未加入任何重金属离子的CD信号进行对比,得到的CD信号与空白样品的CD信号没有明显差别,由此得出其它重金属离子不能识别Pb2+依赖的DNA酶,此方法的特异性良好。 Mn 2+ , Zn 2+ , Mg 2+ , Fe 2+ , Ca 2+ , Hg 2+ , and Cu 2+ were used as detection objects for specificity analysis. The concentration was 5 ng mL -1 , and the operation method was the same as The operation method of Pb 2+ detection is the same. The CD signal of the reaction system is compared with the CD signal of the negative blank sample without adding any heavy metal ions. There is no significant difference between the obtained CD signal and the CD signal of the blank sample. Ions do not recognize Pb 2+ -dependent DNase, and the specificity of this method is good.

(6)添加回收实验 (6) Add recovery experiment

将不同浓度的Pb2+加入到阴性自来水中,用以上方法建立的Pb2+检测传感器进行水样品中的添加回收测定,最终得到的回收率范围在94%-100%,可以进行实际样品的检测。 Add different concentrations of Pb 2+ into the negative tap water, and use the Pb 2+ detection sensor established by the above method to carry out the addition and recovery determination of the water sample. The final recovery rate ranges from 94% to 100%, which can be used for the actual sample. detection.

S1:5'-TCACAGATGA GT-SH-3'; S1: 5'-TCACAGATGA GT-SH-3';

S2:5'-SH-CACGAGTTGA CA-3'; S2: 5'-SH-CACGAGTTGA CA-3';

Sub:5'-ACTCATCTGT GAACTCACTA T(rA)GGAAGAGAT GTGTCAACTC GTG-3'; Sub: 5'-ACTCATCTGT GAACTCACTA T(rA)GGAAGAGAT GTGTCAACTC GTG-3';

17E:5'-CATCTCTTCT CCGAGCCGGT CGAAATAGTG AGT-3'; 17E: 5'-CATCTCTTCT CCGAGCCGGT CGAAATAGTG AGT-3';

  the

Claims (1)

1.一种测定Pb2+的DNA酶组装的手性传感器的构建方法,其特征在于包括:10nm和20nm的银纳米粒子分别用一对DNA探针进行修饰,银纳米粒子在DNA酶的作用下组装成不对称手性二聚体,该银纳米粒子组装体作为检测传感器用于Pb2+的检测,应用CD光谱进行检测;具体步骤为: 1. A method for constructing a chiral sensor that measures Pb 2+ DNase assembly, is characterized in that comprising: the silver nanoparticles of 10nm and 20nm are modified with a pair of DNA probes respectively, and the silver nanoparticles are used in the effect of DNase Assembled into an asymmetric chiral dimer, the silver nanoparticle assembly is used as a detection sensor for the detection of Pb 2+ , and the CD spectrum is used for detection; the specific steps are: (1)10nm、20nm的银纳米粒子分别用一对DNA探针进行修饰 (1) Silver nanoparticles of 10nm and 20nm were modified with a pair of DNA probes respectively 将新合成的10nm、20nm的银纳米粒子分别在10000r/min、8000r/min的转速条件下通过离心浓缩十倍,然后将银纳米粒子分别重悬到包含有50mM NaCl的10mM Tris-HCl缓冲液中,使得10nm、20nm的银纳米粒子的终浓度分别为50 nM、20 nM;然后将DNA探针和银纳米粒子按照10:1的摩尔比例进行偶联,具体如下:1μL的20 μM S1核酸片段加入到100 μL 20 nM的 20 nm银纳米粒子中,1 μL的50 μM S2核酸片段加入到100 μL 50 nM 的10 nm银纳米粒子中,孵育12h后,成功偶联的银纳米粒子离心三次,将未偶联的DNA去除,最后纳米粒子均重悬到50 μL pH 8.2,包含100 mM NaCl 的25 mM Tris-醋酸缓冲液中,最终得到偶联好的银纳米粒子; Concentrate the newly synthesized 10nm and 20nm silver nanoparticles by centrifugation at 10000r/min and 8000r/min respectively, and then resuspend the silver nanoparticles into 10mM Tris-HCl buffer containing 50mM NaCl In the method, the final concentrations of 10nm and 20nm silver nanoparticles were 50 nM and 20 nM respectively; then DNA probes and silver nanoparticles were coupled at a molar ratio of 10:1, specifically as follows: 1 μL of 20 μM S1 nucleic acid The fragments were added to 100 μL of 20 nM 20 nm silver nanoparticles, and 1 μL of 50 μM S2 nucleic acid fragments were added to 100 μL of 50 nM 10 nm silver nanoparticles. After incubation for 12 hours, the successfully coupled silver nanoparticles were centrifuged three times. , the uncoupled DNA was removed, and finally the nanoparticles were resuspended in 50 μL pH 8.2, containing 100 mM NaCl in 25 mM Tris-acetate buffer, and finally the coupled silver nanoparticles were obtained; S1:5'-TCACAGATGA GT-SH-3'; S1: 5'-TCACAGATGA GT-SH-3'; S2:5'-SH-CACGAGTTGA CA-3'; S2: 5'-SH-CACGAGTTGA CA-3'; (2)银纳米粒子在DNA酶的作用下组装成不对称手性二聚体 (2) Silver nanoparticles are assembled into asymmetric chiral dimers under the action of DNase 整个组装体系包括:70 μL S1修饰的20 nm的银纳米粒子,30 μL S2修饰的10 nm的银纳米粒子,1 μL 100 μM 底物链Sub,2 μL 100 μM 酶链17E;首先将整个反应体系加热到70℃,然后将其自然冷却至室温以促进银纳米粒子的组装;杂交8h后,将组装产物离心三次,获得纯化好的不对称银纳米粒子二聚体;  The whole assembly system includes: 70 μL S1 modified 20 nm silver nanoparticles, 30 μL S2 modified 10 nm silver nanoparticles, 1 μL 100 μM substrate chain Sub, 2 μL 100 μM enzyme chain 17E; The system was heated to 70°C, and then cooled naturally to room temperature to promote the assembly of silver nanoparticles; after hybridization for 8 hours, the assembled product was centrifuged three times to obtain a purified asymmetric silver nanoparticle dimer; Sub:5'-ACTCATCTGT GAACTCACTA T(rA)GGAAGAGAT GTGTCAACTC GTG-3'; Sub: 5'-ACTCATCTGT GAACTCACTA T(rA)GGAAGAGAT GTGTCAACTC GTG-3'; 17E:5'-CATCTCTTCT CCGAGCCGGT CGAAATAGTG AGT-3'; 17E: 5'-CATCTCTTCT CCGAGCCGGT CGAAATAGTG AGT-3'; (3)银纳米粒子组装体作为检测传感器用于Pb2+的检测,应用CD光谱进行检测 (3) The silver nanoparticle assembly is used as a detection sensor for the detection of Pb 2+ , and the CD spectrum is used for detection 在不对称银纳米粒子二聚体中加入一系列不同浓度的Pb2+,在Pb2+的作用下,底物链断裂,从而导致二聚体被分裂成单个的粒子,CD信号的强度降低,将不同目标物浓度下的反应体系用CD光谱进行检测,得到200nm-800nm的CD光谱图,随着Pb2+浓度的增加,不对称银纳米粒子二聚体的数量逐渐减少,CD信号逐渐降低,根据Pb2+浓度与CD信号强度之间的对应关系,绘制Pb2+浓度与CD信号强度的标准曲线,从而应用CD信号对Pb2+的浓度进行定量。 Adding a series of different concentrations of Pb 2+ to the asymmetric silver nanoparticle dimer, under the action of Pb 2+ , the substrate chain is broken, which causes the dimer to be split into individual particles, and the intensity of the CD signal decreases , the reaction system under different target concentrations was detected by CD spectrum, and the CD spectrum of 200nm-800nm was obtained. With the increase of Pb 2+ concentration, the number of asymmetric silver nanoparticle dimers gradually decreased, and the CD signal gradually decreased. According to the corresponding relationship between Pb 2+ concentration and CD signal intensity, draw the standard curve of Pb 2+ concentration and CD signal intensity, so as to use CD signal to quantify the concentration of Pb 2+ .
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CN104020204A (en) * 2014-06-19 2014-09-03 湖南大学 Electrochemical sensor for detecting lead as well as preparation method and application thereof
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