CN103901033A - Method for detecting lead ion concentration in sample - Google Patents
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Abstract
本发明涉及一种检测样品中铅离子浓度的方法，包括：（1）将金纳米粒子和富含G碱基的DNA序列混合，富含G碱基的DNA序列吸附在金纳米粒子的表面并且能特异性识别铅离子；（2）将待测样品与上述混合物混合，在铅离子存在的条件下，富含G碱基的DNA序列会形成G-四联体从金纳米粒子表面解吸附下来；（3）金纳米粒子发生聚集，体系颜色由红色变成蓝色；（4）基于步骤（3）中颜色的变化，确定所述样品中的铅离子浓度。该方法可以提出了一种全新的、简单快速的、高选择性、高灵敏性的生物传感器，用以检测样品中铅离子的浓度。
The invention relates to a method for detecting the concentration of lead ions in a sample, comprising: (1) mixing gold nanoparticles and a DNA sequence rich in G bases, the DNA sequences rich in G bases are adsorbed on the surface of the gold nanoparticles and Can specifically identify lead ions; (2) Mix the sample to be tested with the above mixture, and in the presence of lead ions, the DNA sequence rich in G bases will form G-quadruplexes and desorb from the surface of gold nanoparticles (3) Aggregation of gold nanoparticles occurs, and the color of the system changes from red to blue; (4) Based on the color change in step (3), the concentration of lead ions in the sample is determined. This method can propose a new, simple, fast, highly selective, and highly sensitive biosensor for detecting the concentration of lead ions in a sample.
Description
技术领域 technical field
本发明属于化学领域，涉及一种检测样品中铅离子浓度的方法，特别涉及一种基于金纳米粒子与G-四联体的铅离子检测方法。 The invention belongs to the field of chemistry, and relates to a method for detecting the concentration of lead ions in a sample, in particular to a method for detecting lead ions based on gold nanoparticles and G-quadruplexes.
背景技术 Background technique
纳米材料由于其独特的物理性质和化学性质，使其在生物传感器中的应用已成为国际上的研究前沿和研究热点。金纳米粒子是最早出现，研究最多的纳米材料之一。它在生物标记、传感器构建及生物芯片检测等领域都有重要应用。常用金纳米粒子制备简便而且可控，长期分散性、稳定性好，具有良好的生物相容性，并且具有独特的光学性质，使其在分析检测领域得以广泛应用。 Due to its unique physical and chemical properties, the application of nanomaterials in biosensors has become an international research frontier and research hotspot. Gold nanoparticles are one of the earliest and most studied nanomaterials. It has important applications in the fields of biomarkers, sensor construction and biochip detection. Commonly used gold nanoparticles are easy to prepare and controllable, have good long-term dispersion and stability, good biocompatibility, and unique optical properties, making them widely used in the field of analysis and detection.
铅离子的检测方法主要有：双硫腙分光光度法、原子荧光光谱法、电感耦合等离子体发射光谱法、阳极溶出伏安法、示波极谱法、生物染色剂试纸法等。但这些方法不仅需要大量的预处理，而且对操作人员有很高的技术要求。因此，研究快速、简便的检测铅离子的浓度方法仍然具有重要的现实意义。 The detection methods of lead ions mainly include: dithizone spectrophotometry, atomic fluorescence spectrometry, inductively coupled plasma emission spectrometry, anodic stripping voltammetry, oscillographic polarography, biological dye test paper method, etc. However, these methods not only require a large amount of pretreatment, but also have high technical requirements for operators. Therefore, it is still of great practical significance to study a fast and simple method for detecting the concentration of lead ions.
因此，需要对铅离子的检测方法做进一步的改进。 Therefore, it is necessary to further improve the detection method of lead ions.
发明内容 Contents of the invention
本发明的目的是提供一种检测样品中铅离子浓度的方法，用以实现水中铅离子的简单快速的检测，以克服现有的铅离子检测方法操作不便的问题。 The purpose of the present invention is to provide a method for detecting the concentration of lead ions in a sample, so as to realize the simple and rapid detection of lead ions in water, and to overcome the problem of inconvenient operation of the existing lead ion detection methods.
本发明通过以下技术方案来实现：一种检测样品中铅离子浓度的方法，其特征在于，包括： The present invention is realized by the following technical solutions: a method for detecting the concentration of lead ions in a sample, characterized in that it comprises:
（1）将金纳米粒子和富含G碱基的DNA序列混合，富含G碱基的DNA序列吸附在金纳米粒子的表面并且能特异性识别铅离子； (1) Mix gold nanoparticles with DNA sequences rich in G bases, the DNA sequences rich in G bases are adsorbed on the surface of gold nanoparticles and can specifically recognize lead ions;
（2）将待测样品与上述混合物混合，在铅离子存在的条件下，富含G碱基的DNA序列会形成G-四联体从金纳米粒子表面解吸附下来； (2) Mix the sample to be tested with the above mixture. In the presence of lead ions, the DNA sequence rich in G bases will form a G-quadruplex and desorb from the surface of the gold nanoparticles;
（3）金纳米粒子发生聚集，体系颜色由红色变成蓝色； (3) Gold nanoparticles aggregated, and the color of the system changed from red to blue;
（4）基于步骤（3）中颜色的变化，确定所述样品中的铅离子浓度。 (4) Based on the color change in step (3), determine the lead ion concentration in the sample.
本发明的发明人发现，G-四联体是由一段富含G碱基的DNA序列，在特定的离子强度和pH值条件下，通过单链之间或单链内的对应G碱基之间形成Hoogsteen碱基配对，从而使4条或4段富含G碱基的DNA单链旋聚成一段平行右旋的G-四联体。G-四联体现在已经被广泛的应用于生物医学和生物分析技术领域，如应用于对核酸、蛋白质、金属离子和有机分子的检测。金纳米粒子和富含G碱基的DNA序列存在溶液中时，DNA吸附在金纳米粒子周围对其起到稳定作用。当存在铅离子后，富含G碱基的DNA会形成G-四联体从金纳米粒子表面解吸附下来，随后引起金纳米粒子的聚集，体系颜色由红色变成蓝色。本发明的发明人基于该原理，根据体系颜色的变化，开发了一种简便、快速、高选择性、高灵敏性的生物传感器，用以检测水中铅离子的浓度。根据本发明的实施例，可以利用本发明的方法进行检测的样品的类型并不受特别限制。根据本发明的具体实施例，可以为水溶液，例如饮用水、地下水、污水等。 The inventors of the present invention have found that the G-quadruplex is composed of a DNA sequence rich in G bases, under specific ionic strength and pH conditions, passing between single strands or between corresponding G bases in a single strand Hoogsteen base pairing is formed, so that 4 or 4 DNA single-strands rich in G bases are twisted into a parallel right-handed G-quadruplex. G-quadruplex has been widely used in the fields of biomedicine and bioanalysis technology, such as the detection of nucleic acids, proteins, metal ions and organic molecules. When gold nanoparticles and G base-rich DNA sequences exist in solution, DNA adsorbs around the gold nanoparticles to stabilize it. When lead ions are present, G-base-rich DNA will form G-quadruplexes and desorb from the surface of gold nanoparticles, which will then cause the aggregation of gold nanoparticles, and the color of the system will change from red to blue. Based on this principle, the inventors of the present invention have developed a simple, rapid, highly selective, and highly sensitive biosensor for detecting the concentration of lead ions in water according to the color change of the system. According to the embodiments of the present invention, the types of samples that can be detected by the method of the present invention are not particularly limited. According to a specific embodiment of the present invention, it may be an aqueous solution, such as drinking water, ground water, sewage, and the like. the
根据本发明的实施例，上述方法还可以具有下列附加技术特征： According to an embodiment of the present invention, the above method may also have the following additional technical features:
进一步的，所述的富含G碱基的DNA序列具有如SEQ ID NO：1所示的核苷酸序列。由此，可以进一步提高利用本发明方法进行铅离子浓度检测的效率和灵敏度。 Further, the G base-rich DNA sequence has a nucleotide sequence as shown in SEQ ID NO:1. Thus, the efficiency and sensitivity of lead ion concentration detection by the method of the present invention can be further improved.
进一步的，所述的金纳米粒子的浓度为0.075nM。由此，可以进一步提高利用本发明方法进行铅离子浓度检测的效率和灵敏度。 Further, the concentration of the gold nanoparticles is 0.075nM. Thus, the efficiency and sensitivity of lead ion concentration detection by the method of the present invention can be further improved.
进一步的，所述的富含G碱基的DNA序列的浓度为100nM。由此，可以进一步提高利用本发明方法进行铅离子浓度检测的效率和灵敏度。 Further, the concentration of the G base-rich DNA sequence is 100 nM. Thus, the efficiency and sensitivity of lead ion concentration detection by the method of the present invention can be further improved.
进一步的，所述的待测样品中铅离子浓度为1-1000 nM。由此，可以进一步提高利用本发明方法进行铅离子浓度检测的效率和灵敏度。 Further, the concentration of lead ions in the sample to be tested is 1-1000 nM. Thus, the efficiency and sensitivity of lead ion concentration detection by the method of the present invention can be further improved.
进一步的，所述的待测样品中铅离子浓度为5-500 nM。由此，可以进一步提高利用本发明方法进行铅离子浓度检测的效率和灵敏度。 Further, the concentration of lead ions in the sample to be tested is 5-500 nM. Thus, the efficiency and sensitivity of lead ion concentration detection by the method of the present invention can be further improved.
进一步的，基于下列线性方程，确定所述样品中铅离子的浓度：y=6.233×10-4x+0.0727，y为不同铅离子浓度下的相对吸光度值，x为相应铅离子的浓度。由此，可以进一步提高利用本发明方法进行铅离子浓度检测的效率和灵敏度。 Further, the concentration of lead ions in the sample is determined based on the following linear equation: y=6.233×10 −4 x+0.0727, y is the relative absorbance value under different lead ion concentrations, and x is the concentration of corresponding lead ions. Thus, the efficiency and sensitivity of lead ion concentration detection by the method of the present invention can be further improved.
根据本发明的实施例，基于体系的颜色变化，确定所述样品中的铅离子浓度是通过将所述体系的颜色与标准曲线进行比较而完成的，其中，所述标准曲线是基于已知铅离子浓度分别为1nM、5nM、10nM、50nM、100 nM、200nM、500nM、1000 nM的标准样品进行平行实验而建立的。由此，可以进一步提高利用本发明方法进行铅离子浓度检测的效率和灵敏度。 According to an embodiment of the present invention, based on the color change of the system, determining the concentration of lead ions in the sample is accomplished by comparing the color of the system with a standard curve, wherein the standard curve is based on known lead ion concentrations. Standard samples with ion concentrations of 1nM, 5nM, 10nM, 50nM, 100nM, 200nM, 500nM, and 1000nM were established by parallel experiments. Thus, the efficiency and sensitivity of lead ion concentration detection by the method of the present invention can be further improved.
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。 Additional aspects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
附图说明 Description of drawings
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中： The above and/or additional aspects and advantages of the present invention will become apparent and comprehensible from the description of the embodiments in conjunction with the following drawings, wherein:
图1是利用不同铅离子浓度标准样品进行检测所得到的紫外吸收曲线，其中铅离子浓度分别取1nM、5nM、10nM、50nM、100 nM、200nM、500nM、1000 nM。 Figure 1 is the ultraviolet absorption curve obtained by using standard samples with different lead ion concentrations to detect, where the lead ion concentrations are 1nM, 5nM, 10nM, 50nM, 100nM, 200nM, 500nM, 1000nM.
图2显示了根据本发明一个实施例的特异性分析图。 Figure 2 shows a graph of specificity analysis according to one embodiment of the present invention.
具体实施方式 Detailed ways
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例所描述的内容仅用于说明本发明，而不应当也不会限制权利要求书中所描述的本发明。 The present invention can be better understood from the following examples. However, those skilled in the art can easily understand that the content described in the embodiments is only for illustrating the present invention, and should not and will not limit the present invention described in the claims.
实施例1 相应G-DNA片段的设计与合成。 Example 1 Design and synthesis of corresponding G-DNA fragments.
设计一段能特异识别铅离子，并行成G-四联体的DNA片段，DNA序列通过DNA合成仪制备。 Design a DNA fragment that can specifically recognize lead ions and form a G-quadruplex in parallel, and the DNA sequence is prepared by a DNA synthesizer.
G-DNA：5’-GGAAGGTGTGGAAGG-3’（SEQ ID NO：1） G-DNA: 5'-GGAAGGTGTGGAAGG-3' (SEQ ID NO: 1)
实施例2 金纳米粒子的合成。 Example 2 Synthesis of gold nanoparticles.
实验用的金纳米粒子参照Frens的柠檬酸三钠还原法合成。具体操作步骤如下：将配制的1.0 mM氯金酸溶液50 mL加到锥形瓶瓶中，置于恒温电磁搅拌器上加热至沸腾后持续5分钟，迅速加入0.6 mL，38.8 mM的柠檬酸三钠溶液，继续搅拌加热6分钟，溶液的颜色由淡黄色转变到酒红色，此时金纳米粒子生成，继续加热10分钟后停止，继续搅拌使其冷却，4°C保存备用。 The gold nanoparticles used in the experiment were synthesized by referring to Frens' trisodium citrate reduction method. The specific operation steps are as follows: Add 50 mL of the prepared 1.0 mM chloroauric acid solution into the Erlenmeyer flask, place it on a constant temperature electromagnetic stirrer and heat it to boiling for 5 minutes, then quickly add 0.6 mL, 38.8 mM tris-citric acid Sodium solution, continue to stir and heat for 6 minutes, the color of the solution changes from light yellow to wine red, at this time gold nanoparticles are generated, continue to heat for 10 minutes and stop, continue to stir and make it cool, and store it at 4°C for subsequent use.
实施例3 铅离子标准曲线的建立。 Example 3 Establishment of lead ion standard curve.
依次向离心管中加入金纳米粒子，用Tris-HAc（20mM，pH 7.0）缓冲液定容至2mL，其中金纳米粒子的浓度为0.075nM。向离心管中加入10μM的G-DNA 20 μL，使其最终浓度为100nM，随后依次向离心管中加入NaCl，使其最终浓度为50mM，混合均匀，测其521nm波长处的吸光度。随后向离心管中依次加入适量的铅离子，使其最终浓度分别为1nM、5nM、10nM、50nM、100 nM、200nM、500nM、1000 nM，依次向离心管中加入NaCl，使其最终浓度为50mM，反应30分钟，再测其680nm处的吸光度。根据相对吸光度值与铅离子的浓度的关系，做出铅离子的标准曲线图。该方法的线性范围5nM-500nM，检测限为3nM。标准曲线的线性方程为y=6.233×10-4x+0.0727，y为不同铅离子浓度下的相对吸光度值，x为相应铅离子的浓度，线性相关性>0.99。
Add gold nanoparticles to the centrifuge tube in turn, and use Tris-HAc (20mM, pH 7.0) buffer to make up to 2mL, in which the concentration of gold nanoparticles is 0.075nM. Add 20 μL of 10 μM G-DNA to the centrifuge tube to make the
实施例4 特异性测定。 Example 4 Specificity determination.
依次向离心管中加入金纳米粒子，用Tris-HAc（20mM，pH 7.0）缓冲液定容至2mL，其中金纳米粒子的浓度为0.075nM。向离心管中加入10μM的G-DNA 20 μL，使其最终浓度为100nM，依次向离心管中加入NaCl，使其最终浓度为50mM，随后依次向离心管中加入NaCl，使其最终浓度为50mM，测其吸光度。随后向离心管中依次加入适量的铅离子、汞离子、镉离子、铜离子、镍离子，使其最终浓度为500nM，反应30分钟，再测其吸光度。查看结果显示，该方法有比较好的特异性。
Add gold nanoparticles to the centrifuge tube in turn, and use Tris-HAc (20mM, pH 7.0) buffer solution to make the volume to 2mL, in which the concentration of gold nanoparticles is 0.075nM. Add 20 μL of 10 μM G-DNA to the centrifuge tube to make the
实施例5 实际样品的检测。 Example 5 Detection of actual samples.
向自来水样品中分别添加5nM、10nM、20nM及50 nM的铅离子，采用上述方法确定样品中的铅离子浓度，结果如表1： Add 5nM, 10nM, 20nM and 50nM lead ions to tap water samples respectively, and use the above method to determine the concentration of lead ions in the samples. The results are shown in Table 1:
表1 铅离子水样品的测定 Table 1 Determination of lead ionized water samples
结果显示，基于金纳米粒子与G-四联体的铅离子检测方法测定实际样本中的铅离子的添加回收率在94.50%-103.3%之间，标准差为4.67%，能够完全满足现实生活中对铅离子的检测需求。 The results show that the recovery rate of lead ions in actual samples measured by gold nanoparticles and G-quadruplex detection method is between 94.50% and 103.3%, with a standard deviation of 4.67%, which can fully meet the requirements of real life conditions. Detection requirements for lead ions.
序列表 Sequence Listing
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