阅读说明：本技术 一种检测银离子的方法 (Method for detecting silver ions ) 是由 王卫 李欣 钟华 接贵芬 万均 罗细亮 于 2018-08-13 设计创作，主要内容包括：本发明提出了一种基于核酸外切酶循环放大技术与碱基错配识别技术的纳米金复合材料用于Ag<Sup>+</Sup>检测的方法,该方法可用于食品、环境、医药卫生等领域。本发明利用具有中空、多孔结构的纳米金与可识别Ag<Sup>+</Sup>的生物分子相结合,构建具有“孔帽”的纳米复合材料。当含银离子的样品溶液加入后,银离子因与纳米载体表面的生物分子作用使得生物分子脱离纳米载体表面,纳米载体内的染料分子得以释放,分离后,上清液在一定波长的激发光照射下产生荧光发射,根据荧光发射信号的强弱实现对银离子的检测。同时,为了提高灵敏度,本发明利用核酸外切酶实现了荧光信号的循环放大。本发明方法简单、高效,灵敏,选择性好,方便快捷,成本低廉,应用范围广泛。(The invention provides a nano-gold composite material for Ag based on exonuclease cyclic amplification technology and base mismatch identification technology + The detection method can be used in the fields of food, environment, medicine, health and the like. The invention utilizes nano gold with hollow and porous structure and identifiable Ag + The biological molecules are combined to construct the nano composite material with the pore cap. After the sample solution containing silver ions is added, the silver ions and the biomolecules on the surface of the nano carrier act to enable the biomolecules to be separated from the surface of the nano carrier, dye molecules in the nano carrier are released, after separation, supernatant liquid generates fluorescence emission under the irradiation of exciting light with a certain wavelength, and the detection of the silver ions is realized according to the strength of fluorescence emission signals. Meanwhile, in order to improve the sensitivity, the invention realizes the circular amplification of the fluorescence signal by using exonuclease. The method of the invention has the advantages of simplicity, high efficiency, sensitivity, good selectivity, convenience, rapidness and low cost,the application range is wide.)

1. Detect Ag⁺The method is characterized by utilizing designed and synthesized Ag⁺The biomolecule identified by base mismatch is assembled on the surface of a nano-gold carrier with hollow and porous structure characteristics, and on one hand, the biomolecule is used as a 'hole cap' for plugging the hole opening of the nano-gold carrier and preventing dye molecules in the hole from leaking; on the other hand as Ag⁺The recognition probe of (A) can be reacted with Ag⁺Specific base mismatch recognition reaction occurs to form C-Ag⁺Conformation transformation occurs at the same time of the-C base pair to separate from the surface of the nano-gold carrier, so that a blocked 'pore cap' is opened, and dye molecules in the nano-gold carrier are released; at the same time, designed and synthesized can be mixed with Ag⁺Biomolecules recognized by base mismatches with Ag⁺The conjugate can be cleaved by exonuclease to cleave Ag⁺From mismatched base pairs C-Ag⁺-C is released and recycled back into the solution and optionally Ag⁺By base mismatch recognitionOther biological molecules are combined, so that more dye molecules are released, and finally the Ag is realized by using the circularly amplified fluorescent signal⁺The detection method comprises the following steps:

(1) designed and synthesized to be capable of reacting with Ag⁺Cytosine-rich nucleic acid biomolecules of a certain base length recognized by base mismatches, which are capable of reacting with Ag⁺A specific base mismatch recognition reaction occurs, and the binding product of the silver ion and the silver ion can be cut by exonuclease;

(2) mixing the magnetic beads with a nano-gold carrier solution with a hollow and porous structure, adding a poly (diallyldimethylammonium chloride) solution, carrying out magnetic separation after 10-12h, and cleaning with an MOPS buffer solution;

(3) adding dye molecule solution, adding the Ag which is designed and synthesized in the step (1) and can be identified after 10-12h⁺The nucleic acid biomolecule solution is magnetically separated after 10 to 12 hours, and is washed by MOPS buffer solution;

(4) will contain Ag⁺Adding the sample solution to be tested into the nano-gold composite material prepared in the previous step, diluting with MOPS buffer solution (pH is 7.0), adding exonuclease, and oscillating at the constant temperature of 37 ℃ for 2-3 h;

(5) magnetic separation, taking supernatant, and performing fluorescence detection.

2. A detecting Ag according to claim 1⁺The method of (2), characterized by: the said may be reacted with Ag⁺A nucleic acid biomolecule recognized by a base mismatch, which has a base sequence of 5'-TCC TCC CTC CTT AAG GAA CCA CCC ACC A-3'.

3. A detecting Ag according to claim 1⁺The method of (2), characterized by: the dye molecule is rhodamine B.

4. A detecting Ag according to claim 1⁺The method of (2), characterized by: the exonuclease is Exo III.

Technical Field

The invention relates to a method for detecting Ag⁺In particular to Ag based on the exonuclease cyclic amplification technology and the base mismatch identification technology⁺The method of (1).

Background

Silver ion (Ag) as heavy metal ion⁺) With mercury ions (Hg)²⁺) Similarly, it is a metal ion with high toxicity and is also a widely distributed environmental pollutant. Even at low concentrations, can be severely and permanently toxic to the environment and humans. More seriously, through contaminated water source, Ag⁺Can be continuously accumulated in agricultural products and aquatic products and then enter the food chain of human beings. If exposed to Ag for a long time⁺In the existing environment, it may cause the slow occurrence of degenerative diseases in human body and nervous system. Thus, highly efficient, sensitive and economical Ag was established⁺The detection method has very important significance in the fields of environmental monitoring, food safety, clinical diagnosis and the like.

At present, for Ag⁺The traditional methods for detection mainly include plasma mass spectrometry (ICP-AES), atomic absorption/emission spectrometry, polarography and the like. However, these methods often require cumbersome operations, time-consuming analyses, and expensive, complicated equipment, etc., making them unusable in resource-limited environments, and in addition, they are still subject to improvement in detection sensitivity and selectivity. To overcome these disadvantages, there is an urgent need to develop a simple, sensitive, economical and efficient assay method to satisfy the demands of Ag in biological, medical and environmental fields⁺The detection requirement of (1).

The metal ions can form coordinate bonds with base pairs and can replace hydrogen bonds in conventional Watson-Crick base pairs to form metal-base pairs, and the effect has important significance for quickly and efficiently detecting the metal ions, so that the metal ions are widely concerned. Wherein cytosine (C) may be combined with Ag⁺Formation of C-Ag⁺The combination of-C base pair is stable, and the generation of the special structure can be used for Ag⁺Detection of (3). Furthermore, due to a C-C base mismatchRecognition of Ag⁺To form Ag⁺Qiao-linked base pairing of Ag⁺Has very high specificity and selectivity. According to the invention, Ag⁺The reaction characteristic combines the nucleic acid biomolecule rich in cytosine, is skillfully applied to the field of nano materials with hollow and porous structures, and combines the shearing action of biological enzyme on the basis of a base mismatch identification technology to improve the detection sensitivity, so that the Ag is Ag⁺Provides a novel, specific and efficient detection technology. Adopts nano gold with a hollow and porous structure as a nano carrier and utilizes C-C base mismatching Ag⁺Specific biological recognition function for constructing nano composite material to detect Ag⁺The technology of (A) has not been reported in the literature.

Disclosure of Invention

Aiming at overcoming the defects of the prior art, the method aims at detecting Ag by using the nano-gold composite material based on the exonuclease cyclic amplification technology and the base mismatch identification technology⁺The technique of (a) has not been reported, and therefore, the first object of the present invention: provides and constructs a novel nano-gold composite material based on exonuclease cyclic amplification technology and base mismatch identification technology, specifically, hollow and porous nano-gold is used as a nano-carrier to design and synthesize a nano-gold composite material capable of being coated with Ag⁺The identified biological molecules are assembled on the surface of the nano carrier and are used as a 'pore cap' to plug the orifice of the nano carrier and prevent substances in the pore from leaking; on the other hand as Ag⁺The recognition probe of (A) can be reacted with Ag⁺Specific base mismatch recognition reaction occurs to form C-Ag⁺Conformation transformation occurs at the same time of-C base pair to separate from the surface of the nanocapsule, so that the blocked 'pore cap' is opened, dye molecules in the nanocapsule are released, supernatant is separated, fluorescence emission is generated under the irradiation of exciting light with certain wavelength, and the Ag is realized according to the strength of a fluorescence emission signal⁺Detection of (3). Meanwhile, in order to further increase the sensitivity, the invention realizes the cyclic amplification of the fluorescent signal by utilizing the shearing action of the exonuclease on the basis of the base mismatch recognition.

Due to the coreThe exonuclease can be used for preparing the biological molecule-Ag with double-chain structure⁺Shearing the combination, after shearing, Ag⁺Is released, these Ag⁺Can be subjected to recognition reaction with other recognition probes again and then be sheared … … again, resulting in Ag⁺The materials are recycled, more 'pore caps' are opened, and more substances in pores are released. It is due to the action of the cleaving enzyme, Ag⁺Is recycled, so that the detection sensitivity is obviously enhanced. The detection system based on the exonuclease cyclic amplification technology and the base mismatch identification technology can be used for detecting trace Ag⁺The sample realizes high-sensitivity and high-selectivity detection. Even if the sample contains a very small amount of Ag⁺Satisfactory detection results can also be obtained; second object of the invention: provides a method for detecting trace Ag⁺The preparation method of the nano-gold composite material based on the exonuclease cycle amplification technology and the base mismatch identification technology; the third object of the present invention: provides a method for detecting trace Ag⁺The method of (1).

The invention achieves the purpose through the following technical scheme. The invention provides the detection of Ag⁺The nano-gold composite material takes nano-gold with a hollow and porous structure as a nano-carrier, and utilizes the characteristics of the hollow and porous structure to load guest molecules such as fluorescent dye, preferably rhodamine B. In order to prevent the leakage of fluorescent dye, the invention designs and synthesizes the silver-coated⁺The identified biological molecules are assembled on the surface of the nano carrier to form a 'pore cap' for plugging an orifice, so that the effect of preventing substances in the pore from leaking is achieved; wherein said may be Ag⁺The identified biomolecule is a specially designed and synthesized cytosine-rich nucleic acid biomolecule with a certain base length, the base sequence of the nucleic acid biomolecule is 5'-TCCTCC CTC CTTAAG GAACCA CCCACC A-3', and the biomolecule is used as Ag⁺The recognition probe is assembled on the surface of the hollow and porous nanogold to form a 'pore cap', and the assembly of the 'pore cap' is realized by a method of modifying a positive charge modifier on the surface of a nano carrier in advance, preferably, the positive charge modifier is polydiallylbisMethyl ammonium chloride. In order to open more "pore caps" and release more fluorescent dye, the Ag coated glass designed and synthesized by the invention⁺Recognized biomolecules with Ag⁺The combined product can be acted by exonuclease, namely the invention utilizes the exonuclease to react with the biomolecule-Ag with double-chain structure⁺Shearing the combination to obtain Ag⁺From mismatched base pairs C-Ag⁺-C is released and recycled back into solution and is mixed with other Ag⁺The recognition probe is combined, so that more rhodamine B is released, the cyclic amplification of a fluorescence signal is realized, and preferably, the exonuclease is exonuclease ExoIII.

Preparation of the trace Ag detection material provided by the invention⁺The preparation method of the nano-gold composite material based on the exonuclease cycle amplification technology and the base mismatch identification technology comprises the following steps:

(1) designed and synthesized to be capable of reacting with Ag⁺Cytosine-rich nucleic acid biomolecules of a certain base length recognized by base mismatches, which are capable of reacting with Ag⁺A specific base mismatch recognition reaction occurs, and the binding product of the silver ion and the silver ion can be cut by exonuclease;

(2) mixing the magnetic beads with a nano-gold carrier solution with a hollow and porous structure, adding a poly (diallyldimethylammonium chloride) solution, carrying out magnetic separation after 10-12h, and cleaning with an MOPS buffer solution;

(3) adding dye molecule solution, adding recognizable Ag after 10-12h⁺Performing magnetic separation after 10-12h, and washing with MOPS buffer solution;

wherein, the recognizable Ag⁺The biomolecule of (1), having a base sequence of 5'-TCC TCC CTC CTTAAG GAACCACCCACCA-3', which is mixed with Ag⁺The binding product of (A) can be cleaved by exonuclease to release Ag⁺。

The invention has the beneficial effects that: the invention provides a nano-gold composite material based on exonuclease cyclic amplification technology and base mismatch identification technology, which can identify Ag⁺And a nano-molecule having a hollow, porous structureThe gold materials are combined, and can be coated with Ag by design and synthesis⁺Recognized biological molecules are assembled on the surface of the nano carrier to form a 'pore cap', and Ag can be recognized by using the recognized biological molecules⁺With Ag⁺The base mismatch recognition reaction occurs to form C-Ag⁺Conformation transformation is carried out at the same time of-C base pair to separate from the surface of the nano carrier, so that a blocked 'pore cap' is opened, dye molecules in the nano carrier are released, and in order to further increase the sensitivity, the invention also utilizes exonuclease to carry out double-chain structure on biomolecule-Ag⁺The shearing action of the conjugate realizes the cyclic amplification and detection of the fluorescence signal.

The method makes Ag⁺The detection sensitivity is obviously improved, and the detection on Ag can be realized⁺High sensitivity and high selectivity. The nanogold composite material based on the exonuclease cyclic amplification technology and the base mismatch identification technology has the advantages of simple structure, easiness in synthesis, excellent performance, stability, economy, high efficiency, sensitivity and the like, and cannot be subjected to other common interference substances such as Cd²⁺，Hg²⁺，Pb²⁺，Cu²⁺，Fe³⁺，Zn²⁺The influence of plasma metal ions has high specificity and selectivity. The experimental result shows that compared with other common technical methods, the nanogold composite material based on the exonuclease cyclic amplification technology and the base mismatch identification technology provided by the invention shows high sensitivity and excellent selectivity at 1.0 x 10^-13～8.0×10^{- 11}Detecting Ag within the mol/L concentration range⁺The logarithm of the concentration and the fluorescence signal intensity present a good linear relation, and the detection limit is as low as 1.0 multiplied by 10^-13mol/L. Compared with the literature value, the invention is applied to Ag⁺The detection sensitivity of (2) is improved by nearly 100 times. The invention provides a nano-gold composite material based on an exonuclease cyclic amplification technology and a base mismatch identification technology, a preparation method and a detection technology thereof, and the nano-gold composite material has huge medical application potential and wide application prospect, and can play an important role in the fields of early diagnosis and treatment of major diseases, food, biomedicine, medicine, environment and the like.

Drawings

FIG. 1.Ag⁺Log concentration versus fluorescence signal intensity.

Detailed Description

The following are specific examples related to the present invention, and further description is made on the technical solutions of the present invention, but the scope of the present invention is not limited to these examples. All changes, modifications and equivalents that do not depart from the spirit of the invention are intended to be included within the scope thereof.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

An experimental instrument: THZ-82A gas bath constant temperature oscillator (gold jar medical instrument factory); f-4600 Fluorospectrophotometer (Hitachi, Japan); magnetic separation rack (Tianjin double Si le chromatographic development center).

The experimental reagents include exonuclease Exo Ш (Thermo Scientific, USA), polydiallyl propyl dimethyl ammonium chloride (Shanghai-Arlatin Biotech Co., Ltd.), 3-4 μm sulfhydryl magnetic bead (Tianjin Shuangsi-Si-le chromatography technical development center), rhodamine B (Shanghai-Arlatin Biotech Co., Ltd.), and Agents for testing Ag⁺The identified biomolecules are specially designed and synthesized cytosine-rich nucleic acid biomolecules with a certain base length, wherein the base sequence of the nucleic acid biomolecules is 5'-TCC TCCCTC CTTAAG GAACCACCCACCA-3' (Shanghai Biotechnology, Inc.), and the MOPS buffer solution is 0.01M (pH 7.0, Shanghai Aladdin Biotechnology, Inc.).