A method that can simultaneously detect Hg 2+ and Ag + Method

Abstract

Hg 2+ and Ag + Hg and Hg are two highly toxic and widely distributed environmental pollutants that pose a serious threat to the environment and humans even at low concentrations, causing serious and permanent damage. The World Health Organization has set strict limits on drinking water standards. Therefore, it is necessary to establish an efficient and sensitive method for the simultaneous detection of Hg and Hg. 2+ and Ag + The analytical method is very important for environmental monitoring, food safety and other fields. The present invention proposes a method for simultaneously detecting Hg 2+ and Ag + The method uses a nanocapsule-nucleic acid biomolecule complex based on nuclease cyclic amplification technology to achieve not only the 2+ and Ag + The detection sensitivity of Hg was significantly improved under the premise of ensuring high specificity. 2+ and Ag + Wider linear detection range.

Description

Technical Field

[0001] The present invention relates to Hg 2+ and Ag + Simultaneous detection of Hg 2+ and Ag + The invention relates to a nanocapsule-nucleic acid biomolecule complex based on nuclease cyclic amplification technology and biomolecule recognition technology and a preparation method thereof, belonging to the field of environmental monitoring and nanobiotechnology. Background Art

[0002] Mercury ions (Hg 2+ ) and silver ions (Ag + ) are two highly toxic metal ions and are also widely distributed environmental pollutants. Even at low concentrations, they pose a serious threat to the environment and humans, causing serious and permanent damage. More seriously, through polluted water bodies, Hg 2+ and Ag + It can accumulate in agricultural and aquatic products and enter the human food chain. 2+ and Ag + A polluted environment will lead to the slow onset of degenerative diseases of the human body and nervous system. For this reason, the World Health Organization (WHO) and the U.S. Environmental Protection Agency (EPA) have set strict limits on drinking water standards. Among them, the U.S. Environmental Protection Agency (EPA) has proposed a maximum allowable value of mercury in drinking water of 10nM and a maximum allowable value of Ag in drinking water of 10nM. + The content is 50μg / L. Therefore, a sensitive, accurate and efficient method for the simultaneous detection of Hg 2+ and Ag + Analytical methods are crucial for fields such as environmental monitoring, food safety, and biomedicine.

[0003] Currently, for simultaneous detection of Hg 2+ and Ag + There are far fewer detection methods for Hg than for single ions, and the traditional technologies that can be used mainly include inductively coupled plasma mass spectrometry (ICP-AES), atomic absorption / emission spectrometry and polarography. However, these methods often require complicated operations, time-consuming analysis and expensive and complex instruments and equipment, which limit their use in resource-limited environments. Fluorescence sensing technology has been increasingly studied due to its advantages of sensitivity, simplicity and speed. It is undeniable that the methods reported in the literature have problems such as low detection sensitivity and insufficient selectivity. In order to overcome these shortcomings, there is an urgent need to develop a simple, sensitive, economical and efficient determination method to meet the needs of biological, environmental, medical and other fields for Hg. 2+ and Ag + The need for simultaneous detection.

[0004] According to literature reports, when metal ions enter mismatched DNA base pairs, an attractive force will be generated between the metal ions and the DNA base pairs, binding them together. + After entering the cytosine environment, a specific recognition reaction is formed to form a stable cytosine-Ag + -cytosine base pair coordination complex; with Ag + Similar, Hg 2+ It can also specifically "bridge" with nucleotides, selectively recognize thymine bases and form strong and stable thymine-Hg 2+ -thymine coordination complex.

[0005] It is precisely because of Hg 2+ and Ag + The specific recognition of thymine and cytosine base pairs forms a stable metal-base pair coordination complex, which is of great significance for the high sensitivity and high specificity detection of these two metal ions. 2+ and Ag + This reaction characteristic is combined with nucleic acid biomolecules rich in thymine and cytosine, and cleverly applied to the field of nanomaterials with hollow and porous structures. At the same time, in order to further improve the Hg 2+ and Ag + The detection sensitivity is high, based on the base mismatch recognition technology, combined with the shearing effect of biological enzymes, 2+ and Ag + be recycled, and finally the detection signal is significantly amplified, which is Hg 2+ and Ag + So far, the simultaneous and highly sensitive detection of Hg has been based on enzyme cyclic amplification technology, hollow porous nanomaterials, and the use of base mismatching to recognize the two metal ions to construct nanocapsule-nucleic acid biomolecule complexes for the simultaneous detection of Hg. 2+ and Ag + The technology has not been reported in the literature. Summary of the Invention

[0006] In order to overcome the shortcomings of the existing technology, a nanocapsule-nucleic acid biomolecule complex based on nuclease cyclic amplification technology and molecular recognition technology was used to simultaneously detect Hg 2+ and Ag + Therefore, the first purpose of the present invention is to propose and construct a new type of Hg 2+ and Ag +The nanocapsule-nucleic acid biomolecule complex based on nuclease cyclic amplification technology and molecular recognition technology is designed and synthesized using hollow and porous nanogold as nanocapsules to be able to be Hg 2+ and Ag + The identified nucleic acid biomolecules are assembled onto the surface of the nanocapsules, which serve as pore blocking materials to block the pores of the nanocapsules and prevent the leakage of the substances in the pores; on the other hand, they serve as Hg 2+ and Ag + The recognition molecules can bind to Hg 2+ and Ag + A specific base mismatch recognition reaction occurs to form a stable T-Hg 2+ -T, C-Ag + -C base pairs undergo conformational transformation and detach from the surface of the nanocapsule, thereby opening the blocked pores and releasing the dye molecules in the nanocapsule. The supernatant is separated and produces fluorescence emission under the irradiation of a certain wavelength of excitation light. The Hg 2+ and Ag + In the present invention, in order to further improve the simultaneous detection of Hg 2+ and Ag + The sensitivity of the fluorescence signal is high. Based on molecular recognition, the present invention uses the cleavage effect of exonuclease to achieve cyclic amplification of the fluorescence signal. The cyclic amplification technology uses the effect of exonuclease on the cleavage of the double-stranded base pair-metal ion coordination complex to convert Hg 2+ and Ag + Release, released Hg 2+ and Ag + It can react with the biorecognition molecules on the surface of the nanocapsule again, and then be cut again..., and the cycle repeats, resulting in Hg 2+ and Ag + It is recycled many times, more pores are opened, and more substances in the pores are released. It is because of the action of the shear enzyme that Hg 2+ and Ag + The technology can detect Hg with a content as low as trace amount. 2+ and Ag + Highly sensitive and selective simultaneous detection of samples. Even if the sample also contains trace amounts of Hg 2+ and Ag + , and also can obtain satisfactory detection results. The second purpose of the present invention is to provide a method for simultaneously detecting trace amounts of Hg 2+ and Ag +The third purpose of the present invention is to provide a method for simultaneously detecting Hg 2+ and Ag + method.

[0007] The present invention achieves the purpose of the invention through the following technical solutions. 2+ and Ag + The nanocapsule-nucleic acid biomolecule complex is a nano-gold nanocapsule with a hollow and porous structure. The hollow and porous structural characteristics are used to load guest molecules such as fluorescent dyes inside the nanocapsule. In order to achieve the purpose of Hg 2+ and Ag + In order to detect the target metal ion Hg at the same time, two nucleic acid biorecognition molecules were designed and synthesized respectively. The types and numbers of bases were designed to ensure that they could recognize and recognize only the target metal ion Hg. 2+ or Ag + It has a specific response, but has no response to any other coexisting ions and interfering ions. On this basis, relevant experiments and screening were carried out on the two fluorescent signal molecules used. In order to overcome the mutual interference between the two fluorescent molecules used at the same time and the limitations of the use conditions, comprehensive consideration was given to the properties such as good solubility, strong fluorescence, large Stokes shift, stable optical properties, high signal-to-noise ratio, no photobleaching, little environmental impact, and good biocompatibility. Finally, the fluorescent dyes selected were preferably Rhodamine B and fluorescein Cy5. The results showed that the selection and use of the two fluorescent signal molecules became one of the key conditions of the present invention. In order to achieve the effect of biomolecules on Hg 2+ and Ag + In order to prevent the leakage of fluorescent signal molecules, the present invention has designed and synthesized the Hg 2+ and Ag + The biomolecules identified by the nanocapsule are assembled on the surface of the nanocapsule to block the pores, thereby preventing the leakage of fluorescent molecules in the pores; the ... 2+ and Ag + The biomolecules are specially designed and synthesized nucleic acid biomolecules with a certain base length, which are rich in thymine and cytosine. 2+ The base sequence is: 5'-TTT GTTTGT TGGAAAACC TTC TTT CTTA-3', which specifically recognizes Ag +The base sequence is: 5'-ACC ACC CAC CAAGGAATT CCT CCC TCC T-3'. Since these two sequences respond only to the corresponding metal ions, the detection system can completely avoid mutual interference and ensure the accuracy and specificity of simultaneous detection.

[0008] As a identifiable 2+ and Ag + The two nucleic acid biomolecules are assembled onto the hollow and porous nano-gold surfaces respectively by electrostatic assembly, and the nano-gold surface needs to be pre-modified by using a positive charge modifier, preferably polydiallyldimethylammonium chloride. 2+ and Ag + When present, the nucleic acid biomolecules assembled on the surface of the nanocapsules react with Hg 2+ and Ag + A specific response occurs, and the two ions specifically bind to their corresponding nucleic acid biomolecules, causing their conformation to change and fall off from the surface of the nanocapsule, opening the blocked pores and releasing RhB and Cy5 from the inside of the nanocapsule. The two released fluorescent dyes are excited with corresponding wavelengths, and the fluorescence signals of the two dyes are significantly enhanced. When different concentrations of Hg 2+ and Ag + When the fluorescence signal is 0.05, the fluorescence signal shows a good linear relationship with the added ion concentration. Therefore, the Hg 2+ and Ag + Simultaneous detection.

[0009] In order to further improve the simultaneous detection of Hg 2+ and Ag + The sensitivity of the present invention is based on specific biological recognition. The present invention also uses the cleavage effect of nuclease to achieve cyclic amplification of fluorescence signals. This cyclic amplification technology uses the effect of nuclease exonuclease on the cleavage of base pair-metal ion coordination complex with double-stranded structure to convert Hg 2+ and Ag + Released, and the released Hg 2+ and Ag + It can react with the biorecognition molecules on the surface of the nanocapsule again, and then be cut again..., and the cycle repeats, resulting in Hg 2+ and Ag + It is recycled multiple times, more pores are opened, more fluorescent substances are released, and the fluorescent signal is further significantly enhanced. The preferred exonuclease is exonuclease ExoⅢ.

[0010] A method for preparing the method proposed by the present invention that can simultaneously detect Hg 2+ and Ag + The method for preparing a nanocapsule-nucleic acid biomolecule complex based on nuclease cyclic amplification technology and biomolecule recognition technology comprises the following steps:

[0011] (1) By selecting different types and numbers of bases, two nucleic acid biorecognition molecules were designed and synthesized to ensure that they can recognize and recognize only the target metal ion Hg. 2+ or Ag + It has a specific response and does not respond to any other coexisting ions or interfering ions;

[0012] (2) The magnetic beads were washed with a MOPS buffer solution at pH = 7.0 and then mixed with a solution of a nano-gold carrier having a hollow, porous structure. A polydiallyldimethylammonium chloride solution was added. After 10-12 hours, the beads were magnetically separated and washed with a MOPS buffer solution at pH = 7.0.

[0013] (3) Add fluorescent molecule Rhodamine B solution, and add Hg after 10-12 hours. 2+ The nucleic acid biomolecule solution was magnetically separated after 10-12 hours, and washed with a MOPS buffer solution at pH = 7.0 for later use;

[0014] Wherein, the identifiable Hg 2+ The base sequence of the nucleic acid biomolecule is 5'-TTT GTT TGT TGG AAAACC TTC TTT CTTA-3', which is similar to Hg 2+ The specific binding product can be cleaved by exonuclease, releasing Hg 2+ ;

[0015] (4) Repeat step (2) and add fluorescent molecule Cy5 solution. After 10-12 hours, add Ag-recognizable + The nucleic acid biomolecule solution was magnetically separated after 10-12 hours, and washed with a MOPS buffer solution at pH = 7.0 for later use;

[0016] Wherein, the identifiable Ag + The base sequence of the nucleic acid biomolecule is 5'-ACC ACC CAC CAA GGAATT CCT CCC TCC T-3', which is similar to Ag + The specific binding product can be cleaved by exonuclease, releasing Ag + ;

[0017] (5) The products obtained in step (3) and step (4) were diluted with MOPS buffer solution of pH=7.0, mixed, and magnetically separated to obtain a simultaneous detection of Hg. 2+and Ag + Nanocapsule-nucleic acid biomolecule complex.

[0018] A method for simultaneously detecting Hg 2+ and Ag + The method comprises the following steps:

[0019] (1) Hg 2+ and Ag + The solution is added to the nanocapsule-nucleic acid biomolecule complex prepared by the present invention, diluted with a MOPS buffer solution with a pH of 7.0, and the shearing enzyme EXOⅢ is added, and the mixture is placed in a shaker at 37°C for 1-3 hours;

[0020] (2) Magnetic separation, take the supernatant for fluorescence detection, give the corresponding wavelength excitation, detect Hg 2+ and Ag + The corresponding fluorescence signals of the two dyes.

[0021] Beneficial effects of the present invention: The present invention can simultaneously detect Hg 2+ and Ag + The nanocapsule-nucleic acid biomolecule complex based on nuclease cyclic amplification technology and biomolecule recognition technology can not only achieve Hg 2+ and Ag + Not only can it detect simultaneously, but also on the basis of the original controlled release amplification, through the cleavage action of biological enzymes and the recycling of targets, it finally realizes the simultaneous cyclic amplification and detection of two fluorescent signals.

[0022] The results show that compared with the existing technology, the system can simultaneously detect Hg 2+ and Ag + The detection sensitivity of Hg 2+ and Ag + More importantly, the two nucleic acid biorecognition molecules designed and synthesized are respectively and only sensitive to the target metal ion Hg 2+ or Ag + It has a specific response and has no response to any other coexisting ions and interfering ions. 2+ or Ag + When the system is used, the fluorescence signal corresponding to the metal ions contained in it can be detected sensitively. Therefore, the nanocapsule-nucleic acid biomolecule complex proposed in the present invention is not only used for Hg 2+ and Ag + Simultaneous detection of Hg 2+ or Ag +For the separate detection of samples, the three detection systems can obtain highly sensitive and selective detection results.

[0023] The nanocapsule-nucleic acid biomolecule complex proposed in the present invention has the advantages of simple structure, easy synthesis, excellent performance, stability, wide application range, high efficiency, sensitivity, etc., and is not affected by other common interfering substances such as Cd 2+ , Hg 2+ , Pb 2+ , Cu 2+ , Fe 3+ , Zn 2+ The experimental results show that compared with other common technical methods, the nanocapsule-nucleic acid biomolecule complex proposed by the present invention can effectively inhibit the effects of metal ions such as Hg 2+ and Ag + Simultaneous detection was performed, showing high sensitivity and excellent selectivity, among which, at 1.0×10 -13 ~1.0×10 -7 M concentration range, obtain detection of Hg 2+ The linear equation is: FL(Hg 2+ )=467.79+70.36lgC Hg 2+ (10 -13 M), and the detection limit was 8.24×10 -14 M; in 1.0×10 -13 ~1.0×10 -8 M concentration range, the detection of Ag + The linear equation is: FL(Ag + )=1667.85+189.46lgC Ag + (10 -13 M), and the detection limit was 5.11×10 -14 M. Compared with the literature value, the present invention has a 2+ and Ag + Under the premise of ensuring high specificity, the sensitivity of simultaneous detection has been significantly improved by more than 10 times. At the same time, the sensitivity of Hg 2+ and Ag + The nanocapsule-nucleic acid biomolecule complex, its preparation method, and detection technology proposed in this invention are foreseeable to have enormous medical application potential and broad application prospects, and will play an important role in the early diagnosis and treatment of major diseases, as well as in the fields of food, biomedicine, and environmental monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 .Ag +Concentration and fluorescence intensity curve;

[0025] Figure 2 .Hg 2+ Concentration and fluorescence intensity curve. DETAILED DESCRIPTION

[0026] The following are specific embodiments of the present invention to further describe the technical solution of the present invention, but the scope of protection of the present invention is not limited to these embodiments. Any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the scope of protection of the present invention.

[0027] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.

[0028] Experimental instruments: THZ-82A gas bath constant temperature oscillator (Jintan Medical Instrument Factory); F-4600 fluorescence spectrophotometer (Hitachi, Japan); magnetic separation rack (Tianjin Bestele Chromatography Technology Development Center).

[0029] Reagents: Exonuclease ExoШ (Thermo Scientific, USA); Rhodamine B (Shanghai Aladdin Biochemical Technology Co., Ltd.); Cy5 (Solarbio, Beijing Solarbio Technology Co., Ltd.); polydienylpropyldimethylammonium chloride (Shanghai Aladdin Biochemical Technology Co., Ltd.); 3-4 μm thiol magnetic beads (Tianjin Bestel Chromatography Technology Development Center); Hg-specific 2+ The nucleic acid base sequence is 5'-TTT GTT TGT TGGAAAACC TTC TTT CTTA-3', which specifically recognizes Ag + The nucleic acid base sequence is 5′-ACC ACC CAC CAAGGAATT CCT CCC TCC T-3′ (Shanghai Sangon Biotechnology Co., Ltd.), and the MOPS buffer solution has a pH of 7.0 and a concentration of 0.01 M (Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0030] Example 1:

[0031] A method for preparing the method proposed by the present invention that can simultaneously detect Hg 2+ and Ag + The method of nanocapsule-nucleic acid biomolecule complex based on nuclease cyclic amplification technology and biomolecule recognition technology comprises the following steps:

[0032] (1) By selecting different types and numbers of bases, two nucleic acid biorecognition molecules were designed and synthesized to ensure that they can recognize and recognize only the target metal ion Hg. 2+ or Ag +It has a specific response and does not respond to any other coexisting ions or interfering ions;

[0033] (2) After washing 20 μL of magnetic beads with a pH = 7.0 MOPS buffer solution, 400 μL of a nanogold carrier solution with a hollow, porous structure was added, followed by 200 μL of a 11.664 mg / mL polydiallyldimethylammonium chloride solution. After magnetic separation at 37°C for 10 h, the beads were washed with a pH = 7.0 MOPS buffer solution;

[0034] (3) Add 2 μL of Rhodamine B solution (final concentration 1.0×10 -5 mol / L), diluted to 100 μL with MOPS buffer solution at pH = 7.0, and added 10 μL at 37 ° C for 10 h to identify Hg 2+ Nucleic acid biomolecule solution (final concentration 1.0×10 -6 mol / L), and then magnetically separated at 37°C for 10 h, and washed with MOPS buffer solution at pH 7.0 for later use;

[0035] Wherein, the identifiable Hg 2+ The base sequence of the nucleic acid biomolecule is 5'-TTT GTT TGT TGG AAAACC TTC TTT CTTA-3', which is similar to Hg 2+ The specific binding product can be cleaved by exonuclease, releasing Hg 2+ ;

[0036] (4) Repeat step (2) and add 2 μL of fluorescent molecule Cy5 solution (final concentration 1.0×10 -5 mol / L), diluted to 100 μL with MOPS buffer solution of pH=7.0, and added 10 μL after 37℃ for 10h to identify Ag. + Nucleic acid biomolecule solution (final concentration 1.0×10 -6 mol / L), and then magnetically separated at 37°C for 10 h, and washed with MOPS buffer solution at pH 7.0 for later use;

[0037] Wherein, the identifiable Ag + The base sequence of the nucleic acid biomolecule is 5'-ACC ACC CAC CAA GGAATT CCT CCC TCC T-3', which is similar to Ag + The specific binding product can be cleaved by exonuclease, releasing Ag + ;

[0038] (5) The products obtained in step (3) and step (4) were diluted with MOPS buffer solution of pH=7.0, mixed, and magnetically separated to obtain a simultaneous detection of Hg. 2+ and Ag+ Nanocapsule-nucleic acid biomolecule complex;

[0039] The nano-gold material with a hollow and porous structure was obtained according to the literature method (W. Wang, C. Chen, X. Li, SY Wang and X. L. Luo. Chem. Commun., 2015, 51, 9109–9112.).

[0040] Example 2:

[0041] A method for simultaneously detecting Hg 2+ and Ag + Nanocapsule-nucleic acid biomolecule complex based on exonuclease cyclic amplification technology and biomolecule recognition technology for Hg 2+ and Ag + The simultaneous detection method is as follows:

[0042] (1) 20 μL of Hg 2+ and Ag + The solution is added to the solution of the present invention to detect Hg 2+ and Ag + The nanocapsule-nucleic acid biomolecule complex based on the exonuclease cyclic amplification technology and biomolecule recognition technology was diluted to 200 μL with MOPS buffer solution of pH = 7.0, mixed evenly, and 1 μL EXOⅢ shear enzyme (20 U) was added. The mixture was placed on a shaker for 2 h at a reaction temperature of 37 ° C. Due to the presence of Hg 2+ and Ag + In the presence of 2+ and Ag + A specific response occurs, and the two ions specifically bind to their corresponding nucleic acid biomolecules, causing their conformation to change and fall off from the surface of the nanocapsule, opening the blocked pores, thereby releasing RhB and Cy5 from the inside of the nanocapsule respectively; at the same time, due to the shearing action of the nuclease ExoⅢ, the generated double-stranded specific binding product is sheared and Hg is released. 2+ and Ag + , and the released Hg 2+ and Ag + It can react with the nucleic acid biorecognition molecules on the surface of the nanocapsule again, and then be cut again..., and the cycle repeats, resulting in Hg 2+ and Ag + It is recycled multiple times, more pores are opened, and more fluorescent substances RhB and Cy5 are released, achieving a significant enhancement of the fluorescence signal;

[0043] (2) Magnetic separation, the supernatant was diluted to 2.0 mL for fluorescence detection, and the two released fluorescent dyes were excited with corresponding wavelengths, and the fluorescence signals of the two dyes were significantly enhanced; when different concentrations of Hg 2+ and Ag + When the fluorescence signal is 0.05, the fluorescence signal shows a good linear relationship with the added ion concentration; therefore, the Hg 2+ and Ag + Simultaneous detection of Hg; Detection conditions: for 2+ and Ag + The excitation wavelengths for detection are EX WL: 535nm / 649nm; the emission wavelengths are EMWL: 577nm / 652nm; EX Slit: 5.0nm; EM Slit: 5.0nm; ScanSpeed: 1200nm / min.

[0044] The experimental results show that the system can simultaneously detect Hg 2+ and Ag + The linear detection ranges obtained are: 1.0×10 -13 ~1.0×10 -7 M and 1.0×10 -13 ~1.0×10 -8 M; detection limits are: 8.24×10 -14 M and 5.11×10 - 14 M. Compared with the literature value, the present invention has a 2+ and Ag + Under the premise of ensuring high specificity, the sensitivity of simultaneous detection has been significantly improved by more than 10 times. At the same time, the sensitivity of Hg 2+ and Ag + More importantly, the two nucleic acid biorecognition molecules used in this system are specific to and only specific to the target metal ion Hg 2+ or Ag + It has a specific response and has no response to any other coexisting ions and interfering ions. 2+ or Ag + When the system is used, the accurate content of metal ions can be detected sensitively without any interference. Therefore, the nanocapsule-nucleic acid biomolecule complex proposed by the present invention is used for Hg 2+ and Ag + Simultaneous detection of Hg 2+ or Ag + For the separate detection of samples, the three detection systems can obtain highly sensitive and selective detection results.

[0045] The nanocapsule-nucleic acid biomolecule complex proposed in the present invention has the advantages of simple structure, easy synthesis, excellent performance, stability, wide application range, high efficiency, sensitivity, etc., and is not affected by other common interfering substances such as Cd 2+ , Hg 2+ , Pb 2+ , Cu 2+ , Fe 3+ , Zn 2+ It has high specificity and selectivity and can simultaneously achieve the effect of Hg 2+ and Ag + The nanocapsule-nucleic acid biomolecule complex, its preparation method, and its detection technology proposed in this invention are foreseeable to have enormous medical application potential and broad application prospects, playing an important role in the early diagnosis and treatment of major diseases, as well as in the fields of food, biomedicine, and environmental monitoring. Sequence Listing <110> Qingdao University of Science and Technology <120> A method for simultaneous detection of Hg2+ and Ag+ <141> 2020-02-11 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <400> 1 tttgtttgtt ggaaaacctt ctttctta 28 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <400> 2 accacccacc aaggaattcc tccctcct 28

Claims

1. A method for simultaneous detection of Hg 2+ and Ag + The method is characterized in that The following steps are involved: ①Containing Hg 2+ and Ag + The solution was added to the nanocapsule-nucleic acid biomolecule complex composed of two nucleic acid biorecognition molecules, two fluorescent signal molecules and nanogold with a hollow, porous structure, diluted with MOPS buffer solution, added with shear enzyme EXO Ⅲ, and placed in 37 o C shaker reaction for 1-3 h; ② Magnetic separation, take the supernatant for fluorescence detection, give the corresponding wavelength excitation, detect Hg 2+ and Ag + The corresponding fluorescence signals of the two dyes; The two nucleic acid biorecognition molecules specifically recognize Hg 2+ 5'-TTT GTT TGT TGG AAA ACCTTC TTT CTT A-3' and specifically recognizes Ag + 5'-ACC ACC CAC CAA GGA ATT CCT CCC TCC T-3'; The two fluorescent signal molecules are rhodamine B and fluorescein Cy5; The two nucleic acid biorecognition molecules are assembled onto the surface of the nano-gold with a hollow and porous structure respectively through electrostatic interaction; The preparation method of the nanocapsule-nucleic acid biomolecule complex is as follows: (1) By selecting different types and numbers of bases, two nucleic acid biorecognition molecules were designed and synthesized to ensure that they can recognize and recognize only the target metal ion Hg. 2+ or Ag + It has a specific response and does not respond to any other coexisting ions or interfering ions; (2) The magnetic beads were washed with a MOPS buffer solution at pH 7.0 and then mixed with a gold nanoparticle solution having a hollow, porous structure. Polydiallyldimethylammonium chloride solution was added. After 10-12 h, the beads were magnetically separated and washed with a MOPS buffer solution at pH 7.

0. (3) Add fluorescent molecule Rhodamine B solution, and after 10-12 hours, add Hg 2+ The nucleic acid biomolecule solution was magnetically separated after 10-12 hours, and washed with MOPS buffer solution at pH=7.0 for later use; Wherein, the identifiable Hg 2+ The base sequence of the nucleic acid biomolecule is 5'-TTT GTT TGT TGG AAA ACCTTC TTT CTT A-3', which is similar to Hg 2+ The specific binding product can be cleaved by exonuclease, releasing Hg 2+ ; (4) Repeat step (2) and add fluorescent molecule Cy5 solution. After 10-12 hours, add Ag-recognizable + The nucleic acid biomolecule solution was magnetically separated after 10-12 hours, and washed with MOPS buffer solution at pH=7.0 for later use; Wherein, the identifiable Ag + The base sequence of the nucleic acid biomolecule is 5'-ACC ACC CAC CAA GGA ATTCCT CCC TCC T-3', which is similar to Ag + The specific binding product can be cleaved by exonuclease, releasing Ag + ; (5) The products obtained in step (3) and step (4) were diluted with MOPS buffer solution of pH=7.0, mixed, and magnetically separated to obtain a simultaneous detection of Hg 2+ and Ag + Nanocapsule-nucleic acid biomolecule complex.

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