A method that can simultaneously detect Hg 2+ and Ag + Nanocapsule-nucleic acid biomolecule complex and preparation method thereof

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 simultaneously detecting 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 that can simultaneously detect Hg 2+ and Ag + The nanocapsule-nucleic acid biomolecule complex based on the exonuclease cyclic amplification technology and the preparation method thereof, the nanocapsule-nucleic acid biomolecule complex proposed by the present invention can not only achieve the 2+ and Ag + The detection sensitivity of Hg was significantly improved while 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 discloses 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 ion (Hg 2+ ) and silver ions (Ag + ) are two highly toxic metal ions and 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, 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 in the human body and nervous system. For this reason, the World Health Organization (WHO) and the US Environmental Protection Agency (EPA) have set strict limits on drinking water standards. Among them, the US 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 simultaneous detection of Hg 2+ and Ag + The 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. The traditional technologies that can be used mainly include inductively coupled plasma mass spectrometry (ICP-AES), atomic absorption / emission spectroscopy and polarography. However, these methods often require complicated operations, time-consuming analysis and expensive and complex instruments and equipment, which limits their use in resource-limited environments. Fluorescence sensing technology has been increasingly studied due to its advantages such as 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 is generated between the metal ions and the DNA base pairs, binding them together. + After entering the cytosine environment, a specific recognition reaction is performed 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 because of Hg 2+ and Ag + The specific recognition of thymine and cytosine base pairs respectively forms a metal-base pair coordination complex with a stable structure, 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 Hg 2+ and Ag + The detection sensitivity is high. Based on the base mismatch recognition technology, it is combined with the shearing effect of biological enzymes to make Hg 2+ and Ag + The detection signal is significantly amplified, which is Hg 2+ and Ag + So far, the simultaneous and highly sensitive detection of Hg with nanocapsules and nucleic acid biomolecule complexes based on enzyme cyclic amplification technology, hollow porous nanomaterials, and the biorecognition effect of base mispairing on two metal ions have been used to simultaneously detect 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 for simultaneous detection of 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 recognized by 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 to 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 excitation light of a certain wavelength. 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. On the basis of molecular recognition, the present invention uses the shearing effect of exonuclease to realize the cyclic amplification of the fluorescence signal. The cyclic amplification technology uses the effect of exonuclease on the shearing of the base pair-metal ion coordination complex with a double-stranded structure 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 sheared 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 due to 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 highly selective simultaneous detection of samples. Even if the sample also contains trace amounts of Hg 2+ and Ag + , and satisfactory detection results can also be obtained. The second purpose of the present invention is to provide a method for simultaneously detecting trace amounts of Hg 2+ and Ag + A method for preparing a nanocapsule-nucleic acid biomolecule complex based on nuclease cyclic amplification technology and molecular recognition technology.

[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 hollow, porous nanogold nanocapsule. The hollow, porous structure is used to load guest molecules such as fluorescent dyes inside the nanocapsule. 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. The types and quantities of bases were designed to ensure that they can detect and only detect 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 of 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 show that the selection and use of the two fluorescent signal molecules has become one of the key conditions of the present invention. In order to achieve the effect of biological molecules on Hg 2+ and Ag + In order to prevent the leakage of fluorescent signal molecules, the present invention designs and synthesizes the fluorescent signal molecules that can be specifically recognized by Hg 2+ and Ag + The biomolecules that can be identified are assembled on the surface of the nanocapsule to block the pores, thereby preventing the leakage of fluorescent molecules in the pores; the biomolecules that can be identified are 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 TGG AAA ACC TTC TTT CTT A-3', which specifically recognizes Ag + The base sequence is: 5'-ACC ACC CAC CAAGGA ATT 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 Hg 2+ and Ag +The two nucleic acid biomolecules are assembled onto the hollow and porous nano-gold surfaces 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 nanocapsule 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 the surface of the nanocapsule, opening the blocked pores, thereby 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 100%, the concentration of added ions shows a good linear relationship. Therefore, the intensity of the fluorescence signal can be used to detect 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 high. On the basis of specific biological recognition, the present invention also uses the shearing effect of nuclease exonuclease to realize the cyclic amplification of fluorescence signal. The cyclic amplification technology uses the effect of nuclease exonuclease on the shearing of base pair-metal ion coordination complex with double-stranded structure to convert Hg 2+ and Ag + Release, and the released Hg 2+ and Ag + It can react with the biorecognition molecules on the surface of the nanocapsule again, and then be sheared again..., and the cycle repeats, resulting in Hg 2+ and Ag + It can be recycled many 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 only recognize the target metal ion Hg.2+ or Ag + It has a specific response and has no response to any other coexisting ions or interfering ions.

[0012] (2) washing the magnetic beads with a MOPS buffer solution at pH = 7.0, mixing with a nano-gold carrier solution having a hollow, porous structure, adding a polydiallyldimethylammonium chloride solution, magnetically separating after 10-12 hours, and washing with a MOPS buffer solution at pH = 7.0;

[0013] (3) Add the fluorescent molecule Rhodamine B solution, and add the Hg 2+ The nucleic acid biomolecule solution is magnetically separated after 10-12 hours, and washed with a MOPS buffer solution of 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 CTT A-3', which is similar to Hg 2+ The specific binding product can be cleaved by exonucleases, releasing Hg 2+ ;

[0015] (4) Repeat step (2) and add the fluorescent molecule Cy5 solution. After 10-12 hours, add the Ag-recognizable + The nucleic acid biomolecule solution is magnetically separated after 10-12 hours, and washed with a MOPS buffer solution of 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 exonucleases, releasing Ag + ;

[0017] (5) The products obtained in step (3) and step (4) are diluted with a 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] 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 is it a simultaneous detection based on controlled release amplification, but it is also based on the original controlled release amplification, through the shearing action of biological enzymes and the recycling of targets, and ultimately achieves the simultaneous cyclic amplification and detection of two fluorescent signals.

[0019] 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 + It is particularly important that the two nucleic acid biorecognition molecules designed and synthesized can detect 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 can also be sensitively detected. Therefore, the nanocapsule-nucleic acid biomolecule complex proposed in the present invention can be used for Hg 2+ and Ag + Simultaneous detection of Hg 2+ or Ag + For separate detection of samples, the three detection systems can obtain highly sensitive and highly selective detection results.

[0020] 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 will not be 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 in the present invention has a high specificity and selectivity for Hg 2+ and Ag + Simultaneous detection was performed, showing high sensitivity and excellent selectivity. -13 ~1.0×10 -7 M concentration range, obtain detection Hg 2+ The linear equation is: FL(Hg 2+ )=467.79+70.36lgC Hg 2+ (10 -13 M), the detection limit was 8.24×10-14 M; at 1.0×10 -13 ~1.0×10 -8 M concentration range, obtain detection Ag + The linear equation is: FL(Ag + )=1667.85+189.46lgC Ag + (10 -13 M), the detection limit was 5.11×10 -14 M. Compared with the literature value, the present invention has a 2+ and Ag + While 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 + It is foreseeable that the nanocapsule-nucleic acid biomolecule complex and its preparation method and detection technology proposed in the present invention have great 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, environmental monitoring, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 .Ag + Concentration and fluorescence intensity curve;

[0022] Figure 2 .Hg 2+ Concentration and fluorescence intensity curve. DETAILED DESCRIPTION

[0023] The following are specific embodiments of the present invention, which further describe the technical solution of the present invention, but the protection scope 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 protection scope of the present invention.

[0024] The present invention is specifically described below by way of examples, but the present invention is not limited to the following examples.

[0025] Experimental instruments: THZ-82A gas bath constant temperature oscillator (Jintan Medical Equipment Factory); F-4600 fluorescence spectrophotometer (Hitachi, Japan); magnetic separation rack (Tianjin Bestele Chromatography Technology Development Center).

[0026] Experimental 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 Bestele Chromatography Technology Development Center); specific recognition of Hg 2+ The nucleic acid base sequence is 5'-TTT GTT TGT TGG AAA ACC TTC TTT CTT A-3', which specifically recognizes Ag + The nucleic acid base sequence is 5'-ACC ACC CAC CAA GGA ATT 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.).

[0027] Embodiment 1:

[0028] 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:

[0029] (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 only recognize the target metal ion Hg. 2+ or Ag + It has a specific response and has no response to any other coexisting ions or interfering ions.

[0030] (2) After washing 20 μL of magnetic beads with a MOPS buffer solution at pH = 7.0, 400 μL of a nanogold carrier solution having a hollow, porous structure was added, and 200 μL of a 11.664 mg / mL polydiallyldimethylammonium chloride solution was added. After magnetic separation at 37°C for 10 h, the beads were washed with a MOPS buffer solution at pH = 7.0;

[0031] (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), 37°C for 10 h, followed by magnetic separation, washing with MOPS buffer solution at pH = 7.0, and set aside;

[0032] Wherein, the identifiable Hg 2+ The base sequence of the nucleic acid biomolecule is 5'-TTT GTT TGT TGG AAAACC TTC TTT CTT A-3', which is similar to Hg 2+ The specific binding product can be cleaved by exonucleases, releasing Hg 2+ ;

[0033] (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 at pH = 7.0, and added 10 μL at 37 ° C for 10 h to identify Ag. + Nucleic acid biomolecule solution (final concentration 1.0×10 -6 mol / L), 37°C for 10 h, followed by magnetic separation, washing with MOPS buffer solution at pH = 7.0, and set aside;

[0034] 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 exonucleases, releasing Ag + ;

[0035] (5) The products obtained in step (3) and step (4) are diluted with a 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;

[0036] The nano-gold material with a hollow and porous structure is obtained according to the literature method (W. Wang, C. Chen, X. Li, SY Wang and XL Luo. Chem. Commun., 2015, 51, 9109-9112.).

[0037] Embodiment 2:

[0038] 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 method of simultaneous detection is as follows:

[0039] (1) 20 μL of Hg 2+ and Ag+ The solution is added to the solution of the present invention which can simultaneously 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 at pH = 7.0. After mixing evenly, 1 μL EXOⅢ shearing enzyme (20U) was added and placed in a shaker for 2 hours at a reaction temperature of 37°C. 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 conformations to change and fall off from the surface of the nanocapsule, opening the blocked pores, thereby causing RhB and Cy5 to be released 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 sheared again..., and the cycle repeats, resulting in Hg 2+ and Ag + It is recycled many times, more pores are opened, and more fluorescent substances RhB and Cy5 are released, achieving a significant enhancement of the fluorescence signal;

[0040] (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 1.5 μm, 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 of the detection are EX WL: 535nm / 649nm; the emission wavelengths are EMWL: 577nm / 652nm; EX Slit: 5.0nm; EM Slit: 5.0nm; ScanSpeed: 1200nm / min.

[0041] 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 + While 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 target 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 system can also sensitively detect the exact content of the metal ions contained without any interference. Therefore, the nanocapsule-nucleic acid biomolecule complex proposed in the present invention can be used for Hg 2+ and Ag + Simultaneous detection of Hg 2+ or Ag + For separate detection of samples, the three detection systems can obtain highly sensitive and highly selective detection results.

[0042] 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 will not be 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 2+ and Ag + It is foreseeable that the nanocapsule-nucleic acid biomolecule complex and its preparation method and detection technology proposed in the present invention have great 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, environmental monitoring, etc. Sequence Listing <110> Qingdao University of Science and Technology <120> A nanocapsule-nucleic acid biomolecule complex capable of simultaneously detecting Hg2+ and Ag+ and a preparation method thereof <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 simultaneously detecting Hg 2+ and Ag + The nanocapsule-nucleic acid biomolecule complex is characterized by: The nanocapsule-nucleic acid biomolecule complex includes two nucleic acid biorecognition molecules, two fluorescent signal molecules and nanogold with a hollow and porous structure; 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 GGAATT CCT CCC TCC T-3'; The two fluorescent signal molecules are rhodamine B and fluorescein Cy5; The two nucleic acid biorecognition molecules are respectively assembled onto the surface of the nano-gold with a hollow and porous structure through electrostatic interaction; The steps of the preparation method of the nanocapsule-nucleic acid biomolecule complex are 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 has no response to any other coexisting ions or interfering ions. (2) washing the magnetic beads with a MOPS buffer solution at pH = 7.0, mixing with a nanogold solution having a hollow, porous structure, adding a polydiallyldimethylammonium chloride solution, magnetically separating after 10-12 hours, and washing with a MOPS buffer solution at pH = 7.0; (3) Add the fluorescent molecule Rhodamine B solution, and add the Hg 2+ The nucleic acid biomolecule solution is magnetically separated after 10-12 hours, and washed with a MOPS buffer solution of 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 CTTA-3', which is similar to Hg 2+ The specific binding product can be cleaved by exonucleases, releasing Hg 2+ ; (4) Repeat step 2 and add the fluorescent molecule Cy5 solution. After 10-12 hours, add the Ag-recognizable + The nucleic acid biomolecule solution is magnetically separated after 10-12 hours, and washed with a MOPS buffer solution of 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 exonucleases, releasing Ag + ; (5) The products obtained in step 3 and step 4 are diluted with a 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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