A simultaneous detection of As 3+ , Pb 2+ and Hg 2+ Nanocapsule-nucleic acid molecule complex and preparation method thereof

Abstract

Environmental and water pollution caused by highly toxic heavy metals has always been a serious problem facing mankind. 3+ , Pb 2+ and Hg 2+ As are highly toxic and widely distributed environmental pollutants that pose a serious threat to the environment and humans even at low concentrations. 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 As 3+ , Pb 2+ and Hg 2+ The analysis method is crucial for environmental monitoring, food safety and other fields. The present invention proposes a nanocapsule-nucleic acid molecule complex based on nuclease cyclic amplification technology and molecular recognition technology and a preparation method thereof. The use of the complex can not only achieve As 3+ , Pb 2+ and Hg 2+ Simultaneous detection is achieved, and the detection sensitivity is significantly improved while ensuring high specificity. At the same time, a wider linear detection range is obtained.

Description

Technical Field

[0001] The present invention relates to As 3+ , Pb 2+ , Hg 2+ Simultaneous detection of As 3+ , Pb 2+ and Hg 2+ The invention relates to a nanocapsule-nucleic acid biomolecule complex based on nuclease cyclic amplification technology and biomolecule recognition technology and a preparation method and application thereof, belonging to the field of environmental monitoring and nanobiotechnology. Background Art

[0002] Environmental and water pollution caused by highly toxic heavy metals has always been a major concern for people. They can enter the food chain through the air, water and soil and seriously harm the ecosystem.

[0003] Arsenic is an element with metal-like properties that is widely present in nature. Arsenic and its compounds have been recognized as carcinogens by many international authoritative organizations such as the International Agency for Research on Cancer under the World Health Organization (WHO) and the US Environmental Protection Agency (EPA). Arsenic pollution mainly comes from the discharge of three wastes from the pesticide, fertilizer, metallurgy, mining, pharmaceutical and other industries. The main forms of arsenic in the water environment are As(Ш) in H3AsO3 and As(V) in H3AsO4. Among them, As(Ш) is the most toxic, 100 times more toxic than As(V). Chronic ingestion or long-term exposure to arsenic will cause it to be deposited in large quantities in human skin, lungs, bladder, liver and kidneys, causing various cancers such as lung cancer and skin cancer, as well as cardiovascular diseases and damage to the human circulatory system. Due to the further expansion of arsenic pollution, humans are facing a severe threat of arsenic poisoning. The health hazards caused by arsenic pollution, mainly skin and visceral diseases, have received widespread attention worldwide. For this reason, the US Environmental Protection Agency recently readjusted the arsenic content in drinking water from 50μg / L to 10μg / L. This change has prompted more researchers to develop technologies that can detect arsenic with high sensitivity to meet this new standard.

[0004] Heavy metals that seriously endanger human health and the environment also include lead. Lead pollution mainly comes from related fields such as human life and chemical production, involving products such as paint, coatings, batteries, smelting, electroplating, tableware, coal, water pipes, cosmetics, and hair dyes. Lead is a potential neurotoxin and is also one of the most toxic heavy metal pollutions commonly found in nature. It has a serious impact on the environment and human health. Once it enters the human body, it will be difficult to eliminate and directly damage human brain cells. Even exposure to very low levels of lead ion concentrations (<100μg / L in the blood) can cause a variety of diseases, including memory loss, irritability, anemia, kidney damage, and mental retardation, causing various neurotoxic effects, causing damage to the nervous system and cardiovascular system and developmental disorders, especially for children in the growth and development period. The harm is particularly great. Lead can be inhaled through breathing, ingested through food and drinking water, and absorbed through the skin. The toxic effect of lead has no threshold, that is, lead in the body is toxic, so "zero blood lead" has become the goal of clinical control of lead poisoning in children.

[0005] In addition to arsenic and lead, metallic mercury is also an important environmental pollutant with severe toxicity and widespread distribution. Even at low concentrations, it poses a serious threat to the environment and humans, causing serious and permanent damage. 2+ Wastewater pollutants are an important component of water pollution. The mercury pollutants discharged into the water cannot be biodegraded, but participate in the food chain cycle and can be converted by microorganisms into more toxic methylmercury. After being enriched in large quantities in organisms, they enter the human body through the food chain (especially fish), seriously threatening human health and causing damage to the central nervous system, endocrine system and brain. In addition, it may also damage the human immune system and even cause death. For this reason, the US Environmental Protection Agency has proposed a maximum allowable value of mercury in drinking water of 10nM.

[0006] Therefore, it is necessary to establish a sensitive, accurate, and efficient method for simultaneously detecting As 3+ , Pb 2+ , Hg 2+ The analytical methods are crucial for fields such as environmental monitoring, food safety and biomedicine.

[0007] Currently, for the simultaneous detection of As 3+ , Pb 2+ , Hg 2+There are very few detection methods for As, far fewer than the detection methods for single ions. For the detection of single metal ions, traditional technologies mainly include inductively coupled plasma mass spectrometry (ICP-AES), atomic absorption / emission spectroscopy and polarography. However, these methods often require complex professional operations, time-consuming analysis and expensive 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 weak 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 the environment, biology, medicine and other fields for As. 3+ , Pb 2+ , Hg 2+ The need for simultaneous detection.

[0008] 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. 2+ Able to selectively recognize thymine bases and form strong and stable thymine-Hg 2+ -thymine coordination complex. Similar to mercury ions, lead ions can also specifically interact with nucleotides. Since nucleic acid molecules rich in guanine bases have a strong affinity for lead ions, they bind to lead ions through specific coordination to form a stable Pb 2+ -G-quadruplex can achieve highly selective and sensitive detection of lead ions. 3+ For example, it can specifically bind to its nucleic acid aptamer, but the reports on aptamers are relatively rare.

[0009] It is because of As 3+ For nucleic acid aptamers, Hg 2+ and Pb 2+ The metal ion-nucleic acid molecule coordination complex with high affinity and strong specificity for the functional nucleic acid molecules rich in thymine and guanine bases is formed, which is of great significance for the high sensitivity and high specificity detection of these three metal ions. 3+ , Hg 2+ and Pb 2+ This reaction characteristic is combined with nucleic acid aptamers, thymine-rich and guanine-rich nucleic acid biomolecules, and cleverly applied to the field of nanomaterials with hollow and porous structures. At the same time, in order to further improve As 3+ , Hg 2+ and Pb 2+The detection sensitivity is high. On the basis of biorecognition technology, it is combined with the cleavage of biological enzymes to make the target As 3+ , Hg 2+ and Pb 2+ The detection signal is significantly amplified and is As 3+ , Hg 2+ and Pb 2+ At the same time, high-sensitivity detection provides a new, specific and efficient detection technology.

[0010] So far, the enzyme cyclic amplification technology, hollow porous nanomaterials, and base recognition to construct nanocapsule-nucleic acid biomolecule complexes for simultaneous detection of As 3+ , Hg 2+ and Pb 2+ The technology has not been reported in the literature. Summary of the invention

[0011] 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 As 3+ , Hg 2+ and Pb 2+ Therefore, the first purpose of the present invention is to propose and construct a new type of As 3+ , Hg 2+ and Pb 2+ The nanocapsule-nucleic acid biomolecule complex based on nuclease cyclic amplification technology and molecular recognition technology is specifically to use hollow and porous nanogold as nanocapsules to screen, design and synthesize nanocapsules that are easy to modify, convenient to synthesize, and can be As 3+ , Hg 2+ and Pb 2+ The identified nucleic acid biomolecules are assembled onto the surface of the nanocapsules, on the one hand, 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 are used as As 3+ , Hg 2+ and Pb 2+ The recognition molecule can bind to As 3+ , Hg 2+ and Pb 2+ A specific recognition reaction occurs, in which Hg 2+ and Pb 2+ Can form T-Hg 2+ -T base pair complex, Pb 2 +-G quadruplex complex, while forming metal ion-nucleic acid molecule coordination complex, undergoes conformational transformation and detaches from the surface of nanocapsule, thereby opening the blocked pores, releasing the dye molecules in the nanocapsule, separating the supernatant, and generating fluorescence signals under the irradiation of excitation light of a certain wavelength. According to the strength of the fluorescence emission signal, As 3+ , Hg 2+ and Pb 2+ In the present invention, in order to further improve the simultaneous detection of As 3+ , Hg 2+ and Pb 2+ The sensitivity of the fluorescence signal is improved. On the basis of molecular recognition, the present invention also uses the shearing effect of exonucleases to achieve cyclic amplification of fluorescence signals. The cyclic amplification technology uses the effect of exonucleases on the shearing of metal ion-nucleic acid biomolecule coordination complexes with double-stranded structures to convert As 3+ , Hg 2+ and Pb 2+ Release, released As 3+ , Hg 2+ and Pb 2+ 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 As 3+ , Hg 2+ and Pb 2+ 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 As 3+ , Hg 2+ and Pb 2+ The technology can detect As with trace amounts. 3+ , Hg 2+ and Pb 2+ Highly sensitive and highly selective simultaneous detection of samples. Even if the sample also contains trace amounts of As 3+ , Hg 2+ and Pb 2+ , and satisfactory detection results can also be obtained. The second purpose of the present invention is to provide a method for simultaneously detecting As 3+ , Hg 2+ and Pb 2+ A method for preparing a nanocapsule-nucleic acid biomolecule complex based on nuclease cyclic amplification technology and molecular recognition technology.

[0012] The present invention achieves the purpose of the invention through the following technical solutions. 3+ , Hg 2+ and Pb 2+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. 3+ , Hg 2+ and Pb 2+ In order to detect the target metal ion As, we screened, designed and synthesized three nucleic acid biorecognition molecules respectively. By selecting the types and quantities of bases, we ensured that they could recognize and only recognize the target metal ion As. 3+ , Hg 2+ or Pb 2+ 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 three fluorescent signal molecules used. In order to overcome the mutual interference between the three 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 for use were preferably rhodamine B, fluorescein isothiocyanate FITC and fluorescein Cy5. The results show that the selection and use of these three fluorescent signal molecules has become one of the key conditions of the present invention. In order to achieve the response of biological molecules to As 3+ , Hg 2+ and Pb 2+ In order to prevent the leakage of fluorescent signal molecules, the present invention screened and synthesized As 3+ and Pb 2+ We designed and synthesized biomolecules that can be recognized by Hg 2+ The biomolecules recognized by the nanocapsules are assembled on the surface of the nanocapsules to block the pores and prevent the leakage of fluorescent molecules in the pores. 3+ The base sequence is: 5'-GGTAATACGACT CAC TAT AGG GAG ATA CCA GCT TAT TCA ATTTTA CAG AAC AAC CAA CGT CGC TCC GGG TAC TCC TTC TTCATC GAGATAGTAAGT GCAATC C-3'(P1); the specific recognition of guanine-rich bases Pb 2+ The base sequence is: 5'-GGT GGT GGT GGT TGT GGTGGT GGT GG-3'(P2); it can recognize Hg 2+The thymine-rich biomolecule is a specially designed and synthesized nucleic acid biomolecule with a specific base length, and its base sequence is: 5'-TTT GTT TGT TGG CCC CCC TTC TTTCTTA-3'(P3); since these three 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.

[0013] As can be identified 3+ , Hg 2+ and Pb 2+ The three nucleic acid biomolecules are assembled onto the hollow and porous nano-gold surfaces through electrostatic interactions, and the nano-gold surface needs to be pre-modified with a positive charge modifier, preferably polydiallyldimethylammonium chloride. 3+ , Hg 2+ and Pb 2+ When present, the nucleic acid biomolecules assembled on the surface of the nanocapsule react with As 3+ , Hg 2+ and Pb 2+ A specific response occurs, and the three ions specifically bind to their corresponding nucleic acid biomolecules, causing their conformations to change and fall off the surface of the nanocapsule, opening the blocked pores, thereby releasing RhB, FITC, and Cy5 from the inside of the nanocapsule. The three released fluorescent dyes are excited with corresponding wavelengths, and the fluorescence signals of the three dyes are significantly enhanced. When different concentrations of As are added 3+ , Hg 2+ and Pb 2+ When the fluorescence signal is 0.0447 W / m, the fluorescence signal shows a good response relationship with the added ion concentration. Therefore, the As 3+ , Hg 2+ and Pb 2+ simultaneous detection.

[0014] In order to further improve the simultaneous detection of As 3+ , Hg 2+ and Pb 2+ The sensitivity of the fluorescence signal is high. On the basis of specific biological recognition, the present invention 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 metal ion-nucleic acid biomolecule coordination complex with double-stranded structure to convert As 3+ , Hg 2+ and Pb 2+ Release, and the released As 3+ , Hg 2+ and Pb2+ 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 As 3+ , Hg 2+ and Pb 2+ 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.

[0015] A method for preparing the method for simultaneously detecting As 3+ , Hg 2+ and Pb 2+ The method for preparing a nanocapsule-nucleic acid molecule complex based on nuclease cyclic amplification technology and biomolecule recognition technology comprises the following steps:

[0016] (1) Screen, design and synthesize three nucleic acid biorecognition molecules to ensure that they are specific for the target metal ion As. 3+ , Pb 2+ and Hg 2+ It has a specific response and has no response to any other coexisting ions or interfering ions.

[0017] (2) washing the magnetic beads with a Tris-HCl buffer solution at pH = 7.4, mixing the magnetic beads with a solution of a nano-gold carrier having a hollow, porous structure, adding a polydiallyldimethylammonium chloride solution, performing magnetic separation after 10-12 hours, and washing the magnetic beads with a Tris-HCl buffer solution at pH = 7.4;

[0018] (3) Add the fluorescent molecule Cy5 solution, and after 10-12 hours, add the As 3+ The nucleic acid biomolecule solution is magnetically separated after 10-12 hours, and washed with a Tris-HCl buffer solution of pH=7.4 for later use;

[0019] Among them, the identifiable As 3+ The base sequence of the nucleic acid biomolecule is 5'-GGTAATACGACT CAC TATAGG GAG ATA CCA GCT TAT TCA ATT TTA CAG AAC AAC CAA CGT CGC TCC GGG TAC TCCTTC TTC ATC GAGATAGTAAGT GCAATC C-3'(P1), which is similar to As 3+ The specific binding product can be cleaved by exonucleases, releasing As 3+ ;

[0020] (4) Add Rhodamine B solution to the product of step (2), and after 10-12 hours, add the Pb-recognizable2+ The nucleic acid biomolecule solution is magnetically separated after 10-12 hours, and washed with a Tris-HCl buffer solution of pH=7.4 for later use;

[0021] Wherein, the identifiable Pb 2+ The base sequence of the nucleic acid biomolecule is 5'-GGT GGT GGT GGT TGTGGT GGT GGT GG-3'(P2), which is similar to Pb 2+ The specific binding product can be cleaved by exonuclease, releasing Pb 2+ ;

[0022] (5) Add fluorescein isothiocyanate (FITC) solution to the product of step (2), and add Hg-recognizable 2+ The nucleic acid biomolecule solution is magnetically separated after 10-12 hours, and washed with a Tris-HCl buffer solution of pH=7.4 for later use;

[0023] Wherein, the identifiable Hg 2+ The base sequence of the nucleic acid biomolecule is 5'-TTT GTT TGT TGG CCCCCC TTC TTT CTTA-3'(P3), which is similar to Hg 2+ The specific binding product can be cleaved by exonucleases, releasing Hg 2 + ;

[0024] (6) The products obtained in step (3), step (4) and step (5) are diluted with a Tris-HCl buffer solution at pH = 7.4, mixed, and magnetically separated to obtain a simultaneous detection of As. 3+ , Pb 2+ and Hg 2+ Nanocapsule-nucleic acid molecule complex.

[0025] A method for simultaneously detecting As 3+ , Pb 2+ and Hg 2+ The method comprises the following steps:

[0026] (1) 3+ , Pb 2+ and Hg 2+ The solution is added to the nanocapsule-nucleic acid biomolecule complex prepared by the present invention, and the shearing enzyme EXOⅢ is added, diluted with a Tris-HCl buffer solution with a pH of 7.4, and placed in a 37° C. shaker for reaction for 1-3 hours;

[0027] (2) Magnetic separation, taking the supernatant for fluorescence detection, giving excitation of corresponding wavelengths, and detecting As 3+ , Pb2+ and Hg 2+ The corresponding fluorescence signals of the three dyes.

[0028] Beneficial effects of the present invention: The present invention can simultaneously detect As 3+ , Pb 2+ and Hg 2+ This method can not only achieve As 3+ , Pb 2+ and Hg 2+ Not only can the three fluorescent signals be detected simultaneously, but also on the basis of the original controlled release amplification, the cleavage action of biological enzymes and the recycling of targets are used to finally realize the simultaneous cyclic amplification and detection of three fluorescent signals; more importantly, the present invention overcomes the defects of traditional technologies, and no fluorescent labeling of nucleic acid biological molecules is required, thus completely avoiding the influence on the performance of nucleic acid biological molecules; the present invention uses simple and efficient multiple amplification technology to transform the fluorescent signal from a 1:1 mode to an exponential amplification mode, and the sensitivity is significantly enhanced.

[0029] The results show that compared with the existing technology, the system can simultaneously detect As 3+ , Pb 2+ and Hg 2+ , can simultaneously realize As 3+ , Pb 2+ and Hg 2+ The three nucleic acid biorecognition molecules used are respectively and only for the target metal ion As. 3+ , Pb 2+ and Hg 2+ It has a specific response and has no response to any other coexisting ions or interfering ions. 3+ , Pb 2+ or Hg 2+ 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 As 3+ , Pb 2+ and Hg 2+ Simultaneous detection of As 3+ , Pb 2+ , Hg 2+ Or the separate or simultaneous detection of two of the ion samples, all of these detection systems can obtain highly sensitive and highly selective detection results.

[0030] 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 Cd2+ , Fe 3+ 、Zn 2+ , Cu 2+ The influence of metal ions such as As 3+ , Pb 2+ and Hg 2+ There is no interference between them; they have high specificity and selectivity. The experimental results show that compared with the traditional technical methods, the nanocapsule-nucleic acid biomolecule complex proposed in the present invention is more effective in As 3+ , Pb 2+ and Hg 2+ Simultaneous detection was performed, showing high sensitivity and excellent selectivity, among which As 3+ The concentration is 5.0×10 -12 ~1.0×10 -10 The linear equation is satisfied within the range of mol / L: FL = 460.1193 + 168.7801lgC As 3+ (10 -12 mol / L); Pb 2+ The concentration was 1.0×10 -13 ~8.0×10 -11 The linear equation is satisfied within the range of mol / L: FL = 221.6 + 159.1lgC Pb 2+ (10 -13 mol / L); Hg 2+ The concentration is 5.0×10 -13 ~8.0×10 -11 The mol / L range satisfies the linear equation: FL = 75.88 + 61.04lgC Hg 2+ (10 -13 mol / L). Compared with the literature value, the present invention has a significant 3+ , Pb 2+ and Hg 2+ Under the premise of ensuring high specificity, the sensitivity of simultaneous detection is significantly improved by more than 10 times, and a wider linear detection range for the three ions is obtained. It can be foreseen 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

[0031] Figure 1 .Effect of different temperatures on detection of Pb 2+ , Hg 2+ and As 3+The effect of fluorescence response signal. DETAILED DESCRIPTION

[0032] 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.

[0033] The present invention is specifically described below by way of examples, but the present invention is not limited to the following examples.

[0034] 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).

[0035] Experimental reagents: exonuclease ExoШ (Thermo Scientific, USA); rhodamine B (Shanghai Aladdin Biochemical Technology Co., Ltd.); fluorescein isothiocyanate FITC, fluorescein Cy5 (Solarbio, Beijing Solaibao Technology Co., Ltd.); polydimethylammonium chloride, mercuric sulfate, lead nitrate (Shanghai Aladdin Biochemical Technology Co., Ltd.); 3-4 μm thiol magnetic beads (Tianjin Bestele Chromatography Technology Development Center); sodium arsenite mother solution, 5'-GGTAAT ACG ACT CACTAT AGG GAG ATA CCA GCT TAT TCA ATT TTA CAG AAC AAC CAA CGT CGC TCC GGG TACTCC TTC TTC ATC GAGATA GTAAGT GCAATC C-3' (P1), 5'-GGT GGT GGT GGT TGT GGT GGTGGT GG-3' (P2), 5'-TTT GTT TGT TGG CCC CCC TTC TTT CTTA-3'(P3) (Shanghai Sangon Biotechnology Co., Ltd.); Tris-HCl buffer solution, pH=7.4, concentration 0.05 M (Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0036] Embodiment 1:

[0037] A method for preparing the method for simultaneously detecting As 3+ , Pb 2+ and Hg 2+ The method of nanocapsule-nucleic acid biomolecule complex based on nuclease cyclic amplification technology and biomolecule recognition technology comprises the following steps:

[0038] (1) Screen, design and synthesize three nucleic acid biorecognition molecules to ensure that they are specific for the target metal ion As. 3+ , Pb 2+ and Hg 2+ It has a specific response and has no response to any other coexisting ions or interfering ions.

[0039] (2) After washing 20 μL of magnetic beads with a Tris-HCl buffer solution at pH = 7.4, 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 Tris-HCl buffer solution at pH = 7.4;

[0040] (3) Add 2 μL of fluorescent molecule Cy5 solution (final concentration 1.0×10 -5 mol / L), diluted to 100 μL with Tris-HCl buffer solution at pH 7.4, and added 10 μL at 37°C for 10 h to identify As 3+ Nucleic acid biomolecule solution (final concentration 1.0×10 -7 mol / L), 37°C for 10 h, followed by magnetic separation, washing with a pH=7.4 Tris-HCl buffer solution, and set aside;

[0041] Among them, the identifiable As 3+ The base sequence of the nucleic acid biomolecule is 5'-GGTAATACGACT CACTATAGG GAG ATA CCA GCT TAT TCA ATT TTA CAG AAC AAC CAA CGT CGC TCC GGG TAC TCCTTC TTC ATC GAGATAGTAAGT GCAATC C-3'(P1), which is similar to As 3+ The specific binding product can be cleaved by exonucleases, releasing As 3+ ;

[0042] (4) Add 2 μL of Rhodamine B solution (final concentration 1.0×10 -5 mol / L), diluted to 100 μL with Tris-HCl buffer solution at pH 7.4, and added 10 μL at 37°C for 10 h to identify Pb 2+ Nucleic acid biomolecule solution (final concentration 1.0×10 -6 mol / L), 37°C for 10 h, followed by magnetic separation, washing with a pH=7.4 Tris-HCl buffer solution, and set aside;

[0043] Wherein, the identifiable Pb 2+The base sequence of the nucleic acid biomolecule is 5'-GGT GGT GGT GGT TGTGGT GGT GGT GG-3'(P2), which is similar to Pb 2+ The specific binding product can be cleaved by exonuclease, releasing Pb 2+ ;

[0044] (5) Add 2 μL of fluorescein isothiocyanate FITC solution (final concentration 1.0×10 -4 mol / L), diluted to 100 μL with Tris-HCl buffer solution at pH 7.4, 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 a pH=7.4 Tris-HCl buffer solution, and set aside;

[0045] Wherein, the identifiable Hg 2+ The base sequence of the nucleic acid biomolecule is 5'-TTT GTT TGT TGG CCCCCC TTC TTT CTTA-3'(P3), which is similar to Hg 2+ The specific binding product can be cleaved by exonucleases, releasing Hg 2 + ;

[0046] (6) The products obtained in step (3), step (4) and step (5) are diluted with a Tris-HCl buffer solution at pH = 7.4, mixed, and magnetically separated to obtain a simultaneous detection of As. 3+ , Pb 2+ and Hg 2+ Nanocapsule-nucleic acid biomolecule complex;

[0047] 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.).

[0048] Embodiment 2:

[0049] A method for simultaneously detecting As 3+ , Pb 2+ and Hg 2+ The method comprises the following steps:

[0050] (1) 30 μL of As 3+ , Pb 2+ and Hg 2+The solution was added to the nanocapsule-nucleic acid biomolecule complex proposed in the present invention, 2.25 μL EXOⅢ shearing enzyme (45U) was added, and diluted to 100 μL with Tris-HCl buffer solution with pH = 7.4. After mixing evenly, it was placed on a shaker for reaction for 2 hours at a reaction temperature of 37°C. 3+ , Pb 2+ and Hg 2+ In the presence of As, the nucleic acid biomolecules assembled on the surface of the nanocapsules 3+ , Pb 2+ and Hg 2+ A specific response occurs, and the three 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 releasing the fluorescent substances Cy5, RhB and FITC from the inside of the nanocapsule respectively; at the same time, due to the cleavage of the nuclease ExoⅢ, the generated double-stranded specific binding product is cleaved and As is released. 3+ , Pb 2+ and Hg 2+ , and the released As 3+ , Pb 2+ and Hg 2+ 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 As 3+ , Pb 2+ and Hg 2+ It is recycled many times, more pores are opened, more fluorescent substances are released, and the fluorescent signal is significantly enhanced;

[0051] (2) Magnetic separation, the supernatant was diluted to 2.0 mL for fluorescence detection, and the three released fluorescent dyes were excited at the corresponding wavelengths, and the fluorescence signals of the three dyes were significantly enhanced. When different concentrations of As 3+ , Pb 2+ and Hg 2+ When the fluorescence signal is 0.0447 W, the fluorescence signal shows a good response relationship with the ion concentration; therefore, the As 3+ , Pb 2+ and Hg 2+ Simultaneous detection of As; Detection conditions: for As 3+ , Pb 2+ and Hg 2+ The excitation wavelengths for detection are EXWL: 649nm / 550nm / 488nm; the emission wavelengths are EMWL: 653nm / 580nm / 520nm.

[0052] The experimental results show that the system can be used to simultaneously detect As 3+ , Pb 2+ and Hg 2+ The linear detection ranges obtained are: 5.0×10 -12 ~1.0×10 -10 mol / L、1.0×10 -13 ~8.0×10 -11 mol / L and 5.0×10 -13 ~8.0×10 - 11 mol / L; Compared with the literature value, the present invention has a significant 3+ , Pb 2+ and Hg 2+ 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 As 3+ , Pb 2+ and Hg 2+ Wider linear detection range. Since the three nucleic acid biorecognition molecules used in the present invention are respectively and only for the target metal ion As 3+ , Pb 2+ or Hg 2+ It has a specific response, but does not respond to any other coexisting ions or interfering ions, and is not affected by other common interfering substances such as Cd 2+ , Fe 3+ 、Zn 2+ , Cu 2+ The nanocapsule-nucleic acid biomolecule complex proposed in the present invention can detect As 3+ , Pb 2+ and Hg 2+ , can also be used for As 3+ , Pb 2+ or Hg 2+ Individual detection of samples can obtain highly sensitive and highly selective detection results.

[0053] 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.; more importantly, the present invention overcomes the defects of traditional technology, and no fluorescent labeling of nucleic acid biomolecules is required, thus completely avoiding the influence on the performance of nucleic acid biomolecules; the present invention uses a simple and efficient multiple amplification technology, that is, on the basis of controlled release amplification, through the shearing action of biological enzymes and the recycling of targets, the fluorescence signal is transformed from a 1:1 mode to an exponential amplification mode, and the sensitivity is significantly enhanced.

[0054] It can be foreseen that the nanocapsule-nucleic acid biomolecule complex and its preparation method and detection technology proposed in the present invention have huge development 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 molecule complex for simultaneous detection of As3+, Pb2+ and Hg2+ and a preparation method thereof <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 103 <212> DNA <213> Artificial Sequence <400> 1 ggtaatacga ctcactatag ggagatacca gcttattcaa ttttacagaa caaccaacgt 60 cgctccgggt actccttctt catcgagata gtaagtgcaa tcc 103 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <400> 2 ggtggtggtg gttgtggtgg tggtgg 26 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <400> 3 tttgtttgtt ggcccccctt ctttctta 28

Claims

1. A method for simultaneously detecting As 3+ , Pb 2+ and Hg 2+ The nanocapsule-nucleic acid molecule complex is characterized by: The nanocomplex is constructed from three nucleic acid biorecognition molecules, three fluorescent signal molecules, and gold nanoparticles with a hollow, porous structure; The three nucleic acid biorecognition molecules are 5'-GGTAATACGACT CAC TATAGG GAGATA CCAGCTTAT TCAATT TTACAGAAC AAC CAA CGT CGC TCC GGG TAC TCC TTC TTC ATC GAGATAGTAAGTGCAATC C-3', 5'-GGT GGT GGT GGT TGT GGT GGT GGT GG-3' and 5'-TTT GTT TGT TGG CCCCCC TTC TTT CTTA-3'; The three fluorescent signal molecules are fluorescein Cy5, RhB and fluorescein isothiocyanate FITC; The three nucleic acid biorecognition molecules are respectively assembled onto the surface of the nano-gold with a hollow and porous structure through electrostatic interaction; The nanocapsule-nucleic acid molecule complex simultaneously detects As 3+ , Pb 2+ and Hg 2+ The method comprises the following steps: (1) 30 μL of As 3+ , Pb 2+ and Hg 2+ The solution was added to the nanocapsule-nucleic acid biomolecule complex, 2.25 μL EXOⅢ shearing enzyme 45U was added, and diluted to 100 μL with Tris-HCl buffer solution with pH = 7.4, mixed evenly, and placed on a shaker for reaction for 2 hours at a reaction temperature of 37°C; (2) Magnetic separation, take the supernatant and dilute it to 2.0 mL for fluorescence detection. The detection conditions are: for As 3+ , Pb 2+ and Hg 2+ The excitation wavelengths of the detection are: 649nm / 550nm / 488nm; the emission wavelengths are: 653nm / 580nm / 520nm; The steps for preparing the nanocapsule-nucleic acid molecule complex are as follows: (1) Screen, design and synthesize three nucleic acid biorecognition molecules to ensure that they are specific for the target metal ion As. 3+ , Pb 2+ and Hg 2+ It has a specific response and has no response to any other coexisting ions or interfering ions. (2) washing the magnetic beads with a Tris-HCl buffer solution at pH = 7.4, mixing the magnetic beads with a solution of a nano-gold carrier having a hollow, porous structure, adding a polydiallyldimethylammonium chloride solution, performing magnetic separation after 10-12 hours, and washing the magnetic beads with a Tris-HCl buffer solution at pH = 7.4; (3) Add fluorescein Cy5 solution, and after 10-12 hours, add As 3+ The nucleic acid biomolecule solution is magnetically separated after 10-12 hours, and washed with a Tris-HCl buffer solution of pH=7.4 for later use; Among them, the identifiable As 3+ The base sequence of the nucleic acid biomolecule is 5'-GGTAATACGACT CAC TAT AGGGAG ATA CCA GCT TAT TCA ATT TTA CAG AAC AAC CAA CGT CGC TCC GGG TAC TCC TTCTTCATC GAGATAGTAAGT GCAATC C-3', which is 3+ The specific binding product can be cleaved by exonucleases, releasing As 3+ ; (4) Add Rhodamine B solution to the product of step (2), and after 10-12 hours, add the Pb-recognizable 2+ The nucleic acid biomolecule solution is magnetically separated after 10-12 hours, and washed with a Tris-HCl buffer solution of pH=7.4 for later use; Wherein, the identifiable Pb 2+ The base sequence of the nucleic acid biomolecule is 5'-GGT GGT GGT GGT TGT GGTGGT GGT GG-3', which is similar to Pb 2+ The specific binding product can be cleaved by exonuclease, releasing Pb 2+ ; (5) Add fluorescein isothiocyanate (FITC) solution to the product of step (2), and add Hg-recognizable 2+ The nucleic acid biomolecule solution is magnetically separated after 10-12 hours, and washed with a Tris-HCl buffer solution of pH=7.4 for later use; Wherein, the identifiable Hg 2+ The base sequence of the nucleic acid biomolecule is 5'-TTT GTT TGT TGG CCC CCCTTC TTT CTTA-3', which is similar to Hg 2+ The specific binding product can be cleaved by exonucleases, releasing Hg 2+ ; (6) The products obtained in step (3), step (4) and step (5) are diluted with a Tris-HCl buffer solution at pH = 7.4, mixed, and magnetically separated to obtain the nanocapsule-nucleic acid molecule complex.

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