An intracellular As 3+ , Pb 2+ and Hg 2+ Simultaneous fluorescence imaging method

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+ All are highly toxic and widely distributed environmental pollutants, which pose a serious threat to the environment and human health even at low concentrations. 3+ , Pb 2+ and Hg 2+ The nanocapsule-nucleic acid biomolecule complex proposed and constructed in the present invention has many advantages, such as simple structure, good biocompatibility, ingenious design, simple preparation, stable performance, strong cell membrane permeability, strong controllable intracellular release, high specificity, short response time, etc., which can conveniently and quickly realize intracellular As 3+ , Pb 2+ and Hg 2+ Highly sensitive and selective simultaneous fluorescence imaging.

Description

Technical Field

[0001] The present invention relates to As 3+ , Pb 2+ , Hg 2+ Simultaneous detection and imaging of As in cells using a nanocapsule-nucleic acid biomolecule complex 3+ , Pb 2+ and Hg 2+ The invention relates to simultaneous fluorescence imaging and belongs to the field of environmental monitoring and nano-biotechnology. Background Art

[0002] Environmental and water pollution caused by highly toxic heavy metals has always been a major concern for people. Highly toxic heavy metals 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 lung cancer, skin cancer and other cancers, 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, hollow porous nanomaterials and various functional nucleic acid biomolecules have been used to construct nanocapsule-nucleic acid biomolecule complexes for intracellular As 3+ , Hg 2+ and Pb 2+ At the same time, fluorescence imaging 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 multiple functional nucleic acid biomolecule recognition technology is 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+ Nanocapsule-nucleic acid biomolecule complexes are prepared, specifically using hollow and porous nanogold as nanocapsules, and screening, designing and synthesizing 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 an intracellular As 3+ , Hg 2+ and Pb 2+ Simultaneous fluorescence imaging method.

[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] A method for preparing the intracellular As proposed by the present invention 3+ , Hg 2+ and Pb 2+ A method for simultaneous fluorescence imaging of a nanocapsule-nucleic acid biomolecule complex comprises the following steps:

[0015] (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.

[0016] (2) adding a polydiallyldimethylammonium chloride solution to the nanogold solution having a hollow, porous structure, centrifuging after 10-12 hours, and washing with a Tris-HCl buffer solution of pH=7.4;

[0017] (3) Add fluorescein Cy5 solution, and after 10-12 hours, add As 3+ The nucleic acid biomolecule solution is centrifuged after 10-12 hours, and washed with a Tris-HCl buffer solution of pH=7.4 for later use;

[0018] 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 TTCATC GAGATAGTAAGT GCAATC C-3';

[0019] (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 centrifuged after 10-12 hours, and washed with a Tris-HCl buffer solution of pH=7.4 for later use;

[0020] Wherein, the identifiable Pb 2+ The base sequence of the nucleic acid biomolecule is 5'-GGT GGT GGT GGT TGTGGT GGT GGT GG-3';

[0021] (5) Add fluorescein isothiocyanate (FITC) solution to the product of step (2), and add a Hg-recognizable 2+ The nucleic acid biomolecule solution is centrifuged after 10-12 hours, and washed with a Tris-HCl buffer solution of pH=7.4 for later use;

[0022] Wherein, the identifiable Hg 2+ The base sequence of the nucleic acid biomolecule is 5'-TTT GTT TGT TGG CCCCCC TTC TTT CTTA-3';

[0023] (6) The products obtained in step (3), step (4) and step (5) were diluted with a pH 7.4 Tris-HCl buffer solution, mixed, and centrifuged to obtain intracellular As 3+ , Pb 2+ and Hg 2+Simultaneous Fluorescence Imaging of Nanocapsule-nucleic Acid Biomolecule Complexes

[0024] An intracellular As proposed by the present invention 3+ , Pb 2+ and Hg 2+ The simultaneous fluorescence imaging method comprises the following steps:

[0025] (1) Take an appropriate amount of cell suspension and add As 3+ , Pb 2+ and Hg 2+ The cells were cultured with the solution and then centrifuged to remove the supernatant;

[0026] (2) Add the nanocapsule-nucleic acid biomolecule complex solution proposed in the present invention to incubate the cells, and after 10-50 minutes, give excitation of corresponding wavelengths to perform intracellular As 3+ , Pb 2+ and Hg 2+ Laser confocal scanning imaging, wherein the cells are Hela cells.

[0027] Beneficial effects of the present invention: The present invention provides a method for As 3+ , Pb 2+ and Hg 2+ Simultaneous imaging of the nanocapsule-nucleic acid biomolecule complex not only enables intracellular As 3+ , Pb 2+ and Hg 2+ Simultaneous fluorescence imaging can also be achieved for As 3+ , Pb 2+ and Hg 2+ The simultaneous detection solves the problem that the current detection and imaging technology is insufficient and it is difficult to simultaneously perform fluorescence imaging of multiple ions efficiently, sensitively, economically and quickly. More importantly, the present invention overcomes the defects of traditional technology and no longer requires any fluorescent labeling of nucleic acid biomolecules, completely avoiding the impact on the performance of nucleic acid biomolecules; the present invention establishes a simultaneous detection and imaging technology for multiple serious heavy metal pollutants through simple and efficient controlled release and specific biorecognition technology, and the sensitivity is significantly enhanced.

[0028] 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+ , Pb2+ 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 and imaging of As 3+ , Pb 2+ , Hg 2+ Or the separate or simultaneous detection and imaging of two of the ion samples, all of these systems can obtain highly sensitive and highly selective detection and imaging results.

[0029] 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+ , 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+ The simultaneous detection showed high sensitivity and excellent selectivity. Compared with the literature value, the present invention has a significant difference in the detection of As 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 imaging 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

[0030] Figure 1 .As in Hela cells 3+ , Pb 2+ and Hg 2+Bright-field fluorescence imaging photograph. DETAILED DESCRIPTION

[0031] 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.

[0032] The present invention is specifically described below by way of examples, but the present invention is not limited to the following examples.

[0033] Experimental instruments: THZ-82A gas bath constant temperature oscillator (Jintan Medical Equipment Factory); F-4600 fluorescence spectrophotometer (Hitachi, Japan); Leica TCS SP5 II laser confocal scanner (Leica, Germany).

[0034] Experimental reagents: Rhodamine B (Shanghai Aladdin Biochemical Technology Co., Ltd.); fluorescein isothiocyanate FITC, fluorescein Cy5 (Solarbio, Beijing Solarbio Technology Co., Ltd.); polydimethylammonium chloride, mercuric sulfate, lead nitrate (Shanghai Aladdin Biochemical Technology Co., Ltd.); sodium arsenite mother solution, 5'-GGTAATACGACT CAC TAT AGGGAG ATA CCA GCT TAT TCA ATT TTA CAG AAC AAC CAA CGT CGC TCC GGG TAC TCC TTCTTC ATC GAGATAGTAAGT GCAATC C-3'(P1), 5'-GGT GGT GGT GGT TGT GGT GGT GGT 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.).

[0035] Embodiment 1:

[0036] A kind of As 3+ , Pb 2+ and Hg 2+ A method for preparing a nanocapsule-nucleic acid biomolecule complex for simultaneous fluorescence imaging comprises the following steps:

[0037] (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.

[0038] (2) adding 200 μL of 11.664 mg / mL polydiallyldimethylammonium chloride solution to 400 μL of the nanogold carrier solution having a hollow, porous structure, centrifuging at 37° C. for 10 h, and washing with a Tris-HCl buffer solution at pH 7.4;

[0039] (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), centrifuged at 37°C for 10 h, washed with Tris-HCl buffer solution at pH 7.4, and set aside;

[0040] 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 TTCATC GAGATAGTAAGT GCAATC C-3'(P1);

[0041] (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), centrifuged at 37°C for 10 h, washed with Tris-HCl buffer solution at pH 7.4, and set aside;

[0042] Wherein, the identifiable Pb 2+ The base sequence of the nucleic acid biomolecule is 5'-GGT GGT GGT GGT TGTGGT GGT GGT GGT GG-3'(P2);

[0043] (5) Add 2 μL of fluorescein isothiocyanate FITC solution (final concentration 1.0×10 -4mol / 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), centrifuged at 37°C for 10 h, washed with Tris-HCl buffer solution at pH 7.4, and set aside;

[0044] 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);

[0045] (6) The products obtained in step (3), step (4) and step (5) were diluted with a pH 7.4 Tris-HCl buffer solution, mixed, and centrifuged to obtain intracellular As 3+ , Pb 2+ and Hg 2+ Simultaneous fluorescence imaging of nanocapsule-nucleic acid biomolecule complexes;

[0046] 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.).

[0047] Embodiment 2:

[0048] An intracellular As 3+ , Pb 2+ and Hg 2+ The method of simultaneous fluorescence imaging comprises the following steps:

[0049] (1) Take an appropriate amount of cell suspension, centrifuge, remove the supernatant, and add 300 μL of As 3+ , Pb 2+ and Hg 2+ The final concentration was 1.0×10 -11 The cells were cultured in the solution of M at 37°C for 20.0 min, then centrifuged and the supernatant was removed;

[0050] (2) diluting the nanocapsule-nucleic acid biomolecule complex prepared in Example 1 with a Tris-HCl buffer solution of pH=7.4, adding the solution to the cells treated in the previous step, and incubating the cells in a 37°C constant temperature water bath for 15.0 min;

[0051] (3) The incubated cells were placed on the stage of a confocal microscope and the intracellular As was detected using excitation wavelengths of 649 nm, 559 nm, and 488 nm. 3+ , Pb 2+ and Hg 2+ Laser confocal scanning imaging;

[0052] Wherein, the cells are Hela cells.

[0053] The experimental results show that the present invention proposes a simultaneous detection and imaging technology for multiple serious heavy metal pollutants through simple and efficient controlled release and specific biorecognition technology, and the sensitivity is significantly enhanced. 3+ , Pb 2+ and Hg 2+ Ion concentration as low as 1.0×10 -11 mol / L, clear cell fluorescence imaging photos showing the presence of ions were still obtained.

[0054] The present invention proposes a method for using As in cells 3+ , Pb 2+ and Hg 2+ Simultaneous imaging of the nanocapsule-nucleic acid biomolecule complex not only enables intracellular As 3+ , Pb 2+ and Hg 2+ Simultaneous fluorescence imaging can also be achieved for As 3+ , Pb 2+ and Hg 2+ The simultaneous detection of As solves the problem that the current detection and imaging technology is insufficient and it is difficult to perform fluorescence imaging of multiple ions simultaneously in an efficient, sensitive and fast manner. More importantly, the present invention overcomes the defects of traditional technology and does not require any fluorescent labeling of nucleic acid biomolecules, completely avoiding the impact on the performance of nucleic acid biomolecules. 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. 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 no response to any other coexisting ions and interfering ions, and has high specificity and selectivity. Therefore, the nanocapsule-nucleic acid biomolecule complex proposed in the present invention can not only simultaneously 3+ , Pb 2+ and Hg 2+ Detection and imaging, can also be used to contain only As 3+ , Pb 2+ , Hg 2+ Or the detection and imaging of two of the ions can obtain highly sensitive and highly selective detection and imaging results.

[0055] The nanocapsule-nucleic acid biomolecule complex proposed by the present invention is used for intracellular As 3+ , Pb 2+ and Hg 2+ Simultaneous fluorescence imaging, simple structure, good biocompatibility, ingenious design, simple preparation, stable performance, strong cell membrane permeability, strong controllable intracellular release, and strong sensitivity to As 3+ , Pb 2+ and Hg 2+ It has many advantages, such as strong specificity, short response time, and convenient real-time monitoring, and will not be affected by other common interfering substances such as Cd 2+ , Cu 2+ , Fe 3+ , Zn 2+ The influence of metal ions such as As can be easily and quickly achieved in cells 3+ , Pb 2+ and Hg 2+ Highly sensitive and selective simultaneous fluorescence imaging.

[0056] It can be foreseen that the nanocapsule-nucleic acid biomolecule complex and its preparation method and detection imaging technology proposed in the present invention have huge 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 food hygiene, biomedicine, environmental monitoring and other fields. Sequence Listing <110> Qingdao University of Science and Technology <120> A method for simultaneous fluorescence imaging of As3+, Pb2+ and Hg2+ in cells <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 103 <212> DNA <213> Artificial Sequence <400> 1 ggtaatacga ctcactatag ggagatacca gcttattcaa ttttacagaa caaccaacgt cgctccgggt actccttctt catcgagata gtaagtgcaa tcc <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <400> 2 ggtggtggtg gttgtggtgg tggtgg <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <400> 3 tttgtttgtt ggcccccctt ctttctta

Claims

1. An intracellular As 3+ , Pb 2+ and Hg 2+ A simultaneous fluorescence imaging method, characterized in that The method uses a nanocapsule-nucleic acid biomolecule complex 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 intracellular As 3+ , Pb 2+ and Hg 2+ The steps of the simultaneous fluorescence imaging method are as follows: (1) Take an appropriate amount of cell suspension and add As 3+ , Pb 2+ and Hg 2+ The cells were cultured with the solution and then centrifuged to remove the supernatant; (2) Add the nanocapsule-nucleic acid biomolecule complex solution for incubation, and after 10-50 minutes, give excitation of corresponding wavelengths to perform intracellular As 3+ , Pb 2+ and Hg 2+ Fluorescence imaging of The preparation method of the nanocapsule-nucleic acid biomolecule complex comprises the following steps: (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) adding polydiallyldimethylammonium chloride solution to the nanogold solution having a hollow, porous structure, centrifuging after 10-12 hours, and washing with a Tris-HCl buffer solution of pH=7.4; (3) Add fluorescein Cy5 solution, and after 10-12 hours, add As 3+ The nucleic acid biomolecule solution is centrifuged 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'; (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 centrifuged 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'; (5) Add fluorescein isothiocyanate (FITC) solution to the product of step (2), and add Hg-recognizable 2+ The nucleic acid biomolecule solution is centrifuged 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'; (6) The products obtained in step (3), step (4) and step (5) are diluted with a Tris-HCl buffer solution of pH = 7.4, mixed, and centrifuged to obtain the nanocapsule-nucleic acid biomolecule complex.

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