WO2022179183A1 - 一种核酸功能化金属纳米探针及其制备方法 - Google Patents

一种核酸功能化金属纳米探针及其制备方法 Download PDF

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WO2022179183A1
WO2022179183A1 PCT/CN2021/129724 CN2021129724W WO2022179183A1 WO 2022179183 A1 WO2022179183 A1 WO 2022179183A1 CN 2021129724 W CN2021129724 W CN 2021129724W WO 2022179183 A1 WO2022179183 A1 WO 2022179183A1
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nucleic acid
metal
pathogenic
probe
pathogenic microorganism
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French (fr)
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王雪梅
国增超
曾嘉瑜
姜晖
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东南大学
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    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/58—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing copper, silver or gold
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
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    • C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6432—Quenching
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of biological materials, in particular to a nucleic acid-functionalized metal nanoprobe, a method for in situ construction and preparation thereof, and a method for treating infectious diseases.
  • Bacteria and viruses in nature are everywhere.
  • the temperature of the human body is about 37°C and the moist internal mucosa is an ideal place for bacteria to set up camp.
  • new or sudden diseases such as highly pathogenic avian influenza, atypical pneumonia and viral pneumonia have emerged continuously, which has brought great difficulties to clinical diagnosis and treatment. Therefore, for pathogenic microorganisms, early diagnosis and treatment and early control are the keys to effectively reduce physical damage and reduce the threat to human health.
  • detection methods for pathogenic microorganisms such as plate culture, enzyme-linked immunosorbent assay, polymerase chain reaction (PCR), sequencing, etc., they usually require complicated and time-consuming processes, and sometimes false positives or low sensitivity may occur. series of questions.
  • metal nanoprobes Due to the unique optical, electrical, magnetic and other physical and chemical properties of metal nanomaterials, nanotechnology has received extensive attention in recent years in the diagnosis and treatment of pathogenic microorganisms.
  • the synthesis of metal nanoprobes usually requires macromolecular substances such as proteins and nucleic acids as templates to stabilize the structure of metal nanoprobes, which is crucial for the application of nanoprobes.
  • macromolecular substances such as proteins and nucleic acids as templates to stabilize the structure of metal nanoprobes, which is crucial for the application of nanoprobes.
  • the conditions of these synthetic methods are relatively harsh, such as the need to maintain high temperature and narrow acid-base range.
  • the current research is still mainly on the use of metal nanomaterials as drug carriers.
  • nucleic acids such as DNA have the advantages of low cytotoxicity and high biocompatibility. Because of their precise base complementary pairing, they can be used as good in vivo self-assembly materials.
  • exogenous genes often enter pathogenic microbial cells through special treatments such as transfection, transformation, etc., which greatly reduces the possibility of exogenous genes as templates to target pathogenic bacteria in vivo.
  • exogenous nucleic acid molecules into pathogenic microbial cells and combining metal cations to generate functionalized nanoprobes in situ can not only eliminate the side effects caused by nanoparticles, but also effectively combine bioimaging technology with therapeutic methods It can realize the integration of diagnosis and treatment, and reduce the damage caused by the use of excessive exogenous substances to the body.
  • the Chinese authorized patent publication number CN1435493A discloses a solid-phase nucleic acid detection probe and a preparation method thereof, which is an oligonucleotide probe fixed on a solid substrate and a microarray chip made by this method.
  • a non-labeled oligonucleotide probe for detecting nucleic acid sequence information the probe is fixed with a fluorescence quenching material 3 on a solid substrate 1 through an arm molecule 2, and a fluorescent group is prepared on the surface of the fluorescence quenching material 3. 5.
  • An oligonucleotide probe consisting of the stem portion 6 of the oligonucleotide probe molecule and the loop portion 7 of the oligonucleotide probe, and one end of the oligonucleotide probe is fixed on the surface of the fluorescence quenching material 3
  • the bases near the other end of the oligonucleotide probe are labeled with fluorescent group 5, and the sequences near the two ends of the oligonucleotide probe have 3 to 15 bases as complementary sequences, which can make the oligonucleotide
  • the sequences near both ends of the nucleotide probe can form hybridization, and the base sequence in the middle part of the oligonucleotide probe is the complementary sequence of the nucleic acid sequence to be detected.
  • the patent application with publication number CN105021585A discloses a method for detecting food-borne pathogenic bacteria based on a metal-organic framework material-nucleic acid aptamer fluorescence sensor, which utilizes the fluorescence quenching properties of the metal-organic framework material and the adsorption of nucleic acid aptamers.
  • the probe fluorescence is quenched, and the target bacteria are added to the system, and the fluorescent probe-labeled nucleic acid aptamer leaves the metal-organic framework material and combines with the target bacteria , so that the fluorescent signal of the probe is enhanced, combined with the high affinity and high specific recognition ability of the nucleic acid aptamer, and the method of the present invention is constructed.
  • Salmonella as the model analyte, the fluorescence intensity of the probe has a good linear relationship with the logarithm of the concentration of the target bacteria.
  • the relative standard deviation (RSD) of the standard addition experiment was in the range of 3.6% to 7.5%, and the recovery rate was in the range of 90.0% to 106.0%.
  • the invention has the advantages of high accuracy, sensitivity and specificity for the detection of food-borne pathogenic bacteria.
  • the fluorescein-modified Salmonella and aptamers When fluorescein-modified Salmonella and aptamers are adsorbed to the surface , Since the electron transfer induced by the material quenches the fluorescence of fluorescein, if there is Salmonella in the solution, the Salmonella and its aptamer will be specifically bound and desorbed from the surface of the material, and the electron transfer process between the material and fluorescein will be cut off and the fluorescence will be reduced. The fluorescence recovery of the element is obtained.
  • the method was used for the detection of Salmonella in shrimp meat samples, and the recovery rate of standard addition was 90.0%-108.0%.
  • the sensor has good selectivity and sensitivity to Salmonella.
  • the purpose of the present invention is to provide a nucleic acid functionalized metal nanometer.
  • Probes are mixed with metal cations and nucleic acid molecules to generate DNA or RNA composite nanostructures, which are incubated with pathogenic microorganisms.
  • the composite nanostructures enter the cells of pathogenic microorganisms through electrostatic adsorption, and then use them as templates.
  • Fluorescent nanoprobes are synthesized by in situ self-assembly of unique intracellular microenvironments such as high levels of oxidative and reducing substances. It can achieve rapid detection and precise killing, and has the characteristics of strong targeting effect and simple and easy operation.
  • the probe mixes a metal soluble salt solution with good biocompatibility with exogenous DNA or RNA molecules to generate DNA or RNA composite nanostructures; incubated with pathogenic microorganisms, the composite nanostructures enter the pathogenic microorganism cells through electrostatic adsorption , using the unique microenvironment of pathogenic microorganisms to promote the in situ synthesis of intelligent biological probes.
  • the present invention is based on a relatively high level of oxidative and reducing substances in pathogenic microorganism cells, delivering exogenous nucleic acid molecules into pathogenic microorganisms, and in situ self-assembly of programmable nucleic acid biomolecules.
  • Nanoprobes. needle as follows:
  • Nucleic acid-functionalized metal nanoprobes are the combination of nucleic acid molecules and metal cations with good biocompatibility through electrostatic adsorption to form a combination of gene-metal cations, which are incubated in situ with pathogenic microorganisms, pathogenic bacteria and virus cells. Self-assembled, nanoscale, with an average diameter of 2.3 nm.
  • the preparation method of nucleic acid functionalized metal nanoprobe the steps are as follows:
  • Step 1 Fully mix the nucleic acid fragment with the diluted nucleic acid inserter SYBR Green I (2.5x), and place it at room temperature for 30 minutes in the dark;
  • Nucleic acid fragments include DNA fragments or RNA fragments or related gene fragments synthesized by chemical methods;
  • RNA fragments are self-assembled RNA fragments formed after denaturation and gradient annealing.
  • Step 2 adding a metal soluble salt solution with excellent biocompatibility into the above solution, and after thorough mixing, a mixed solution A of metal cations and nucleic acid molecules is obtained;
  • Described metal soluble salt is one or any combination of solutions in water-soluble Mn 2+ , chloroauric acid, copper chloride, magnesium chloride, zinc gluconate, silver nitrate or ferrous chloride;
  • the final concentration is 10 ⁇ mol/L ⁇ 300 ⁇ mol/L.
  • the preferred concentration is 100 ⁇ mol/L.
  • the metal cation used is ferrous chloride
  • the obtained nucleic acid-functionalized metal nanoprobe can play a targeting and guiding role under the action of an external magnetic field.
  • the excellent biocompatibility means that the metal cations obtained by the hydrolysis of the metal soluble salt will not cause damage to normal cells of the human body, and have no toxic and side effects on normal cells.
  • Step 3 Mix the mixture A obtained in step 2 with the pathogenic microbial cells, and place the mixture on a constant temperature shaker for 0.5h-12h to obtain a mixture B.
  • the pathogenic microorganisms are Escherichia coli and Staphylococcus aureus.
  • Step 4 Centrifuge the mixture B at a speed of 2000-5000 r/min to extract the incubated pathogenic microbial cells, and wash the incubated cells 3-5 times with sterile water.
  • Step 5 Take the cleaned and incubated pathogenic microbial cells and excite them with a laser confocal fluorescence microscope for fluorescence imaging detection, and then act on the pathogenic microorganisms in combination with external physical intervention methods such as magnetic field and near-infrared to achieve targeting.
  • the present invention has the following advantages and effects:
  • nucleic acid molecules and metal cations with good biocompatibility form a combination of gene-metal cations through electrostatic adsorption, and in situ self-assembly with pathogenic microorganisms, pathogenic bacteria and virus cells to synthesize metal fluorescence or magnetic nanoparticles.
  • the pathogenic bacteria cells were then fluorescently imaged using confocal fluorescence microscopy excitation.
  • the present invention can realize precise target labeling and real-time rapid detection of pathogenic microorganisms and pathogenic bacteria, and has the characteristics of high specificity, simplicity and ease of operation.
  • the nucleic acid functionalized metal nanoprobe of the present invention combined with physical interventions such as photoelectric, magnetic field, near-infrared, etc., has a good antibacterial effect, and can be used as a target for infectious diseases such as lung infection, intestinal infection and influenza to intervention.
  • Example 1 is a transmission electron microscope micrograph of the nanoprobes obtained in Example 1.
  • the diameters of the nanoprobes range from 1.7 to 2.6 nm, and the average diameter is 2.3 nm.
  • Figure 2 is a comparison chart of the fluorescence intensity of metal cations with different concentrations. As the concentration of metal cations increases, the signal intensity increases. When the concentration reaches 100 ⁇ mol/L, the signal intensity reaches the maximum, and continues to increase the concentration, the signal intensity is basically unchanged. Therefore, the optimal metal cation concentration is 100 ⁇ mol/L.
  • Figure 3 is a comparison chart of the bactericidal effect of different concentrations of metal cations. As the concentration of metal cations increases, the bactericidal effect increases. When the concentration reaches 100 ⁇ mol/L, almost all bacteria are killed.
  • Figure 4 is a comparison chart of the fluorescence intensities of different metal cation solutions. Except for the blank group, the signal intensities of the other experimental groups are slightly different, but they all have good signal effects.
  • Fig. 5 is the bactericidal effect comparison chart of different metal cation solutions, except blank group, other experimental groups all show strong bactericidal effect.
  • the reagents and instruments involved in the embodiments of the present invention are all commercially available products, which can be purchased through commercial channels.
  • the metal cation solutions used in this experiment were all tested for cytotoxicity, and they were all common metal cation solutions with good biocompatibility.
  • the screening experiments of metal cation species are as follows: The cytotoxicity of different metal cations to mammalian cells was evaluated by MTT assay, represented by human normal hepatocytes (L02). L02 cells were seeded in a 96-well plate at a density of 0.5 ⁇ 10 5 cells/mL, 100 ⁇ L per well, and cultured at 37° C. for 8 h (5.0% CO 2 ). Then, 100 ⁇ L of culture medium containing different metal cations was added to each well, and the culture was continued for 24 h. Then, 20 ⁇ L of 5 mg/mL MTT solution was added to each well, and the culture was continued for 4 h.
  • MTT assay represented by human normal hepatocytes (L02). L02 cells were seeded in a 96-well plate at a density of 0.5 ⁇ 10 5 cells/mL, 100 ⁇ L per well, and cultured at 37° C. for 8 h (5.0% CO 2 ). Then, 100 ⁇ L of
  • the culture was completed, 150 ⁇ L of dimethyl sulfoxide was added to each well and shaken horizontally for 10 min to completely dissolve the MTT reaction.
  • the UV absorption intensity at 490 nm was measured by a microplate reader, and the measured value was corrected with a blank well containing no cells and only medium.
  • the cell viability (%) was expressed as the ratio of the measured value of the test group to the measured value of the control group (without gold nanoclusters).
  • the preparation method of nucleic acid functionalized metal nanoprobe the steps are as follows:
  • the glass slide was placed under a laser confocal fluorescence microscope for excitation. Further, the above mixture was irradiated with 808 nm laser for 5-10 min, cultured on solid medium, and counted. The results showed that the Escherichia coli in the experimental group exhibited strong fluorescence and were almost all killed, the normal control group had weak fluorescence and most of them were killed, while the blank group had no change.
  • test method is the same as in Example 1, except that the influence of different nucleic acid fragments on the results is investigated
  • the investigated nucleic acid fragments are: 1. DNA molecules, 2. self-assembled RNA fragments formed after denaturation and gradient annealing (the RNA molecules are rapidly cooled on ice after being denatured at 90° C. for 1 min. Then they are subjected to gradient annealing treatment, namely 70°C-50°C, 50°C-37°C, 37°C-4°C).
  • RNA fragments like DNA molecules, can also be used for the synthesis of metal nanoprobes, and show good detection and killing effects.
  • test method is the same as in Example 1, except that the influence of different metal cations on the results is investigated
  • the investigated metal cations are (concentration is 100 ⁇ mol/L): 1 chloroauric acid, 2 zinc gluconate, 3 silver nitrate, 4 mixture of chloroauric acid and ferrous chloride, 5 zinc gluconate and ferrous chloride mixture.
  • the test method is the same as in Example 1, except that the influence of different concentrations of metal cations on the results is investigated.
  • concentrations of the investigated metal cations are respectively: 0 ⁇ mol/L, 10 ⁇ mol/L, 50 ⁇ mol/L, 100 ⁇ mol /L, 200 ⁇ mol/L, 300 ⁇ mol/L.
  • the concentration has a certain influence on the effect of metal cations, and with the increase of the concentration, the effect of detection and treatment is enhanced.
  • test method is the same as that of Example 1, except that in step (3), mixture A is incubated with different bacterial species, and the investigated bacterial species are: 1 Escherichia coli, 2 Staphylococcus aureus.
  • nucleic acid-functionalized metal nanoprobe obtained in the present invention is used for targeted intervention of related diseases such as pulmonary infection, intestinal infection, and infectious diseases such as influenza.
  • Sample preparation refer to the preparation method of Example 1, except that the metal salt is co-incubated with an equal concentration mixed solution of zinc gluconate solution and ferrous chloride solution.
  • mice The samples obtained from co-incubation were injected in situ from the tail vein or the lesion site of skin-infected mice or pneumonia-infected mice, and a control group (nucleic acid fragments without metal cations) was set at the same time, and a small animal in vivo imager was used.
  • the above-mentioned experimental mice were subjected to real-time multi-modality (fluorescence, MRI, ultrasound, CT, etc.) in situ imaging observation, and at the same time, the lesions were physically intervened by photothermal treatment methods, and then the changes and ablation of the lesions were recorded.
  • the imaging signal could quickly identify the lesion site of the mice in the experimental group and be enriched in this area, and the related symptoms disappeared after physical therapy.

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Abstract

本发明公开了一种核酸功能化金属纳米探针及其制备方法，该探针将生物相容性良好的金属阳离子与外源性的DNA或RNA分子混合生成DNA或RNA复合纳米结构；与致病微生物共同孵育，复合纳米结构通过静电吸附进入致病微生物细胞，利用致病微生物所具有的独特的高水平的氧化性及还原性物质的微环境促使原位合成智能生物探针。实现对致病微生物的精准靶向标记与即时快速检测，用于肺部感染、肠道感染以及流感等感染性疾病的靶向干预。结合光电、磁热、近红外线等物理干预，能够实现相关病灶多模态的实时动态与高特异性的快速准确示踪和精准杀灭。

Description

一种核酸功能化金属纳米探针及其制备方法
技术领域
本发明涉及生物材料领域，尤其涉及一种核酸功能化金属纳米探针、其原位构建制备方法及其用于治疗感染性疾病的方法。
背景技术
自然界的细菌与病毒无处不在，人体37℃左右的温度和湿润的体内黏膜，更是细菌安营扎寨的理想场所。近年来，诸如高致病性禽流感、非典型肺炎以及病毒性肺炎等新发或突发疾病不断涌现，给临床诊断和治疗带来了极大的困扰。因此，对于致病微生物而言，尽早诊治、及早控制是有效降低身体损害、减少对人类健康威胁的关键。虽然已有的致病微生物检测方法很多，如平板培养、酶联免疫法、聚合酶链反应(PCR)、测序等，但通常需要复杂耗时的过程，有时会出现假阳性或灵敏度低等一系列问题。除了诊断问题，治疗问题也变得越来越严重。自从青霉素发明以来，抗生素在对抗各种病原微生物方面做出了巨大贡献。然而，随着抗生素的滥用，致病微生物不得不对抗生素产生突变并产生耐药性，导致许多耐药菌株的出现，这大大降低了抗生素的治疗效果。因此，迫切需要建立简单、灵敏、可靠的新型致病微生物检测技术，并开发安全、高效、有效的治疗新策略。
由于金属纳米材料独特的光、电、磁等理化性质，使得纳米技术近年来在致病微生物诊疗等方面得到了人们的广泛关注。金属纳米探针的合成通常需要大分子物质如蛋白质、核酸等作为模板稳定金属纳米探针的结构，这对纳米探针的应用至关重要。然而，这些合成方法的条件相对苛刻，如需要保持高温和狭窄的酸碱范围等。另外，目前的研究还主要停留在将金属纳米材料作为药物载体使用上。近年来，金属纳米探针的原位自组装合成策略被频繁报道，这种方法不仅不需要添加任何化学试剂来防止纳米探针团聚，而且可以实现对靶细胞的实时检测。DNA等核酸作为生物材料，其具有细胞毒性低，生物相容性高等优点，因其精确的碱基互补配对，可作为很好的体内自组装材料。但是外源基因往往通过特殊处理如转染、转化等方法才能进入病原微生物细胞，大大降低了外源基因作为模板在体内靶向病原菌的可能性。因此，将外源核酸分子递送进入致病微生物细胞，并结合金属阳离子原位生成功能化的纳米探针，不仅可以排除纳米粒子带来的副作用，而且将生物成像技术与和治疗手段有效的联合起来，实现诊断治疗一体化，减少过多外源物质的使用对机体造成的伤害。
公开号为CN1435493A的中国授权专利公开了固相化核酸检测探针及其制备方法，是一种固定在固体基片上寡核苷酸探针及其用此法制作的微阵列芯片，是一种检测核酸序列信息的非标记的寡核苷酸探针，该探针在固体基片1上通过手臂分子2固定有荧光淬灭材料3，在荧光淬灭材料3表面上制备有由荧光基团5、寡核苷酸探针分子的茎杆部6、寡核苷酸探针的环部7组成的寡核苷酸探针，寡核苷酸探针的一端固定在荧光淬灭材料3表面上，寡核苷酸探针的另一端附近的碱基标记有荧光基团5，寡核苷酸探针两端附近的序列分别有3 至15个碱基为互补序列，可使该寡核苷酸探针两端附近的序列能够形成杂交，寡核苷酸探针中间部分的碱基序列为被检测的核酸序列的互补序列。
公开号为CN105021585A的专利申请公开了一种基于金属有机骨架材料－核酸适体荧光传感器检测食源性致病菌的方法，利用金属有机骨架材料的荧光淬灭特性以及对核酸适体的吸附性，当荧光探针标记的核酸适体被吸附到金属有机骨架材料上，探针荧光被淬灭，在体系中加入目标菌，荧光探针标记的核酸适体离开金属有机骨架材料与目标菌结合，从而使探针荧光信号增强，结合核酸适体的高亲和力和高特异性识别能力，构建了本发明方法。以沙门氏菌为模型分析物，探针的荧光强度与目标菌浓度的对数呈良好的线性关系，线性范围为18～3.2×104cfu/mL，检出限达5cfu/mL(S/N＝3)，加标实验的相对标准偏差(RSD)介于3.6％～7.5％，回收率在90.0％～106.0％范围内。本发明用于食源性致病菌的检测具有准确、灵敏、特异性高等优点。
蒋晓华等在“金属有机骨架材料-核酸适配体荧光法检测沙门氏菌”一文中(《分析测试学报》2018年5期)基于金属有机骨架材料(Uio-66-NH2)的荧光猝灭特性以及对核酸适配体的吸附性,结合核酸适配体的高亲和力与高特异性识别能力,构建了针对沙门氏菌检测的荧光生物传感器,当有荧光素修饰的沙门氏菌、适配体被材料吸附到表面时,由于材料诱导电子转移猝灭了荧光素的荧光,若溶液中存在沙门氏菌,则沙门氏菌与其适配体特异性结合后从材料表面脱附,材料与荧光素之间的电子转移过程被切断,荧光素的荧光恢复.基于此原理构建的荧光传感器的信号与沙门氏菌浓度的对数在101～105cfu/mL范围内呈良好的线性关系,检出限(S/N＝3)为7cfu/mL,将该方法用于虾肉样品中沙门氏菌的检测,加标回收率为90.0％～108.0％,该传感器对沙门氏菌有较好的选择性与灵敏度。
发明内容
发明目的：为了克服现有技术在金属纳米探针合成时所需的复杂制备过程以及在致病微生物诊断及治疗过程中带来的毒副作用，本发明的目的是提供一种核酸功能化金属纳米探针，将金属阳离子与核酸分子混合培育生成DNA或RNA复合纳米结构，与致病微生物共同孵育，复合纳米结构通过静电吸附进入致病微生物细胞，然后以其为模板，依托致病微生物较高水平的氧化性及还原性物质等独特的胞内微环境原位自组装合成荧光纳米探针。实现快速检测与精准杀灭，具有靶向作用强及简便易行等特点。
该探针将生物相容性良好的金属可溶性盐溶液与外源性的DNA或RNA分子混合生成DNA或RNA复合纳米结构；与致病微生物共同孵育，复合纳米结构通过静电吸附进入致病微生物细胞，利用致病微生物独特的微环境促使原位合成智能生物探针。
技术方案：为了解决上述技术问题，本发明基于病原微生物胞内较高水平的氧化性及还原性物质，将外源核酸分子递送到病原微生物体内，原位自组装可编程核酸生物分子的纳米探针，具体如下：
核酸功能化的金属纳米探针，是将核酸分子与生物相容性良好的金属阳离子通过静电吸附作用形成基因-金属阳离子的结合物，与致病微生物、致病菌以及病毒细胞共孵育原位自组装得到，为纳米级别，其平均直径为2.3nm。
核酸功能化的金属纳米探针的制备方法，步骤如下：
步骤一、将核酸片段与稀释后的核酸插入剂SYBR GreenⅠ(2.5x)充分混合，置于室温下避光反应30min；
核酸片段包括DNA片段或RNA片段或通过化学方法合成的相关基因片段；
RNA片段为经过变性及梯度退火后形成的自组装RNA片段。
步骤二、将生物相容性优良的金属可溶性盐溶液加入到上述溶液中，充分混合后得到金属阳离子与核酸分子的混合液A；
所述的金属可溶性盐为水溶性Mn 2+、氯金酸、氯化铜、氯化镁、葡萄糖酸锌、硝酸银或氯化亚铁中的一种或任意几种溶液的组合；金属可溶性盐的终浓度10μmol/L～300μmol/L。较佳浓度是100μmol/L。当所采用的金属阳离子是氯化亚铁时，所得核酸功能化的金属纳米探针可以在外界磁场的作用下发挥靶向引导作用。所述的生物相容性优良是指金属可溶性盐水解所得的金属阳离子不会引起人体正常细胞的损伤，对正常细胞无毒副作用。
步骤三、将步骤二中获得的混合液A与致病微生物细胞相混合，置于恒温摇床继续孵育0.5h-12h得到混合液B。
所述的致病微生物是大肠杆菌、金黄色葡萄球菌。
步骤四、将混合液B在2000-5000r/min的速率下离心提取出孵育后的致病微生物细胞，用无菌水清洗孵育后的细胞3-5次。
步骤五、取清洗孵育后的致病微生物细胞利用激光共聚焦荧光显微镜激发，进行荧光成像检测，然后结合诸如磁场、近红外等外界物理干预方式作用于致病微生物，实现靶向作用。
与现有技术方法相比，本发明具有以下优点和效果：
(1)本发明将核酸分子与生物相容性良好的金属阳离子通过静电吸附作用形成基因-金属阳离子的结合物，与致病微生物、致病菌以及病毒细胞共孵育原位自组装合成金属荧光或磁性纳米颗粒。然后利用激光共聚焦荧光显微镜激发，对致病菌细胞进行荧光成像。
(2)本发明对致病微生物、致病菌可实现精准靶向标记与即时快速检测，具有高特异性及简便易行等特点。
(3)本发明核酸功能化的金属纳米探针，同时结合光电、磁场、近红外等物理干预，具有良好的抗菌效果，可用于对肺部感染、肠道感染以及流感等感染性疾病的靶向干预。
附图说明
图1为实施例1所得纳米探针的透射电镜显微图，纳米探针的直径范围为1.7-2.6nm,平均直径为2.3nm。
图2为不同浓度金属阳离子的荧光强度对比图，随着金属阳离子浓度的升高，信号强度增强，当浓度达到100μmol/L时，信号强度达到最大，继续增大浓度，信号强度基本不变，因此最佳金属阳离子浓度是100μmol/L。
图3为不同浓度金属阳离子的杀菌效果对比图，随着金属阳离子浓度的升高，杀菌效果增强，当浓度达到100μmol/L时，几乎所有的细菌均被杀死。
图4为不同金属阳离子溶液的荧光强度对比图，除空白组外，其余实验组的信号强度虽略有差异，但均具有良好的信号效果。
图5为不同金属阳离子溶液的杀菌效果对比图，除空白组外，其余实验组的均表现 出强烈的杀菌效果。
具体实施方式
为了进一步理解本发明，下面将结合本发明具体实施例，对本发明中的技术方案进行清楚、完整地描述。
如无特殊说明，本发明实施例中所涉及的试剂、仪器均为市售产品，均可以通过商业渠道购买获得。
本实验所用的金属阳离子溶液均进行了细胞毒性实验，其都是生物相容性良好的常见金属阳离子溶液。
金属阳离子种类的筛选实验如下：以人正常肝细胞(L02)为代表，采用MTT实验评估了不同的金属阳离子对哺乳动物细胞的细胞毒性。将L02细胞按0.5×10 5个/mL的密度接种于96孔板中，每孔100μL，37℃培养8h(5.0％CO 2)。然后，分别向每孔继续加入100μL含有不同金属阳离子的培养基，继续培养24h。然后往每个孔中加入20μL5mg/mL的MTT溶液，继续培养4h。培养完成后，往每孔加入150μL的二甲基亚砜，水平振荡10min，以使得MTT的反应物完全溶解。采用酶标仪测定490nm处的紫外吸收强度，并以未加细胞仅含培养基的空白孔校正测量值。细胞存活率(％)以试验组的测量值与对照组(未加金纳米簇)测量值的比值表示。
结果表明，氯金酸、葡萄糖酸锌、硝酸银、氯化亚铁或者氯化亚铁与氯金酸、氯化亚铁与葡萄糖酸锌的混合金属盐均表现出良好的生物相容性，细胞存活率均在90％以上。
实施例1
核酸功能化的金属纳米探针的制备方法，步骤如下：
(1)将DNA分子与稀释后的核酸插入剂SYBRGreenⅠ(2.5x)充分混合，置于室温下避光反应30min。
(2)将步骤(1)的反应产物与100μmol/L氯金酸溶液充分混合获得混合液A。
(3)将混合液A与大肠杆菌于恒温摇床继续共同孵育1-12h，得到混合液B。同时设置正常对照组(只添加有与实验组相同浓度的氯金酸溶液)和空白组(不做任何处理大肠杆菌悬浮液)。孵育完成后，取少量上述混合液滴加到载玻片上，用盖玻片封片。
(4)将载玻片置于激光共聚焦荧光显微镜下进行激发。进一步地，将上述混合液用808nm激光照射5-10min，于固体培养基上进行培养，计数。结果显示，实验组的大肠杆菌展现出强烈的荧光现象并且几乎全部被杀死，正常对照组荧光较弱并且大部分被杀死，而空白组无任何变化。
同时，以正常L02细胞作为实验对象，考察了混合液A对人正常细胞的作用。结果显示，与大肠杆菌实验组相比较，并未检测到荧光信号且细胞生存状态良好，表明金属纳米探针并不能在人体正常细胞内合成，具有较好的靶向特异性。
实施例2不同核酸片段对试验结果的影响
试验方法同实施例1，不同之处在于考察不同核酸片段对于结果的影响
所考察的核酸片段分别是：①DNA分子、②经过变性及梯度退火后形成的自组装RNA片段(将RNA分子于90℃变性1min后迅速置于冰上冷却。然后将其进行梯度退火处理，即70℃-50℃，50℃-37℃，37℃-4℃)。
结果如下：与DNA分子实验组相比较，添加有RNA片段的实验组荧光强度虽略有下降，但其信号也非常强烈并也表现出良好的抗菌效果。
可见：RNA片段与DNA分子一样，也可以用于金属纳米探针的合成，并展现出良好的检测及灭杀效果。
实施例3不同金属阳离子对试验结果的影响
试验方法同实施例1，不同之处在于考察不同金属阳离子对于结果的影响
所考察的金属阳离子分别是(浓度是100μmol/L)：①氯金酸、②葡萄糖酸锌、③硝酸银、④氯金酸与氯化亚铁的混合物、⑤葡萄糖酸锌与氯化亚铁的混合物。
结果如下：由图4、图5可知，不同的的金属阳离子溶液所表现出的现象虽略有不同，但都表现出强烈的信号强度，并展现出良好的抗菌效果。
可见：实验所用的金属阳离子具有良好的检测及杀菌效果。
实施例4不同金属阳离子浓度对试验结果的影响
试验方法同实施例1，不同之处在于考察金属阳离子的不同浓度对于结果的影响，所考察的金属阳离子(氯金酸)的浓度分别是：0μmol/L、10μmol/L、50μmol/L、100μmol/L、200μmol/L、300μmol/L。
结果如下：，随着金属阳离子(氯金酸)浓度的升高，信号强度增强，当浓度达到100μmol/L时，信号强度不再增加(见图2)。并且随着金属阳离子浓度的增加，杀菌效果逐步增强(见图3)。
可见：浓度对金属阳离子的作用有一定的影响，并且随着浓度的升高，检测及治疗的效果增强。
实施例5核酸功能化的金属纳米探针对不同致病菌种的影响
试验方法同实施例1，不同之处在于步骤(3)中混合物A与不同菌种共孵育，考察菌种是：①大肠杆菌、②金黄色葡萄球菌。
结果如下：①大肠杆菌、②金黄色葡萄球菌展现出强烈的荧光现象并且几乎全部被杀死。可见：本发明所得核酸功能化的金属纳米探针用于相关疾病如肺部感染、肠道感染以及流感等感染性疾病的靶向干预。
实施例6动物实验
1、样品制备：参照实施例1的制备方法，不同之处在于金属盐是萄糖酸锌溶液和氯化亚铁溶液的等浓度混合溶液共孵育。
2、实验过程：将共孵育所得样品从皮肤感染小鼠或肺炎感染小鼠的尾静脉或病灶部位原位注射，同时设置对照组(未添加金属阳离子的核酸片段)，采用小动物活体成像仪，将上述实验小鼠进行实时多模态(荧光、MRI、超声、CT等)原位成像观察，同时，利用光热等治疗手段对病变部位进行物理干预，进而记录病灶的变化和消融情况，与对照组相比，成像信号能快速识别实验组小鼠的病变部位并富集在该区域，经物理治疗后相关病症消失。
以上所述仅是本发明的优选实施方式，应理解这些实施例仅用于说明本发明而不用于限制本发明的范围，对于本领域的技术人员来说，在不脱离本发明原理的前提下，对本发明的各种修改和润饰均落于本申请所附权利要求所限定的范围内。

Claims (5)

  1. 核酸功能化的金属纳米探针，其特征是，将核酸分子与生物相容性良好的金属阳离子通过静电吸附作用形成基因-金属阳离子的结合物，与致病微生物共孵育原位自组装得到荧光纳米探针，粒径为1.7-2.6nm。
  2. 权利要求1所述的核酸功能化的金属纳米探针的制备方法，其特征是，步骤如下：
    步骤一、将核酸片段与核酸插入剂SYBR Green Ⅰ充分混合，置于室温下避光反应30min；
    步骤二、将金属可溶性盐溶液加入到步骤一的反应液中，充分混合后得到金属阳离子与核酸分子的混合液A；
    步骤三、将步骤二中获得的混合液A与致病微生物相混合，置于恒温摇床继续孵育0.5h-12h得到混合液B；
    步骤四、将混合液B离心提取出孵育后的致病微生物，用无菌水清洗孵育后的细胞3-5次；
    步骤五、取清洗孵育后的致病微生物细胞利用激光共聚焦荧光显微镜激发，进行荧光成像检测，然后在外界物理干预下作用于致病微生物。
  3. 根据权利要求1所述的核酸功能化的金属纳米探针的制备方法，其特征是，步骤一中，核酸片段包括DNA片段或RNA片段或通过化学方法合成的相关基因片段；RNA片段为经过变性及梯度退火后形成的自组装RNA片段。
  4. 根据权利要求1所述的核酸功能化的金属纳米探针的制备方法，其特征是，步骤二中，所述的金属可溶性盐为水溶性Mn 2+、氯金酸、氯化铜、氯化镁、葡萄糖酸锌、硝酸银或氯化亚铁中的一种或任意几种溶液的混合物；金属可溶性盐的浓度10μmol/L—300μmol/L。
  5. 根据权利要求1所述的核酸功能化的金属纳米探针的制备方法，其特征是，步骤五中，所述的外界物理干预是磁场或红外线加热，所述的致病微生物是大肠杆菌或者金黄色葡萄球菌。
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