WO2020168607A1 - 纳米复合物及其免标记适体电化学γ-干扰素传感器的制备方法 - Google Patents

纳米复合物及其免标记适体电化学γ-干扰素传感器的制备方法 Download PDF

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WO2020168607A1
WO2020168607A1 PCT/CN2019/078997 CN2019078997W WO2020168607A1 WO 2020168607 A1 WO2020168607 A1 WO 2020168607A1 CN 2019078997 W CN2019078997 W CN 2019078997W WO 2020168607 A1 WO2020168607 A1 WO 2020168607A1
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interferon
aptamer
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pamam
electrochemical
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金辉
高小惠
桂日军
王宗花
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青岛大学
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    • G—PHYSICS
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
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    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
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    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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    • G01N27/28—Electrolytic cell components
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    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
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    • C12N2310/351—Conjugate
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • C—CHEMISTRY; METALLURGY
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    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/555—Interferons [IFN]
    • G01N2333/57—IFN-gamma

Definitions

  • the invention belongs to the technical field of electrochemical biosensor and nanocomposite material preparation, and specifically relates to a method for preparing a dendrimer/gold nanoparticle/molybdenum disulfide nanocomposite and a label-free aptamer electrochemical sensor.
  • the sensor can be used for gamma -Highly sensitive and selective detection of interferon.
  • Gamma-interferon is a cell secretion factor, which is the only member of type II interferon, also known as macrophage activating factor.
  • the secretion of ⁇ -interferon is related to many diseases, such as inflammatory bowel disease, genital herpes simplex virus infection, Alzheimer's disease and so on.
  • Sensitive detection of ⁇ -interferon can be used to study the vitality of immune response and the diagnosis of infectious diseases.
  • Enzyme-linked immunosorbent assay is used to qualitatively or quantitatively detect ⁇ -interferon to determine whether the human body is infected with Mycobacterium tuberculosis.
  • Cytokines are usually detected by antibody immunoassay methods, which are time-consuming and require multiple washings and multi-step processing to obtain results. Due to complex cleaning and labeling, the detection of antibodies is difficult to complete the dynamic monitoring of cell secretions. In addition, the efficiency of the enzyme-catalyzed reaction will cause fluctuations in the output signal, which in turn leads to unsatisfactory reproducibility of the detection results and prolonged analysis time.
  • nucleic acid aptamers As an effective alternative to antibodies, nucleic acid aptamers have the advantages of thermal and chemical stability, reproducibility and easy modification, etc., which have attracted a wide range of research interests from scientific and technological workers.
  • a series of aptamer sensors have been developed based on the technology of nucleic acid aptamer-ligands that specifically bind to target molecules.
  • the distinguishing feature of this sensor is that oligonucleotides are designed as beacons, which directly generate signals when the target analyte binds to it. , Without marking and cleaning steps.
  • Aptamer biosensors have a simple detection process and show broad application prospects, especially suitable for real-time and dynamic detection of biological samples.
  • Nucleic acid aptamer electrochemical sensor is based on aptamer as a molecular recognition element. It is fixed to the signal converter by a specific method and connected to a device through an electronic wire; combined with electrochemical methods, it is used for qualitative and quantitative detection of the analyte .
  • electrochemical aptamer sensors Compared with traditional electrochemical analysis methods, electrochemical aptamer sensors have the advantages of high sensitivity, wide detection range, simple production, good selectivity, accuracy and reproducibility in the detection of biomolecules.
  • researchers have developed an immunoglobulin detection strategy based on nucleic acid aptamer-ligands. The redox probe (methylene blue or ferrocene) labeled ⁇ -interferon aptamer is assembled on the electrode through a series of electrode surface modification.
  • the electrochemical signal change of the measuring electrode is used to quantify the concentration of gamma-interferon.
  • Liu et al. prepared an aptamer functionalized electrode to detect cell secreted factors, including ⁇ - and ⁇ -interferon (Y. Liu, Y. Liu, Z. Matharu, A. Rahimian, A. Revzin, Detecting multiple cell-secreted cytokines from the same aptamer-functionalized electrode, Biosensors and Bioelectronics 2015, 64:43-50).
  • Abnous et al. developed an electrochemical aptamer sensor based on a triple helix molecular switch for the detection of gamma-interferon, and methylene blue was used as a redox probe (K.
  • Zhao Rui et al. disclosed a biosensor for detecting human beta interferon and a preparation method of its special polypeptide.
  • the identification element is a polypeptide or its derivative containing a specific amino acid sequence ( Zhao Rui, Luo Jia, Zhang Qundan, Liu Guoquan.
  • Chinese invention patent. Publication number CN101221185) Yang Zhanjun and others developed an impedance immunosensor based on zinc oxide nanomaterials for label-free electrochemical immunoassay of bovine gamma interferon (Yang Zhanjun, Qin Baiya, Chen Xiang.
  • Chinese invention patent. Publication number CN104090116A In recent years, research on label-free aptamer sensors has attracted widespread attention. Compared with the traditional aptamer-labeled sensor, the label-free aptamer sensor has the advantages of simple manufacture, label-free aptamer, and low cost. Based on this, the present invention designs a label-free aptamer electrochemical sensor based on the dendrimer/gold nanoparticle/molybdenum disulfide novel nanocomposite, which is used for the efficient detection of ⁇ -interferon.
  • the purpose of the present invention is to overcome the above-mentioned defects in the prior art, and design a dendritic macromolecule/gold nanoparticle/molybdenum disulfide nanocomposite based on the advantages of simple preparation method, low cost, high sensitivity and good selectivity.
  • the label-free aptamer electrochemical sensor can be used for the highly sensitive and selective detection of ⁇ -interferon.
  • the preparation process of a label-free aptamer electrochemical ⁇ -interferon sensor based on dendrimer/gold nanoparticles/molybdenum disulfide nanocomplexes includes the following steps:
  • MoS 2 molybdenum disulfide
  • the ultrasonic power described in step (1) is 150-200W, the frequency is 20-50kHz, the ultrasonic time is 5-10h, and the concentration of the homogeneous dispersion of MoS 2 is 1-2mg mL -1 ;
  • step (2) the concentration of chloroauric acid is 10-50mM, the mass concentration of PAMAM is 0.1-1%, the concentration of sodium borohydride is 0.5-1M, and the concentration of PAMAM/AuNPs dispersion is 1-10mg mL -1 ;
  • the ultrasonic treatment time in step (3) is 10-60 min, the stirring treatment time is 6-12h, and the mass concentration ratio of MoS 2 to PAMAM/AuNPs is (1:10)-(1:2);
  • the concentration of the aptamer described in step (4) is diluted to 1 ⁇ 5 ⁇ M; the heating temperature is 50 ⁇ 100°C; the heat treatment time is 1 ⁇ 6h;
  • the concentration of ⁇ -interferon in step (5) is 0 ⁇ 1000 pg mL -1 ; the detection limit of ⁇ -interferon is 1 ⁇ 3 fg mL -1 .
  • the effect of the present invention is: drop-coating PAMAM/AuNPs/MoS 2 composite on the surface of bare glassy carbon electrode to prepare modified electrode, ⁇ -interferon aptamer chain sulfhydryl terminal is connected to AuNPs surface through Au-S bond, and PAMAM/ AuNPs/MoS 2 -Modified electrode of aptamer.
  • MoS 2 was selected as the electrochemical reaction substrate, and AuNPs grown on PAMAM were used as the label-free aptamer chain binding sites to construct a label-free electrochemical aptamer sensor based on the nanocomposite.
  • ⁇ -interferon When ⁇ -interferon is present, ⁇ -interferon specifically binds to the aptamer chain on the sensor, resulting in the destruction of the aptamer hairpin structure, and the aptamer chain is opened and stretched, which can effectively adsorb MB in the electrolyte and cause MB redox signal Significantly enhanced. Fitting the linear relationship between MB oxidation peak current intensity and ⁇ -interferon concentration, and constructing a label-free aptamer electrochemical sensor for ⁇ -interferon detection.
  • the method of the present invention has simple operation, low cost, high sensitivity and good selectivity, and can be developed into a novel label-free aptamer electrochemical sensor for the high sensitivity and sensitivity of interferon- ⁇ in biological samples. Highly selective detection.
  • Fig. 1 is a schematic diagram of the preparation process of the dendrimer/gold nanoparticle/molybdenum disulfide nanocomposite and the label-free aptamer electrochemical sensor according to the present invention and the principle diagram of the detection of ⁇ -interferon;
  • Figure 2 (a) shows the electrochemical square wave voltammetry curve corresponding to the presence of different ⁇ -interferon concentrations using the label-free aptamer electrochemical sensor of the present invention
  • Figure 2(b) shows the oxidation current peak intensity of methylene blue in the presence of different ⁇ -interferon concentrations, fitting the linear relationship between the different current peak intensities and the ⁇ -interferon concentration.
  • the invention relates to the preparation of an electrochemical sensor based on dendrimer/gold nanoparticle/molybdenum disulfide nanocomposite and its label-free aptamer and the electrochemical detection of ⁇ -interferon.
  • the preparation process and principle schematic diagram are as follows As shown in Figure 1, the specific process steps are:
  • the PAMAM/AuNPs dispersion was added dropwise to the MoS 2 dispersion, the mass ratio of the two was 1:2, and the PAMAM/AuNPs/MoS 2 nanocomposite was prepared by the water bath ultrasonic treatment for 30 minutes, and then the magnetic stirring treatment for 12 hours.
  • the crosslinking agent Nafion was dropped on the polished and polished surface of the bare glassy carbon electrode, and the nanocomposite dispersion was dropped to prepare a PAMAM/AuNPs/MoS 2 modified electrode.
  • the aptamer ⁇ -interferon nucleic acid is diluted to 5 ⁇ M with buffer, then heated to 90°C, maintained at the heat treatment for 5h and cooled to room temperature.
  • the aptamer forms a secondary hairpin structure, and the sulfhydryl group at the end of the aptamer chain is connected to AuNPs through Au-S bonds. surface.
  • the preparation of electrochemical sensor based on dendrimer/gold nanoparticle/molybdenum disulfide nanocomposite and label-free aptamer and the electrochemical detection of ⁇ -interferon the preparation process and schematic diagram of the principle are the same as the embodiment 1.
  • the specific process steps are:
  • the PAMAM/AuNPs dispersion was added dropwise to the MoS 2 dispersion, the mass ratio of the two was 1:4, and the PAMAM/AuNPs/MoS 2 nanocomposite was prepared by the water bath ultrasonic treatment for 20 minutes and then the magnetic stirring treatment for 6 hours.
  • the crosslinking agent Nafion was dropped on the polished and polished surface of the bare glassy carbon electrode, and the nanocomposite dispersion was dropped to prepare a PAMAM/AuNPs/MoS 2 modified electrode.
  • the aptamer ⁇ -interferon nucleic acid was diluted with buffer to 2 ⁇ M, then heated to 80°C, maintained heat treatment for 3h and then cooled to room temperature.
  • the aptamer formed a secondary hairpin structure, and the sulfhydryl group at the end of the aptamer chain was connected to AuNPs through Au-S bonds. surface.
  • the preparation of electrochemical sensor based on dendrimer/gold nanoparticle/molybdenum disulfide nanocomposite and label-free aptamer and the electrochemical detection of ⁇ -interferon the preparation process and schematic diagram of the principle are the same as the embodiment 1.
  • the specific process steps are:
  • the PAMAM/AuNPs dispersion was added dropwise to the MoS 2 dispersion, the mass ratio of the two was 1:5, and the PAMAM/AuNPs/MoS 2 nanocomposite was prepared by the water bath ultrasonic treatment for 60 min and then the magnetic stirring treatment for 12 h.
  • the crosslinking agent Nafion was dropped on the polished and polished surface of the bare glassy carbon electrode, and the nanocomposite dispersion was dropped to prepare a PAMAM/AuNPs/MoS 2 modified electrode.
  • the aptamer ⁇ -interferon nucleic acid was diluted with buffer to 5 ⁇ M, then heated to 90°C, maintained at the heat treatment for 6h and then cooled to room temperature.
  • the aptamer formed a secondary hairpin structure, and the sulfhydryl group at the end of the aptamer chain was connected to AuNPs through Au-S bonds. surface.

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Abstract

一种基于树状大分子/金纳米粒/二硫化钼纳米复合物的免标记适体电化学γ-干扰素传感器的制备方法。滴涂复合物于电极表面制备改性电极，γ-干扰素适体链终端巯基通过Au-S键与金纳米粒连接，制得复合物-适体的改性电极。当γ-干扰素存在时，γ-干扰素与传感器上适体链特异性结合，导致适体发卡结构被打开伸展，能够有效吸附电解液中的亚甲基蓝MB，引起MB氧化还原信号显著增强。拟合MB氧化峰电流强度与γ-干扰素浓度间的线性关系，构建免标记适体电化学γ-干扰素传感器。这种方法操作简单、成本低廉、灵敏度高、选择性好，将成为一种新的免标记适体电化学传感器，用于生物样品中γ-干扰素的高灵敏和高选择性检测。

Description

纳米复合物及其免标记适体电化学γ-干扰素传感器的制备方法 技术领域：
本发明属于电化学生物传感器和纳米复合材料制备技术领域，具体涉及树状大分子/金纳米粒/二硫化钼纳米复合物及其免标记适体电化学传感器的制备方法，该传感器可用于γ-干扰素的高灵敏和高选择性检测。
背景技术：
γ-干扰素是一种细胞分泌因子，属于II型干扰素的唯一成员，也被称为巨噬细胞激活因子。γ-干扰素的分泌失调与多种疾病相关，如炎症性肠病、生殖器单纯疱疹病毒感染、阿尔茨海默病等。对γ-干扰素的敏感检测可用于研究免疫反应的活力和传染病的诊断。采用酶联免疫吸附实验定性或定量检测γ-干扰素，可判断人体是否感染结核分枝杆菌。细胞因子通常采用抗体免疫分析法检测，这些分析方法耗时较长，需要多次清洗和多步处理来获得结果。由于复杂的清洗和标记，抗体的检测难以完成对细胞分泌物的动态监测。此外，酶催化反应的效率会引起输出信号的波动，进而导致检测结果的重现性不理想和分析时间延长。
作为抗体的有效替代物，核酸适-配体具有热及化学稳定性，可再生性和易于修饰等优点，吸引了科技工作者广泛的研究兴趣。当前，基于核酸适-配体特异性结合靶分子的技术发展了一系列适体传感器，该传感器显著的特征是将寡核苷酸设计成信标，当目标分析物与之结合后直接产生信号，无需标记和清洗步骤。适配异构体生物传感器具备简易的检测过程，展现出广阔的应用前景，尤其适用于对生物样品的实时和动态检测。
核酸适体电化学传感器是以适体作为分子识别原件.采用特定方法将其固定到信号转换器上，通过电子导线连接成装置；再结合电化学方法，用于待测物的定性和定量检测。相比传统电化学分析方法，电化学适体传感器在生物分子检测方面具备灵敏度高，检测范围宽，制作简便，良好的选择性，准确性和重现性等优点。研究者开发了基于核酸适-配体的免疫球蛋白检测策略，将氧化还原探针(亚甲基蓝或二茂铁)标记的γ-干扰素适体，通过一系列电极表面改性将其组装在电极上，测定 电极的电化学信号变化用于定量γ-干扰素的浓度。Liu等制备了适体功能化电极用于检测细胞分泌因子，包括γ-和α-干扰素(Y.Liu,Y.Liu,Z.Matharu,A.Rahimian,A.Revzin,Detecting multiple cell-secreted cytokines from the same aptamer-functionalized electrode,Biosensors andBioelectronics 2015,64:43–50)。Abnous等开发了基于三螺旋分子开关的电化学适体传感器用于γ-干扰素的检测，亚甲基蓝作为氧化还原探针(K.Abnous,N.M.Danesh,M.Ramezani,M.Alibolandi,K.Y.Hassanabad,A.S.Emrani,A.Bahreyni,S.M.Taghdisi,A triple-helix molecular switch-based electrochemical aptasensor for interferon-gamma using a gold electrode and Methylene Blue as a redox probe,MicrochimicaActa 2017,184:4151–4157)。
有关干扰素检测的传感器已有相关专利报道，例如赵睿等公开了一种检测人β干扰素的生物传感器及其专用多肽的制备方法，识别元件为含有特定氨基酸序列的多肽或其衍生物(赵睿,罗佳,章群丹,刘国诠.一种检测人β干扰素的生物传感器及其专用多肽.中国发明专利.公开号CN101221185)。杨占军等基于氧化锌纳米材料开发了阻抗型免疫传感器，用于牛伽马干扰素的无标记电化学免疫分析(杨占军,秦坯芽,陈祥.基于氧化锌纳米材料的牛伽马干扰素阻抗型免疫传感器的制备方法.中国发明专利.公开号CN104090116A)。近年来，免标记适体传感器相关研究引起了广泛的关注。相比传统适体标记型传感器，免标记适体传感器具备制作简便，适体免标记，成本低等优点。基于此，本发明设计了基于树状大分子/金纳米粒/二硫化钼新型纳米复合物的免标记适体电化学传感器，用于γ-干扰素的高效检测。目前，尚未有树状大分子/金纳米粒/二硫化钼纳米复合物，以及基于纳米复合物的免标记适体电化学γ-干扰素传感器的国内外文献和专利报道。
发明内容：
本发明的目的在于克服上述现有技术存在的缺陷，设计一种制备方法简单、成本低廉、灵敏度高、选择性好等优点的基于树状大分子/金纳米粒/二硫化钼纳米复合物的免标记适体电化学传感器，其制备的传感器可用于γ-干扰素的高灵敏和高选择性检测。
为了实现上述目的，本发明涉及的一种基于树状大分子/金纳米粒/二硫化钼纳 米复合物的免标记适体电化学γ-干扰素传感器的制备工艺包括以下步骤：
1.纳米复合物及其免标记适体电化学γ-干扰素传感器的制备方法，其特征在于，该方法具体包括以下步骤：
(1)将二硫化钼(MoS 2)粉末加入乙醇和蒸馏水的混合溶剂中，在水浴中超声处理，然后离心分离，去除沉淀物，制得MoS 2均质分散液备用；
(2)配制氯金酸水溶液，加入聚乙二胺树状大分子(PAMAM)水溶液中，磁力搅拌混合均匀，滴加硼氢化钠水溶液，制备金纳米粒负载的树状大分子(PAMAM/AuNPs)，通过透析处理，除去残留反应物；
(3)向MoS 2分散液中滴加PAMAM/AuNPs分散液，先水浴超声处理，再磁力搅拌处理，反应制得PAMAM/AuNPs/MoS 2纳米复合物；
(4)在抛光打磨处理的裸玻碳电极表面滴加交联剂Nafion，滴涂纳米复合物分散液，制得PAMAM/AuNPs/MoS 2改性的电极；γ-干扰素核酸适体用缓冲液稀释，加热至一定温度后冷却至室温，适体形成二级发卡结构，适体链巯基终端通过Au-S键连接至AuNPs表面；
(5)在浸没了PAMAM/AuNPs/MoS 2-适体改性电极的电解液中，加入氧化还原探针亚甲基蓝MB，随着靶分子γ-干扰素加入量的增大，MB的电化学信号峰逐渐增强，拟合MB氧化峰电流强度与γ-干扰素浓度之间的线性关系，构建用于γ-干扰素检测的免标记适体电化学传感器。
步骤(1)中所述的超声功率为150～200W，频率为20～50kHz，超声时间为5～10h,MoS 2均质分散液浓度为1～2mg mL -1；
步骤(2)中所述的氯金酸浓度为10～50mM,PAMAM质量浓度为0.1～1％，硼氢化钠浓度为0.5～1M，PAMAM/AuNPs分散液浓度为1～10mg mL -1；
步骤(3)中所述的超声处理时间为10～60min，搅拌处理时间为6～12h，MoS 2与PAMAM/AuNPs质量浓度比为(1:10)～(1:2)；
步骤(4)中所述的适体浓度稀释至1～5μM；加热温度为50～100℃；热处理时间为1～6h；
步骤(5)中所述的γ-干扰素浓度为0～1000pg mL -1；γ-干扰素的检测限为1～3fg mL -1。
本发明的效果是：滴涂PAMAM/AuNPs/MoS 2复合物于裸玻碳电极表面制备改性电极，γ-干扰素适体链巯基终端通过Au-S键连接至AuNPs表面，制得PAMAM/AuNPs/MoS 2-适体的改性电极。选用MoS 2为电化学反应基底，以PAMAM上生长的AuNPs作为免标记适体链结合位点，构建基于该纳米复合物的免标记电化学适体传感器。当γ-干扰素存在时，γ-干扰素与传感器上适体链特异性结合，导致适体发卡结构破坏，适体链被打开伸展，可有效吸附电解液中的MB，引起MB氧化还原信号显著增强。拟合MB氧化峰电流强度与γ-干扰素浓度之间的线性关系，构建用于γ-干扰素检测的免标记适体电化学传感器。与现有技术相比，本发明方法操作简单、成本低廉、灵敏度高、选择性好，可发展成为一种新颖的免标记适体电化学传感器用于生物样品中γ-干扰素的高灵敏和高选择性检测。
附图说明：
图1为本发明涉及的树状大分子/金纳米粒/二硫化钼纳米复合物及其免标记适体电化学传感器的制备过程与γ-干扰素检测的原理示意图；
图2(a)为使用本发明的免标记适体电化学传感器测定不同γ-干扰素浓度存在下对应的电化学方波伏安曲线；
图2(b)为不同γ-干扰素浓度存在下，对应亚甲基蓝的氧化电流峰强度，拟合不同电流峰强度与γ-干扰素浓度之间的线性关系。
具体实施方式：
下面结合附图并通过具体实施例对本发明进行详细说明。
实施例1：
本发明涉及的一种基于树状大分子/金纳米粒/二硫化钼纳米复合物及其免标记适体电化学传感器的制备与γ-干扰素的电化学检测，其制备工艺与原理示意图如图1所示，具体工艺步骤为：
将30毫克MoS 2粉末加入乙醇/蒸馏水(体积比1:1)的混合溶剂中，在水浴中超声处理8h(180W,40kHz)，然后在3000转/分钟转速下离心20min，去除沉淀物，制得MoS 2均质分散液(1.5mg mL -1)备用。配制氯金酸水溶液25mM，取10mL加入到聚 乙二胺树状大分子(PAMAM,1wt％,15mL)水溶液，磁力搅拌1h混合均匀，再滴加硼氢化钠水溶液(0.5M)，直到溶液从淡黄色变成红棕色，产物经透析除去残留反应物，制得金纳米粒负载的树状大分子(PAMAM/AuNPs,1mg mL -1)备用。向MoS 2分散液中滴加PAMAM/AuNPs分散液，二者质量比为1:2，先水浴超声处理30min，再磁力搅拌处理12h，反应制得PAMAM/AuNPs/MoS 2纳米复合物。
在抛光打磨处理的裸玻碳电极表面滴加交联剂Nafion，滴涂纳米复合物分散液，制得PAMAM/AuNPs/MoS 2改性的电极。γ-干扰素核酸适体用缓冲液稀释至5μM，然后加热至90℃，维持热处理5h后冷却至室温，适体形成二级发卡结构，适体链终端的巯基通过Au-S键连接至AuNPs表面。在浸没了PAMAM/AuNPs/MoS 2-适体改性电极的电解液中，含有10mM缓冲液Tris-HCl，加入氧化还原探针亚甲基蓝MB(1mM)，随着靶分子γ-干扰素加入量的增大，MB电化学信号峰逐渐增强，拟合MB氧化峰电流强度与γ-干扰素浓度之间的线性关系，构建用于γ-干扰素检测的免标记适体电化学传感器。如图2所示，γ-干扰素浓度的线性检测范围为0.01～1000ng mL -1，检测限为3fg mL -1。
实施例2：
本实施例中基于树状大分子/金纳米粒/二硫化钼纳米复合物及其免标记适体电化学传感器的制备与γ-干扰素的电化学检测，其制备工艺与原理示意图同实施例1，具体工艺步骤为：
将30毫克MoS 2粉末加入乙醇/蒸馏水(体积比1:1)的混合溶剂中，在水浴中超声处理6h(150W,30kHz)，然后在3000转/分钟转速下离心20min，去除沉淀物，制得MoS 2均质分散液(1mg mL -1)备用。配制氯金酸水溶液15mM，取10mL加入到聚乙二胺树状大分子(PAMAM,0.5wt％,15mL)水溶液，磁力搅拌1h混合均匀，再滴加硼氢化钠水溶液(0.5M)，直到溶液从淡黄色变成红棕色，产物经透析除去残留反应物，制得金纳米粒负载的树状大分子(PAMAM/AuNPs,2mg mL -1)备用。向MoS 2分散液中滴加PAMAM/AuNPs分散液，二者质量比为1:4，先水浴超声处理20min，再磁力搅拌处理6h，反应制得PAMAM/AuNPs/MoS 2纳米复合物。
在抛光打磨处理的裸玻碳电极表面滴加交联剂Nafion，滴涂纳米复合物分散液，制得PAMAM/AuNPs/MoS 2改性的电极。γ-干扰素核酸适体用缓冲液稀释至2μM， 然后加热至80℃，维持热处理3h后冷却至室温，适体形成二级发卡结构，适体链终端的巯基通过Au-S键连接至AuNPs表面。在浸没了PAMAM/AuNPs/MoS 2-适体改性电极的电解液中，含有10mM缓冲液Tris-HCl，加入氧化还原探针亚甲基蓝MB(1mM)，随着靶分子γ-干扰素加入量的增大，MB电化学信号峰逐渐增强，拟合MB氧化峰电流强度与γ-干扰素浓度之间的线性关系，构建用于γ-干扰素检测的免标记适体电化学传感器。如图2所示，γ-干扰素浓度的线性检测范围为0.01～500ng mL -1，检测限为2.5fg mL -1。
实施例3：
本实施例中基于树状大分子/金纳米粒/二硫化钼纳米复合物及其免标记适体电化学传感器的制备与γ-干扰素的电化学检测，其制备工艺与原理示意图同实施例1，具体工艺步骤为：
将30毫克MoS 2粉末加入乙醇/蒸馏水(体积比1:1)的混合溶剂中，在水浴中超声处理10h(200W,50kHz)，然后在3000转/分钟转速下离心20min，去除沉淀物，制得MoS 2均质分散液(2mg mL -1)备用。配制氯金酸水溶液50mM，取10mL加入到聚乙二胺树状大分子(PAMAM,1wt％,15mL)水溶液，磁力搅拌1h混合均匀，再滴加硼氢化钠水溶液(1M)，直到溶液从淡黄色变成红棕色，产物经透析除去残留反应物，制得金纳米粒负载的树状大分子(PAMAM/AuNPs,10mg mL -1)备用。向MoS 2分散液中滴加PAMAM/AuNPs分散液，二者质量比为1:5，先水浴超声处理60min，再磁力搅拌处理12h，反应制得PAMAM/AuNPs/MoS 2纳米复合物。
在抛光打磨处理的裸玻碳电极表面滴加交联剂Nafion，滴涂纳米复合物分散液，制得PAMAM/AuNPs/MoS 2改性的电极。γ-干扰素核酸适体用缓冲液稀释至5μM，然后加热至90℃，维持热处理6h后冷却至室温，适体形成二级发卡结构，适体链终端的巯基通过Au-S键连接至AuNPs表面。在浸没了PAMAM/AuNPs/MoS 2-适体改性电极的电解液中，含有10mM缓冲液Tris-HCl，加入氧化还原探针亚甲基蓝MB(1mM)，随着靶分子γ-干扰素加入量的增大，MB电化学信号峰逐渐增强，拟合MB氧化峰电流强度与γ-干扰素浓度之间的线性关系，构建用于γ-干扰素检测的免标记适体电化学传感器。如图2所示，γ-干扰素浓度的线性检测范围为0.01～800ng mL -1，检测限为2fg mL -1。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (6)

  1. 纳米复合物及其免标记适体电化学γ-干扰素传感器的制备方法，其特征在于，该方法具体包括以下步骤：
    (1)将二硫化钼(MoS 2)粉末加入乙醇和蒸馏水的混合溶剂中，在水浴中超声处理，然后离心分离，去除沉淀物，制得MoS 2均质分散液备用；
    (2)配制氯金酸水溶液，加入聚乙二胺树状大分子(PAMAM)水溶液中，磁力搅拌混合均匀，滴加硼氢化钠水溶液，制备金纳米粒负载的树状大分子(PAMAM/AuNPs)，通过透析处理，除去残留反应物；
    (3)向MoS 2分散液中滴加PAMAM/AuNPs分散液，先水浴超声处理，再磁力搅拌处理，反应制得PAMAM/AuNPs/MoS 2纳米复合物；
    (4)在抛光打磨处理的裸玻碳电极表面滴加交联剂Nafion，滴涂纳米复合物分散液，制得PAMAM/AuNPs/MoS 2改性的电极；γ-干扰素核酸适体用缓冲液稀释，加热至一定温度后冷却至室温，适体形成二级发卡结构，适体链巯基终端通过Au-S键连接至AuNPs表面；
    (5)在浸没了PAMAM/AuNPs/MoS 2-适体改性电极的电解液中，加入氧化还原探针亚甲基蓝MB，随着靶分子γ-干扰素加入量的增大，MB的电化学信号峰逐渐增强，拟合MB氧化峰电流强度与γ-干扰素浓度之间的线性关系，构建用于γ-干扰素检测的免标记适体电化学传感器。
  2. 根据权利要求1所述的纳米复合物及其免标记适体电化学γ-干扰素传感器的制备方法，其特征在于，步骤(1)中所述的超声功率为150～200W，频率为20～50kHz，超声时间为5～10h，MoS 2均质分散液浓度为1～2mg mL -1；
  3. 根据权利要求1所述的纳米复合物及其免标记适体电化学γ-干扰素传感器的制备方法，其特征在于，步骤(2)中所述的氯金酸浓度为10～50mM，PAMAM质量浓度为0.1～1％，硼氢化钠浓度为0.5～1M，PAMAM/AuNPs分散液浓度为1～10mg mL -1；
  4. 根据权利要求1所述的纳米复合物及其免标记适体电化学γ-干扰素传感器的制备方法，其特征在于，步骤(3)中所述的超声处理时间为10～60min，搅拌处理时间为6～12h，MoS 2与PAMAM/AuNPs质量浓度比为(1:10)～(1:2)；
  5. 根据权利要求1所述的纳米复合物及其免标记适体电化学γ-干扰素传感器的制备方法，其特征在于，步骤(4)中所述的适体浓度稀释至1～5μM；加热温度为50～100℃；热处理时间为1～6h；
  6. 根据权利要求1所述的纳米复合物及其免标记适体电化学γ-干扰素传感器的制备方法，其特征在于，步骤(5)中所述的γ-干扰素浓度为0～1000pg mL -1；γ-干扰素的检测限为1～3fg mL -1。
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