WO2020114124A1 - 一种基于金纳米颗粒和二碳化钛MXenes的双重催化鲁米诺电化学发光生物传感器 - Google Patents

一种基于金纳米颗粒和二碳化钛MXenes的双重催化鲁米诺电化学发光生物传感器 Download PDF

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WO2020114124A1
WO2020114124A1 PCT/CN2019/112594 CN2019112594W WO2020114124A1 WO 2020114124 A1 WO2020114124 A1 WO 2020114124A1 CN 2019112594 W CN2019112594 W CN 2019112594W WO 2020114124 A1 WO2020114124 A1 WO 2020114124A1
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mxenes
biosensor
aptamer
electrode
ecl
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PCT/CN2019/112594
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French (fr)
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王宗花
张慧欣
刘洋
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青岛大学
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    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals

Definitions

  • the present disclosure relates to a two-dimensional nanomaterial-Ti 3 C 2 MXenes using its reducibility to reduce chloroauric acid (HAuCl 4 ) to form a gold nanoparticle-MXenes (AuNPs-MXenes) complex.
  • the AuNPs-MXenes complex can double catalyze Luminol's electrochemiluminescence (ECL) and the use of the multi-site effect of sodium alginate (SA) to construct an ECL biosensor to detect exosomes.
  • Exosomes are vesicles with a double membrane structure with a diameter of 30-100 nm, which can be secreted into the microenvironment by various cells through exocytosis. Exosomes are present in most body fluids, including blood, urine, saliva, and breast milk. They are secreted by most mammalian cells and carry genetic material, including mRNA, carbohydrates, proteins (CD63, CD81, CD9, EpCAM, etc.) and DNA, which can promote cell-cell communication. According to reports, exosomes play an important role in anti-tumor immune response, tumor diagnosis and other processes, and are promising biomarkers for early cancer diagnosis. Therefore, the highly sensitive method for exosome detection is not only valuable for clinical diagnosis, but also provides insights into the basic biochemical process of tumor growth and metastasis, and also helps to further study the relevant mechanisms of anti-tumor immune response .
  • ECL as a powerful analytical technique, has been widely used in the detection of proteins, DNA, enzymes and other substances due to its high sensitivity, rapidity, easy control and low cost. Therefore, based on these advantages of ECL, It can be expected to be used for exosome detection and analysis of exosome activity.
  • the present disclosure relates to a dual-catalyzed luminol-based ECL biosensor and application based on titanium carbide-MXenes and AuNPs.
  • the present disclosure relates to technical solutions in the following aspects:
  • a two-dimensional transition metal carbide-nucleic acid aptamer (MXenes-Aptamer) probe is provided, which is characterized in that the probe includes nanosheets Ti 3 C 2 MXenes ⁇ Amino acids and CD63 protein nucleic acid aptamers modified with carboxyl groups, the nanosheets Ti 3 C 2 MXenes are connected to amino acids through Ti-N bonds, and amino acids are connected to nucleic acid aptamers through amide bonds.
  • MXenes-Aptamer transition metal carbide-nucleic acid aptamer
  • a method for preparing the MXenes-Aptamer probe includes: mixing nanosheets Ti 3 C 2 MXenes and amino acids in water, mixing, stirring, and separating to obtain For the precipitate, the MXenes-Aptamer probe can be obtained by performing an amide reaction with the aptamer.
  • a biosensor electrode used in conjunction with the probe including: a matrix electrode modified with polyacrylamide (PAM) on its surface, sodium alginate (SA) and Amino acid aptamer of CD63 protein modified with amino groups;
  • PAM polyacrylamide
  • SA sodium alginate
  • Amino acid aptamer of CD63 protein modified with amino groups including:
  • the substrate electrode modified with PAM on the surface and the amino acid-modified CD63 protein nucleic acid aptamer are both connected to SA through an amide reaction.
  • a method for preparing the biosensor electrode includes: dropping a PAM solution onto a base electrode, drying; and then immersing the base electrode in 1-(3-bi Incubate in a mixed solution of methylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and SA; then immerse the substrate electrode in the nucleic acid aptamer solution Conduct incubation in order to obtain biosensor electrodes.
  • EDC methylaminopropyl-3-ethylcarbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • SA N-hydroxysuccinimide
  • an ECL biosensor in a fifth typical embodiment of the present disclosure, is provided, the biosensor includes the MXenes-Aptamer probe and the biosensor electrode, and when exosomes are present, the MXenes-Aptamer The probe, the biosensor electrode and the exosome form a sandwich structure (MXenes-Aptamer probe-exosome-biosensor electrode), and the sandwich structure is immersed in a solution of chloroauric acid (HAuCl 4 ) in situ The AuNPs-MXenes-Aptamer probe-exosome-biosensor electrode is formed, which is the ECL biosensor.
  • HUACl 4 chloroauric acid
  • an ECL kit which includes the MXenes-Aptamer probe, the biosensor electrode, luminol, and HAuCl 4 solution.
  • the application of the MXenes-Aptamer probe, the biosensor electrode or the ECL biosensor or the kit in the detection of exosomes using the ECL method is provided .
  • a method for detecting exosomes for non-diagnostic purposes includes using the MXenes-Aptamer probe, the biosensor electrode or the ECL biological The step of detecting exosomes by the sensor or the kit using the ECL method.
  • This application combines a multi-site identification strategy and the reducibility of the new two-dimensional nanomaterial-Ti 3 C 2 MXenes to form AuNPs–MXenes-Aptamer as ECL signal probes, designing a highly sensitive ECL sensor platform to detect Hela exosomes.
  • SA polymer molecules can provide a multi-site recognition interface to capture more aptamers to increase the efficiency of exosome capture, which helps to improve the sensitivity of biosensors, and AuNPs-MXenes-Aptamer can double Catalyze Luminol to enhance the ECL signal. Based on this double amplification strategy, highly sensitive and selective biosensors have been obtained to detect exosomes.
  • ECL biosensors can also be used to detect exosomes in serum. The sensor can be used as a viable tool for detecting exosomes, and provides strong evidence for the clinical diagnosis of exosomes.
  • FIG. 1 is a schematic diagram of the assembly and mechanism of the disclosed ECL sensor.
  • Fig. 2 is an SEM image of Ti 3 AlC 2 .
  • Figure 3 is a TEM image of MXenes.
  • Fig. 4 is an XRD diagram before and after the peeling of MXenes.
  • Figure 5 is the signal response of ECL sensors with different concentrations of exosomes; ah stands for: 10 2 , 5 ⁇ 10 2 , 10 3 , 2.5 ⁇ 10 3 , 5 ⁇ 10 3 , 10 4 , 5 ⁇ 10 4 , 10 5 particles / ⁇ L.
  • an MXenes-Aptamer probe is provided, The probe is characterized in that the probe includes a nanosheet Ti 3 C 2 MXenes, an amino acid and a carboxyl-modified CD63 protein nucleic acid aptamer, the nanosheet Ti 3 C 2 MXenes is connected to an amino acid through a Ti-N bond, and the amino acid is through an amide bond Connected to nucleic acid aptamers.
  • the nucleic acid aptamer specifically binds to the CD63 protein on the surface of exosomes.
  • the nucleic acid aptamer of the CD63 protein includes, but is not limited to the following sequence: 5'--TTTTTTCACCCC ACCTCGCCTCCCG TGACACTAATATGCTA, such as SEQ ID NO. 1 shown.
  • the design of the probe can retain the excellent properties of MXenes itself: large specific surface area, better conductivity, high catalytic properties and super reducibility. It can also have amino groups on the surface of MXenes to facilitate modification on the electrode . This is the use of the interaction between amino acids and MXenes to form a Ti-N bond, so that the surface of MXenes is modified with amino groups. Compared with the previous study through electrostatic adsorption, the current modification method is more stable than electrostatic adsorption, which can make the surface of MXenes carry amino groups for the next modification.
  • the amino acid is glycine or leucine, and it has been experimentally verified that glycine or leucine can better bind Ti 3 C 2 MXenes.
  • a method for preparing the MXenes-Aptamer probe includes: mixing nanosheets Ti 3 C 2 MXenes and amino acids in water, mixing, stirring, and separating to obtain For the precipitate, the MXenes-Aptamer probe can be obtained by performing an amide reaction with the aptamer.
  • the feeding ratio of Ti 3 C 2 MXenes, amino acids and water is (0.5 to 1) mg: (4 to 6) mg: (10 to 40) mL; stirring at room temperature for 18 to 36 hours.
  • the temperature of the amide reaction is 35-40°C, and the time is 0.5-1.5h.
  • Ti 3 C 2 MXenes has good electron transfer ability, excellent catalytic ability, good biocompatibility and super reducibility.
  • the large specific surface area of Ti 3 C 2 MXenes also makes it a good carrier for loading more biomolecules, thereby significantly improving the sensitivity of biosensors.
  • the preparation method of the nanosheet Ti 3 C 2 MXenes includes the following steps: adding LiF to hydrochloric acid with a concentration of 8 to 10 mol/L, stirring, and then adding Ti 3 AlC 2 powder to this mixture and adding 30 Stir at ⁇ 40°C for 18 ⁇ 36h; then centrifuge and wash the solution to make pH ⁇ 6, discard the supernatant, add water to the precipitate, centrifuge, discard the supernatant, continue to add water to the precipitate Under protection, sonicate for 0.5 to 1.5 hours. Finally, centrifuge the solution to retain the supernatant, which is the nano-sheet Ti 3 C 2 MXenes dispersion.
  • a biosensor electrode used in conjunction with the probe including: a substrate electrode modified with PAM on the surface, an SA and an amino acid-modified CD63 protein nucleic acid aptamer ;
  • the substrate electrode modified with PAM on the surface and the nucleic acid aptamer are both connected to SA through an amide reaction.
  • the base electrode is a glassy carbon electrode.
  • Sodium alginate (C 6 H 7 O 6 Na) n is mainly composed of the sodium salt of alginic acid, which is dependent on ⁇ -D-mannuronic acid (M unit) and ⁇ -L-guluronic acid (G unit) ⁇ -1,4-glycosidically linked copolymer composed of different proportions of GM, MM and GG fragments, the surface contains many carboxyl groups, can provide more recognition sites, and its good hydrophilicity and low adhesion, The performance is excellent and it is biocompatible, so the present disclosure can significantly improve the sensitivity and selectivity of the biosensor by utilizing its excellent properties.
  • Mw>Mn Mw>Mn.
  • the coefficient of Mw/Mn is the dispersibility index, and the classic range of the index of sodium alginate products is 1.5 to 2.5.
  • a method for preparing the biosensor electrode includes: dropping a PAM solution onto a substrate electrode, incubating to be dried; and then immersing the substrate electrode in EDC, NHS and Incubate in the SA mixed solution; then incubate the substrate electrode into the nucleic acid aptamer solution to obtain the biosensor electrode.
  • the volume ratio of EDC, NHS and SA is 0.8-1.2: 0.8-1.2: 1.8-2.2
  • the concentration of EDC is 80-120mM
  • the concentration of NHS is 200-400mM
  • the concentration of SA is 5-15mg/ mL; incubate at 35 ⁇ 40°C for 1 ⁇ 3h.
  • the substrate electrode is then put into a nucleic acid aptamer solution for incubation, and the incubation conditions are: 35 to 40°C for 1 to 3 hours.
  • the weight average molecular weight of the polyacrylamide is: MW ⁇ 3 million.
  • an ECL biosensor in a fifth typical embodiment of the present disclosure, includes the MXenes-Aptamer probe and the biosensor electrode, and when exosomes are present, the MXenes-Aptamer The probe, the biosensor electrode and the exosome form a sandwich structure (MXenes-Aptamer probe-exosome-biosensor electrode), and the sandwich structure is immersed in HAuCl 4 solution to form AuNPs-MXenes- in situ Aptamer probe-exosome-biosensor electrode is ECL biosensor.
  • an ECL kit is provided, the kit at least including the MXenes-Aptamer probe, the biosensor electrode, HAuCl 4 solution, and luminol.
  • the application of the MXenes-Aptamer probe, the biosensor electrode or the ECL biosensor or the kit in detecting exosomes using ECL is provided.
  • a method for detecting exosomes for non-diagnostic purposes includes using the MXenes-Aptamer probe, the biosensor electrode or the ECL biological The step of detecting exosomes by the sensor or the kit using the ECL method.
  • the detection method using the kit includes: immersing the biosensor electrode in the solution of the exosome to be tested, attaching the exosome to the biosensor electrode, and then immersing the biosensor electrode attached to the exosome In the MXenes-Aptamer probe solution, the probe is attached to the exosome of the biosensor electrode, thereby forming a biosensor that carries the exosome with the probe and the biosensor electrode, and then the biosensor is immersed in the HAuCl 4 solution In the process, AuNPs are generated on the surface of the electrode complex, and then prepared as a probe with AuNPs attached to the surface and an ECL biosensor with biosensor electrodes carrying exosomes, and the ECL biosensor is tested in Lumino solution. .
  • This detection method is used to detect exosomes for non-diagnostic purposes, and to study the related mechanisms of anti-tumor immune responses.
  • Ti 3 C 2 MXenes can be used as a catalyst and a reducing agent, using its reducibility to reduce HAuCl 4 to form AuNPs-MXenes complex for improving Emin of Luminol, so we developed a Multi-site identification and AuNPs-MXenes catalyzed Luminol ECL's highly sensitive biosensor platform for exosome detection.
  • the detection limit is 30/microliter, which is relatively lower than the traditional method of enzyme-linked immunoassay and the ECL biosensor obtained by the applicant's previous research.
  • the detection of other exosomes has been successful, which shows that the platform is feasible.
  • the biosensor combined with SA-based multi-site recognition and AuNPs-MXenes-Aptamer nanoprobe provides a powerful tool for evaluating the expression of exosome surface proteins and opens up the physiology of exosomes during the metabolic process New insights into function and clinical diagnosis and drug screening.
  • the assembly and mechanism of the ECL biosensor are shown in Figure 1.
  • the surface of the base electrode glassy carbon electrode (GCE) is modified with PAM. Because the surface of the PAM contains amino groups, the SA with many carboxyl groups can be modified on the surface according to the amide reaction. Increasing the active site for aptamer binding allows more aptamers to be modified on the electrode, thereby capturing more exosomes.
  • MXenes-Aptamer was synthesized as a probe to combine with exosomes to form a sandwich structure. Finally, MXenes super reducibility was used to immerse the assembled electrode in HAuCl 4 to form AuNPs-MXenes-Aptamer complex on the electrode surface.
  • the formed AuNPs–MXenes-Aptamer can double catalyze the electroluminescence of Luminol, and use the strength of the generated ECL signal to reflect the number of captured exosomes, so as to achieve the purpose of detecting exosomes.
  • a strong ECL signal indicates that there are more AuNPs–MXenes-Aptamer on the electrode surface, thus indirectly indicating that there are more exosomes on the electrode, which can reflect how much the sensor captures exosomes according to the strength of the ECL signal .
  • the aptamers are 5'-COOH-TTTTTTCAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TA, 5'-NH 2 -TTTTTTCAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TA was obtained from Shanghai Shengong Biological Engineering Technology Service Co., Ltd. Ti 3 AlC 2 (98%) was purchased from Forsman Science Co., Ltd. (Beijing, China). Polyacrylamide (PAM) and Luminol were purchased from Sigma-Aldrich. HAuCl 4 ⁇ 3H 2 O (48%, w/w) was obtained from Shanghai Reagent (Shanghai, China).
  • SA sodium alginate
  • EDC 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride
  • NHS N-hydroxyl Succinimide sodium salt
  • ethylenediamine EDA
  • LiFS ethylenediamine
  • Electrochemical workstation electrochemiluminescence workstation, using a three-electrode system (reference electrode: Ag/AgCl electrode, counter electrode: platinum wire electrode, working electrode: glassy carbon electrode), scanning electron microscope (SEM), transmission electron microscope (TEM) ), ultraviolet spectrophotometer, Fourier infrared spectrometer, etc.
  • reference electrode Ag/AgCl electrode
  • counter electrode platinum wire electrode
  • working electrode glassy carbon electrode
  • SEM scanning electron microscope
  • TEM transmission electron microscope
  • ultraviolet spectrophotometer Fourier infrared spectrometer, etc.
  • the glassy carbon electrode (GCE) was polished with 0.3 ⁇ m Al 2 O 3 powder on chamois leather, and then ultrasonically cleaned with ethanol and water for 3 min, and the electrode surface was blown dry with pure nitrogen.
  • GCE working electrode
  • Ag/AgCl reference electrode
  • platinum wire counter electrode
  • potassium ferricyanide solution -0.2 ⁇ 0.6V, 100mV/s
  • scan CV to stable. Repeat this way until the redox potential difference of GCE is about 80mV, wash the glassy carbon electrode with water, and blow dry with nitrogen.
  • GCE after PAM modification treatment Take 6 ⁇ L of PAM with a concentration of 0.1 mg/mL onto the surface of GCE, incubate at 37°C to dry, and then add 20 mg of SA to 2 mL of 2-(N-morpholine)ethanesulfonic acid (MES , Buffer 0.1M, pH 6.0), and mix this solution with 2mL EDC/NHS solution (100mM/300mM, volume ratio is 1:1) for 1 hour, then immerse the PAM/GCE electrode into the above mixed solution (120 ⁇ L) 1 hour, then immerse the electrode in 1 ⁇ M (40 ⁇ L) Aptamer (5'-NH 2 -TTTTTTCAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TA), incubate at 37°C for 2h, wash and blow dry to get Aptamer/ SA/PAM/GCE.
  • MES 2-(N-morpholine)ethanesulfonic acid
  • Aptamer/SA/PAM/GCE was immersed in Hela exosomes with different concentrations in a 37°C environment for 2h. Wash and blow dry to get exosomes/Aptamer/SA/PAM/GCE. After the electrodes that have captured the exosomes were washed with distilled water and dried, they were placed in a probe solution and incubated at 37°C for 2 hours. After the reaction was completed, they were washed with distilled water and dried with nitrogen.
  • the assembled electrodes (MXenes- Aptamer/exosomes/Aptamer/SA/PAM/GCE) is soaked in 2mg/mL HAuCl 4 solution for 1h, and then the electrode is rinsed and blown dry to obtain the prepared ECL biosensor.
  • the sensor platform can be used to monitor the exosomes secreted by different cells.
  • This application also uses this method to detect hepatic cancer cell (HepG2) exosomes and ovarian cancer cell (OVCAR) exosomes.
  • FIG. 2 is an SEM schematic diagram of Ti 3 AlC 2 before peeling.
  • Fig. 3 is a TEM image of MXenes after peeling. It can be seen from the figure that MXenes formed nanosheets after peeling.
  • Figure 4 shows the XRD pattern of MXenes before and after stripping.
  • Figure 5 shows the detection of ECL signals generated by different concentrations of exosomes. As can be seen from Figure 5, as the concentration of exosomes increases, the ECL signal gradually increases. In the range of exosome concentration of 10 2 -10 5 / microliter, the size of ECL signal is linearly related to the logarithm of the concentration of exosomes.
  • GCE after PAM modification treatment Take 6 ⁇ L of PAM with a concentration of 0.1mg/mL onto the surface of GCE, incubate at 37°C to dry, and then add 20mg of SA to 2mL of 2-(N-morpholine)ethanesulfonic acid (MES Buffer 0.1M, pH 6.0), and mix this solution with 2mL EDC/NHS solution (100mM/300mM, volume ratio is 1:1) for 1 hour, and then immerse the PAM/GCE electrode into the above mixed solution (120 ⁇ L) After 1 hour, the electrode was immersed in 0.8 ⁇ M (40 ⁇ L) Aptamer, incubated at 37° C.
  • MES Buffer 0.1M, pH 6.0 2-(N-morpholine)ethanesulfonic acid
  • Aptamer/SA/PAM/GCE was immersed in exosomes of different concentrations in a 25°C environment for 1 hour. Wash and blow dry to get exosomes/Aptamer/SA/PAM/GCE.
  • GCE after PAM modification treatment Take 6 ⁇ L of PAM with a concentration of 0.1mg/mL onto the surface of GCE, incubate at 37°C to dry, and then add 20mg of SA to 2mL of 2-(N-morpholine)ethanesulfonic acid (MES Buffer 0.1M, pH 6.0), and mix this solution with 2mL EDC/NHS solution (100mM/300mM, volume ratio is 1:1) for 1 hour, and then immerse the PAM/GCE electrode in the above mixed solution (120 ⁇ L) 1 hour, then immerse the electrode in 1.2 ⁇ M (40 ⁇ L) Aptamer, incubate at 37°C for 2h, wash and blow dry to get Aptamer/SA/PAM/GCE. Submerge Aptamer/SA/PAM/GCE in different concentrations In the exosomes, in a 50°C environment for 30 minutes. Wash and blow dry to get exosomes/Aptamer/SA/PAM/G
  • the electrodes with captured exosomes were washed with distilled water and dried, they were placed in the probe solution and incubated at 37°C for 1 hour. After the reaction was completed, the electrodes were washed with distilled water, dried with nitrogen, and then the assembled electrodes were soaked in 2 mg /mL of HAuCl 4 solution for 1 h to form AuNPs-MXenes-Aptamer/exosomes/Aptamer/SA/PAM/GCE, and then rinse and dry the electrode to obtain the prepared ECL biosensor.

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Abstract

一种基于金纳米颗粒(AuNPs)和二维过渡金属碳化物（MXenes）的双重催化鲁米诺的电化学发光(ECL)生物传感器，形成AuNPs-MXenes-Aptamer作为ECL信号探针，设计高灵敏的ECL传感平台检测宫颈癌细胞分泌的外泌体。海藻酸钠聚合物分子可以提供多位点识别界面以捕获更多适配体以提高外泌体的捕获效率，这有助于改善生物传感器的灵敏度并且AuNPs-MXenes可以双重催化鲁米诺的ECL发射。

Description

一种基于金纳米颗粒和二碳化钛MXenes的双重催化鲁米诺电化学发光生物传感器 技术领域
本公开涉及一种二维纳米材料-Ti 3C 2 MXenes利用其还原性，还原氯金酸(HAuCl 4)形成金纳米颗粒-MXenes(AuNPs-MXenes)复合物，AuNPs-MXenes复合物可以双重催化鲁米诺的电化学发光(ECL)以及利用海藻酸钠(SA)的多位点效应构建ECL生物传感器检测外泌体的方法。
背景技术
这里的陈述仅提供与本公开有关的背景信息，而不必然构成现有技术。
外泌体是一种直径在30～100nm、具有双层膜结构的囊泡，可由多种细胞通过胞吐方式分泌至微环境中。外泌体存在于大多数人体液中，包括血液、尿液、唾液和母乳。它们由大多数哺乳动物细胞分泌，携带的遗传物质，包括mRNA、碳水化合物、蛋白质(CD63、CD81、CD9和EpCAM等)和DNA，这些物质可以促进细胞-细胞通讯。据报道，外泌体在抗肿瘤免疫应答、肿瘤诊断和其他过程中发挥重要作用，并且是早期癌症诊断有希望的生物标志物。因此，用于外泌体检测的高灵敏的方法不仅对临床诊断有价值，而且还能提供关于肿瘤生长和转移的基本生化过程的见解，同时还有助于进一步研究抗肿瘤免疫应答的相关机制。
迄今为止，已经开发了用于外泌体检测的各种方法，包括蛋白印迹法，流式细胞术或酶联免疫吸附法。这些方法具有一定的缺点，如昂贵的仪器、复杂的技术技能和耗时的操作等。因此，开发简单、灵敏和可靠的外泌体检测方法是一项巨大的挑战。近年来，ECL作为一种强大的分析技术，由于其高灵敏，快速性，易控制性和低成本，已被广泛用于蛋白质、DNA、酶等物质的检测，因此，基于ECL的这些优点，它可以有望应用于外泌体检测，分析外泌体的活性。
发明内容
针对背景技术，本公开涉及一种基于二碳化钛-MXenes和AuNPs的双重催化鲁米诺的ECL生物传感器与应用。
具体来讲，本公开涉及以下方面的技术方案：
在本公开的第一个典型的实施方式中，提供一种二维过渡金属碳化物-核酸适配体(MXenes-Aptamer)探针，其特点是：该探针包括纳米片Ti 3C 2 MXenes、氨基酸和修饰有羧基的CD63蛋白核酸适配体，所述纳米片Ti 3C 2 MXenes通过Ti-N键与氨基酸相连，氨基酸通过酰胺键与核酸适配体相连。
在本公开的第二个典型的实施方式中，提供所述MXenes-Aptamer探针的制备方法，该方法包括：将纳米片Ti 3C 2 MXenes和氨基酸置于水中混合均匀后，搅拌，分离得到沉淀物，将得到的沉淀物与适配体进行酰胺反应即可得到MXenes-Aptamer探针。
在本公开的第三个典型的实施方式中，提供一种与所述探针配合使用的生物传感器电极，包括：表面修饰有聚丙烯酰胺(PAM)的基体电极、海藻酸钠(SA)和修饰有氨基的CD63蛋白核酸适配体；
所述表面修饰有PAM的基体电极和修饰有氨基的CD63蛋白核酸适配体均通过酰胺反应与SA连接。
在本公开的第四个典型的实施方式中，提供所述生物传感器电极的制备方法，该方法包括：将PAM溶液滴到基体电极上，干燥；然后将基体电极浸泡在1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)、N-羟基琥珀酰亚胺(NHS)和SA的混合溶液中进行孵化；随后将基体电极浸入核酸适配体溶液中进行孵化，得到生物传感器电极。
在本公开的第五个典型的实施方式中，提供一种ECL生物传感器，该生物传感器包括所述MXenes-Aptamer探针和所述生物传感器电极，当外泌体存在时，所述MXenes-Aptamer探针、所述生物传感器电极和外泌体形成三明治结构(MXenes-Aptamer探针-外泌体-生物传感器电极)，并将该三明治结构浸泡在氯金酸(HAuCl 4)溶液中，原位形成AuNPs-MXenes-Aptamer探针-外泌体-生物传感器电极，即为ECL生物传感器。
在本公开的第六个典型的实施方式中，提供一种ECL的试剂盒，该试剂盒包括所述MXenes-Aptamer探针、所述生物传感器电极、鲁米诺和HAuCl 4溶液。
在本公开的第七个典型的实施方式中，提供所述MXenes-Aptamer探针、所述生物传感器电极或所述的ECL生物传感器或所述试剂盒在采用ECL方法检测外泌体中的应用。
在本公开的第八个典型的实施方式中，提供一种非诊断目的的检测外泌体的方法，该方法包括采用所述MXenes-Aptamer探针、所述生物传感器电极或所述的ECL生物传感器或所述试剂盒在采用ECL方法检测外泌体的步骤。
与本发明人知晓的相关技术相比，本公开其中的一个技术方案具有如下有益效果：
本申请结合多位点识别策略和新型二维纳米材料-Ti 3C 2 MXenes的还原性，形成AuNPs–MXenes-Aptamer作为ECL信号探针，设计高灵敏的ECL传感平台检测Hela外泌体。在该策略中，SA聚合物分子可以提供多位点识别界面以捕获更多适配体以提高外泌体的捕获效率，这有助于改善生物传感器的灵敏度，并且AuNPs-MXenes-Aptamer可以双重催化鲁米诺，使ECL信号增强。基于这种双重扩增策略，获得了高灵敏和高选择性的生物传感器来检测外泌体，另外，ECL生物传感器也可用于血清中的外泌体检测。该传感器可作为检 测外泌体的可行性工具，为外泌体的临床诊断等提供了有力的证据。
附图说明
构成本公开一部分的说明书附图用来提供对本公开的进一步理解，本公开的示意性实施例及其说明用于解释本公开，并不构成对本公开的不当限定。
图1为本公开ECL传感器的组装及机理示意图。
图2是Ti 3AlC 2的SEM图。
图3是MXenes的TEM图。
图4是MXenes剥离前后的XRD图。
图5是不同浓度外泌体的ECL传感器的信号响应；a-h代表:10 2，5×10 2，10 3，2.5×10 3，5×10 3，10 4，5×10 4，10 5particles/μL。
具体实施方式
应该指出，以下详细说明都是示例性的，旨在对本公开提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本公开所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本公开的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作和/或它们的组合。
正如背景技术所介绍的，需要一种高灵敏和高选择性的生物传感器来检测外泌体，针对此，在本公开的第一个典型的实施方式中，提供一种MXenes-Aptamer探针，其特点是：该探针包括纳米片Ti 3C 2 MXenes、氨基酸和修饰羧基的CD63蛋白核酸适配体，所述纳米片Ti 3C 2 MXenes通过Ti-N键与氨基酸相连，氨基酸通过酰胺键与核酸适配体相连。
本公开中核酸适配体特异性结合的是外泌体表面的CD63蛋白。在本公开的一个或一些实施方式中，所述CD63蛋白的核酸适配体的包括但不仅仅限于以下序列：5'--TTTTTTCAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TA，如SEQ ID NO.1所示。
该探针的设计既能保留MXenes自身的优良性质：大的比表面积，较好的导电性，高的催化性质以及超强的还原性，还能使MXenes表面带有氨基，便于修饰在电极上。这是利用氨基酸和MXenes之间的相互作用力结合，形成Ti-N键，使MXenes表面修饰氨基。与之前研究的通过静电吸附作用相比，利用当前这种修饰方式，比静电吸附作用更加稳定，可以使MXenes表面带有氨基，进行下一步的修饰。
在本公开的一个或一些具体的实施方式中，所述氨基酸为甘氨酸或亮氨酸，经过实验 验证，甘氨酸或亮氨酸可以与Ti 3C 2 MXenes更好的结合。
在本公开的第二个典型的实施方式中，提供所述MXenes-Aptamer探针的制备方法，该方法包括：将纳米片Ti 3C 2 MXenes和氨基酸置于水中混合均匀后，搅拌，分离得到沉淀物，将得到的沉淀物与适配体进行酰胺反应即可得到MXenes-Aptamer探针。
进一步的，所述Ti 3C 2 MXenes、氨基酸和水的投料比例(0.5～1)mg：(4～6)mg：(10～40)mL；室温下搅拌18～36h。
进一步的，所述酰胺反应的温度为35～40℃，时间为0.5～1.5h。
Ti 3C 2 MXenes具有良好的电子转移能力，优异的催化能力，良好的生物相容性以及超强的还原性。此外，Ti 3C 2 MXenes大的比表面积还使其成为加载更多生物分子的良好载体，从而显著提高生物传感器的灵敏度。进一步的，所述纳米片Ti 3C 2 MXenes的制备方法，包括以下步骤：将LiF加入浓度为8～10mol/L的盐酸中，搅拌，随后将Ti 3AlC 2粉末加入此混合物中并在30～40℃下搅拌18～36h；随后将溶液离心洗涤，使pH≥6，弃去上清液，在沉淀中加入水，离心，弃去上清液，继续在沉淀中加入水，在氮气的保护下，超声0.5～1.5h，最后，将溶液离心，保留上清液，即为纳米片Ti 3C 2 MXenes分散液。
在本公开的第三个典型的实施方式中，提供一种与所述探针配合使用的生物传感器电极，包括：表面修饰有PAM的基体电极、SA和修饰有氨基的CD63蛋白核酸适配体；
所述表面修饰有PAM的基体电极和核酸适配体均通过酰胺反应与SA连接。
进一步的，所述基体电极为玻碳电极。
海藻酸钠(C 6H 7O 6Na)n主要由海藻酸的钠盐组成，由β-D-甘露糖醛酸(M单元)与α-L-古洛糖醛酸(G单元)依靠β-1,4-糖苷键连接并由不同比例的GM、MM和GG片段组成的共聚物，表面含有许多羧基，可以提供更多的识别位点，而且其亲水性好，附着力低，性能优异，并且具有生物相容性，因此本公开利用其优异的性质可以显著提高生物传感器的灵敏度和选择性。在多分散性分子群中，通常Mw>Mn。Mw/Mn的系数为分散性指数，海藻酸钠商品的指数经典范围为1.5～2.5。
在本公开的第四个典型的实施方式中，提供所述生物传感器电极的制备方法，该方法包括：将PAM溶液滴到基体电极上，孵化待干；然后将基体电极浸泡在EDC、NHS和SA的混合溶液中进行孵化；随后将基体电极进入核酸适配体溶液中进行孵化，得到生物传感器电极。
进一步的，所述EDC、NHS和SA的体积比例为0.8～1.2：0.8～1.2：1.8～2.2，EDC的浓度为80-120mM，NHS的浓度为200-400mM，SA的浓度为5～15mg/mL；在35～40℃孵化1～3h。
进一步的，随后将基体电极进入核酸适配体溶液中进行孵化，孵化条件为：35～40℃下孵化1～3h。
进一步的，所述聚丙烯酰胺的重均分子量为：MW≥300万。
在本公开的第五个典型的实施方式中，提供一种ECL生物传感器，该生物传感器包括所述MXenes-Aptamer探针和所述生物传感器电极，当外泌体存在时，所述MXenes-Aptamer探针、所述生物传感器电极和外泌体形成三明治结构(MXenes-Aptamer探针-外泌体-生物传感器电极)，并将该三明治结构浸泡在HAuCl 4溶液中，原位形成AuNPs-MXenes-Aptamer探针-外泌体-生物传感器电极，即为ECL生物传感器。
在本公开的第六个典型的实施方式中，提供一种ECL的试剂盒，该试剂盒至少包括所述MXenes-Aptamer探针、所述生物传感器电极、HAuCl 4溶液和鲁米诺。
在本公开的第七个典型的实施方式中，提供所述MXenes-Aptamer探针、所述生物传感器电极或所述的ECL生物传感器或所述试剂盒在采用ECL检测外泌体中的应用。
在本公开的第八个典型的实施方式中，提供一种非诊断目的的检测外泌体的方法，该方法包括采用所述MXenes-Aptamer探针、所述生物传感器电极或所述的ECL生物传感器或所述试剂盒在采用ECL方法检测外泌体的步骤。
采用所述试剂盒进行检测的方法包括：将所述生物传感器电极浸泡至待测外泌体溶液中，使外泌体附着在生物传感器电极上，随后将附着外泌体的生物传感器电极浸泡至MXenes-Aptamer探针溶液中，使探针附着在生物传感器电极的外泌体上，从而组成探针和生物传感器电极夹载外泌体的生物传感器，再将所述生物传感器浸泡在HAuCl 4溶液中，使AuNPs在电极表面的复合物上生成，进而制备成表面附着AuNPs的探针和生物传感器电极夹载外泌体的ECL生物传感器，对该ECL生物传感器在鲁米诺溶液中进行ECL检测。
具体方法包括：
(1)标准溶液的配制：配制一组不同浓度的外泌体标准溶液；
(2)工作曲线的绘制：将所述生物传感器电极分别浸泡至不同浓度的外泌体标准溶液中，使外泌体附着在生物传感器电极上，随后将附着外泌体的生物传感器电极浸泡至MXenes-Aptamer探针溶液中，使探针附着在生物传感器电极的外泌体上，从而组成探针和生物传感器电极夹载外泌体的生物传感器，再将所述生物传感器浸泡在HAuCl 4溶液中，使AuNPs在电极表面的复合物上生成，进而制备成表面附着有AuNPs的探针和生物传感器电极夹载外泌体的ECL生物传感器，在光电倍增管为600V下对该ECL生物传感器在鲁米诺溶液中进行ECL检测，得到不同浓度的外泌体标准溶液的ECL强度，再根据外泌体标准溶液的浓度以及相应的ECL强度，绘制线性关系曲线；
(3)样品的检测：根据步骤(2)中的方法测定待测外泌体溶液的ECL强度，再根据所述线性关系曲线，得到待测的外泌体的浓度。
该检测方法用于非诊断目的的检测外泌体，可研究抗肿瘤免疫应答的相关机制等。
在本公开中，发现Ti 3C 2 MXenes可以作为催化剂和还原剂，利用其还原性，还原HAuCl 4形成AuNPs-MXenes复合物，用于改进鲁米诺的ECL，从而我们开发了一种基于SA的多位点识别与AuNPs-MXenes催化鲁米诺ECL的高灵敏生物传感器平台用于外泌体检测。检测限为30个/微升，该检测限相对低于酶联免疫的传统方法以及申请人前期研究得到的ECL生物传感器。此外，基于双重扩增策略，其他外泌体的检测都取得了成功，这表明该平台是可行的。因此，基于SA的多位点识别和AuNPs-MXenes-Aptamer纳米探针结合的生物传感器为评估外泌体表面蛋白的表达提供了强有力的工具，并开启了对外泌体在代谢过程中的生理功能以及临床诊断的新见解和药物筛选。
电化学发光传感器的组装及实验原理：
ECL生物传感器的组装及机理如图1所示，基体电极玻碳电极(GCE)表面修饰PAM，由于PAM表面含有氨基，从而根据酰胺反应，可以在其表面修饰带有很多羧基的SA，这样可以增加适配体结合的活性位点，使更多的适配体修饰在电极上，从而捕获更多的外泌体。接着合成MXenes-Aptamer作为探针与外泌体结合，形成三明治结构，最后利用MXenes超强的还原性，使组装完成的电极浸泡在HAuCl 4中，在电极表面形成AuNPs-MXenes-Aptamer复合物，形成的AuNPs–MXenes-Aptamer可以双重催化鲁米诺的电化学发光，利用产生的ECL信号的强弱来反映捕获外泌体的多少，从而达到检测外泌体的目的。ECL信号较强则表示在电极表面的AuNPs–MXenes-Aptamer较多，从而间接的说明在电极上的外泌体较多，可以根据ECL信号的强弱来反映出此传感器捕获外泌体的多少。
为了使得本领域技术人员能够更加清楚地了解本公开的技术方案，以下将结合具体的实施例详细说明本公开的技术方案。
实验试剂与材料
适配体为5'-COOH-TTTTTTCAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TA，5'-NH 2-TTTTTTCAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TA获自上海生工生物工程技术服务有限公司。Ti 3AlC 2(98％)购自福斯曼科学有限公司(中国北京)。聚丙烯酰胺(PAM)和鲁米诺购自Sigma-Aldrich。HAuCl 4·3H 2O(48％，w/w)获自Shanghai Reagent(中国上海)。海藻酸钠(SA)购自阿拉丁(中国)产品编号S100128，1-(3-(二甲基氨基)丙基)-3-乙基碳化二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺钠盐(NHS)，乙二胺(EDA)均购自北京化工有限公司(中国北京)，氟化锂购自上海阿拉丁生物技术有限公司，其他分 析级试剂均来自国药化学试剂公司(中国)。
实验仪器
电化学工作站，电化学发光工作站，使用三电极体系(参比电极：Ag/AgCl电极，对电极：铂丝电极，工作电极：玻碳电极)，扫描电子显微镜(SEM)，透射电子显微镜(TEM)，紫外分光光度仪，傅里叶红外光谱仪等。
实施例1
1、MXenes-glycine-Aptamer纳米探针的合成
将0.8g LiF加入10mL，9mol/L的盐酸中，搅拌5分钟，随后将Ti 3AlC 2(0.5g)粉末加入此混合物中并在35℃下搅拌24小时。随后将溶液离心洗涤7-8次，使pH≥6，弃去上清液，在沉淀中加入40-50mL去离子水，以3500rpm离心60分钟，弃去上清液，继续在沉淀中加入40-50mL去离子水，在氮气的保护下，超声1h，最后，将溶液以3500rpm离心60分钟，保留上清液(即为纳米片Ti 3C 2 MXenes分散液)，并在4℃下保存备用。
将5mg的甘氨酸(glycine)溶于20mL去离子水中在室温下搅拌。然后，逐滴加入10mL浓度为0.075mg/mL的Ti 3C 2 MXenes分散液并在室温下搅拌24小时，随后以12000r离心20分钟，弃去上清液，沉淀标记为MXenes-glycine，备用。
EDC(400mM)和NHS(100mM)和Aptamer(1uM，5'-COOH-TTTTTT CAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TA)混合物在37℃下活化1小时。此后，将200μL获得的Ti 3C 2 MXenes-glycine在37℃下加入Aptamer溶液中，反应1h，最后，将混合物以12000rpm离心10分钟，弃去上清液，洗涤并加入去离子水。
2、电极的组装及肽链在电极上的共价反应
(1)玻碳电极表面预处理
将玻碳电极(GCE)用0.3μm的Al 2O 3粉末在麂皮上进行打磨抛光处理，然后分别用乙醇、水超声清洗3min，用纯净氮气将电极表面吹干。
清洗吹干的GCE作工作电极，Ag/AgCl作参比电极，铂丝作对电极，在铁氰化钾溶液中，-0.2～0.6V，100mV/s，扫描CV至稳定。如此反复，直至GCE的氧化还原电势差在80mV左右，将玻碳电极用水洗净，氮气吹干。
(2)电化学发光生物传感器的组装
PAM修饰处理后的GCE：取浓度为0.1mg/mL的PAM 6μL滴到GCE表面，37℃下孵化待干，接着，将20mg SA添加到2mL 2-(N-吗啉)乙磺酸(MES，缓冲液0.1M，pH 6.0)中，并将此溶液与2mL EDC/NHS溶液(100mM/300mM,体积比为1:1)混合1小时，然后将PAM/GCE电极浸入上述混合溶液(120μL)中1小时，随后将电极浸没在1μM(40 μL)Aptamer(5'-NH 2-TTTTTTCAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TA)中，37℃下孵化2h，洗净吹干后得到Aptamer/SA/PAM/GCE。
将Aptamer/SA/PAM/GCE浸没在不同浓度的Hela外泌体中，在37℃的环境中2h。洗净吹干后得到exosomes/Aptamer/SA/PAM/GCE。将已捕获外泌体的电极用蒸馏水洗净吹干后，置于探针溶液中37℃下孵化2h，待反应完全之后用蒸馏水进行清洗，氮气吹干，然后将组装好的电极(MXenes-Aptamer/exosomes/Aptamer/SA/PAM/GCE)浸泡在2mg/mL的HAuCl 4溶液中1h，随后将电极冲洗吹干，即得到制备好的ECL生物传感器。
由于CD63蛋白在外泌体上是普遍存在的，只是含量有不同，因此可利用该传感器平台监测不同细胞分泌的外泌体。本申请采用该方法还检测了肝癌细胞(HepG2)外泌体和卵巢癌细胞(OVCAR)外泌体等。
3、实验结果的讨论
图2为剥离前的Ti 3AlC 2的SEM示意图。
图3为剥离后的MXenes的TEM图。由图可见经过剥离，形成纳米片的MXenes。
图4为剥离前后MXenes的XRD图。
图5为检测不同浓度外泌体产生的ECL信号。由图5可见，随着外泌体浓度的增加，ECL信号逐渐增大。在外泌体的浓度为10 2-10 5个/微升范围内，ECL信号的大小与外泌体的浓度的对数呈线性关系。
实施例2
1、MXenes-glycine-Aptamer纳米探针的合成
同实施例1。
2、电极的组装及传感器的组装
(1)玻碳电极表面预处理
同实施例1
(2)电化学发光生物传感器的组装
PAM修饰处理后的GCE：取浓度为0.1mg/mL的PAM 6μL滴到GCE表面，37℃下孵化待干，接着，将20mg SA添加到2mL 2-(N-吗啉)乙磺酸(MES缓冲液0.1M，pH 6.0)中，并将此溶液与2mL EDC/NHS溶液(100mM/300mM,体积比为1:1)混合1小时，然后将PAM/GCE电极浸入上述混合溶液(120μL)中1小时，随后将电极浸没在0.8μM(40μL)Aptamer中，37℃下孵化2h，洗净吹干后得到Aptamer/SA/PAM/GCE。将Aptamer/SA/PAM/GCE浸没在不同浓度的外泌体中，在25℃的环境中1h。洗净吹干后得到exosomes/Aptamer/SA/PAM/GCE。
将已捕获外泌体的电极用蒸馏水洗净吹干后，置于探针溶液中37℃下孵化2h，待反应完全之后用蒸馏水进行清洗，氮气吹干，然后将组装好的电极浸泡在2mg/mL的HAuCl 4溶液中1h，形成AuNPs-MXenes-Aptamer/exosomes/Aptamer/SA/PAM/GCE，随后将电极冲洗吹干，即得到制备好的ECL生物传感器。
实施例3
1、MXenes-glycine-Aptamer纳米探针的合成
同实施例1
2、电极的组装及传感器的组装
(1)玻碳电极表面预处理
同实施例1。
(2)电化学发光生物传感器的组装
PAM修饰处理后的GCE：取浓度为0.1mg/mL的PAM 6μL滴到GCE表面，37℃下孵化待干，接着，将20mg SA添加到2mL 2-(N-吗啉)乙磺酸(MES缓冲液0.1M，pH 6.0)中，并将此溶液与2mL EDC/NHS溶液(100mM/300mM,体积比为1:1)混合1小时，然后将PAM/GCE电极浸入上述混合溶液(120μL)中1小时，随后将电极浸没在1.2μM(40μL)Aptamer中,37℃下孵化2h，洗净吹干后得到Aptamer/SA/PAM/GCE.将Aptamer/SA/PAM/GCE浸没在不同浓度的外泌体中，在50℃的环境中30分钟。洗净吹干后得到exosomes/Aptamer/SA/PAM/GCE。
将已捕获外泌体的电极用蒸馏水洗净吹干后，置于探针溶液中37℃下孵化1h，待反应完全之后用蒸馏水进行清洗，氮气吹干，然后将组装好的电极浸泡在2mg/mL的HAuCl 4溶液中1h，形成AuNPs-MXenes-Aptamer/exosomes/Aptamer/SA/PAM/GCE，随后将电极冲洗吹干，即得到制备好的ECL生物传感器。
上述实施例为本公开较佳的实施方式，但本公开的实施方式并不受上述实施例的限制，其他的任何未背离本公开的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本公开的保护范围之内。
Figure PCTCN2019112594-appb-000001

Claims (10)

  1. 一种二维过渡金属碳化物-核酸适配体(MXenes-Aptamer)探针，其特征是：该探针包括纳米片Ti 3C 2 MXenes、氨基酸和修饰有羧基的CD63蛋白核酸适配体，所述纳米片Ti 3C 2 MXenes通过Ti-N键与氨基酸相连，氨基酸通过酰胺键与核酸适配体相连。
  2. 如权利要求1所述的MXenes-Aptamer探针，其特征是：所述核酸适配体的序列为：5'-COOH-TTTTTTCAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TA。
  3. 权利要求1或2所述的MXenes-Aptamer探针的制备方法，其特征是，该方法包括以下步骤：将纳米片Ti 3C 2 MXenes和氨基酸置于水中混合均匀后，搅拌，分离得到沉淀物，将得到的沉淀物与适配体进行酰胺反应即可得到MXenes-Aptamer探针；
    进一步的，所述氨基酸为甘氨酸或亮氨酸；
    进一步的，所述Ti 3C 2 MXenes、氨基酸和水的投料比例为(0.5～1)mg：(4～6)mg：(10～40)mL；室温下搅拌18～36h；
    进一步的，所述酰胺反应的温度为35～40℃，时间为0.5～1.5h。
  4. 一种与权利要求1或2所述的探针配合使用的生物传感器电极，其特征是，包括：表面修饰有聚丙烯酰胺(PAM)的基体电极、海藻酸钠(SA)和修饰有氨基的CD63蛋白核酸适配体；
    所述表面修饰有PAM的基体电极和修饰有氨基的CD63蛋白核酸适配体均通过酰胺反应与SA连接；
    进一步的，所述基体电极为玻碳电极(GCE)；
    进一步的，所述核酸适配体的序列为：5'-NH 2-TTTTTTCAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TA。
  5. 权利要求4所述的生物传感器电极的制备方法，其特征是，该方法包括：将PAM溶液滴到基体电极上，孵化待干；然后将基体电极浸泡在1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)、N-羟基琥珀酰亚胺(NHS)和SA的混合溶液中进行孵化；随后将基体电极进入核酸适配体溶液中进行孵化，得到生物传感器电极。
  6. 如权利要求5所述的制备方法，其特征是：所述EDC、NHS和SA的体积比例为0.8～1.2：0.8～1.2：1.8～2.2，EDC的浓度为80-120mM，NHS的浓度为200-400mM，SA的浓度为5～15mg/mL；在35～40℃孵化1～3h；
    进一步的，随后将基体电极进入核酸适配体溶液中进行孵化，孵化条件为：35～40℃孵化1～3h。
  7. 一种电化学发光(ECL)生物传感器，其特征是：该生物传感器包括权利要求1或 2所述的MXenes-Aptamer探针和权利要求4所述的生物传感器电极，当外泌体存在时，所述MXenes-Aptamer探针、所述生物传感器电极和外泌体形成三明治结构，并将该三明治结构浸泡在氯金酸(HAuCl 4)溶液中，原位形成金纳米颗粒(AuNPs)-MXenes-Aptamer探针-外泌体-生物传感器电极，即为ECL生物传感器。
  8. 一种ECL的试剂盒，其特征是：该试剂盒至少包括权利要求1或2所述的MXenes-Aptamer探针、权利要求4所述的生物传感器电极、HAuCl 4溶液和鲁米诺。
  9. 权利要求1或2所述的MXenes-Aptamer探针、权利要求4所述的生物传感器电极或权利要求7所述的ECL生物传感器或权利要求8所述的试剂盒在采用ECL方法检测外泌体中的应用。
  10. 一种非诊断目的的检测外泌体的方法，其特征是，该方法包括：
    将权利要求4所述的生物传感器电极浸泡至待测外泌体溶液中，使外泌体附着在生物传感器电极上，随后将附着外泌体的生物传感器电极浸泡至权利要求1或2所述的MXenes-Aptamer探针溶液中，使探针附着在生物传感器电极的外泌体上，从而组成探针和生物传感器电极夹载外泌体的生物传感器，再将所述生物传感器浸泡在HAuCl 4溶液中，使AuNPs在电极表面的复合物上生成，进而制备成表面附着有AuNPs的探针和生物传感器电极夹载外泌体的ECL生物传感器，对该ECL生物传感器在鲁米诺溶液中进行ECL检测。
PCT/CN2019/112594 2018-12-03 2019-10-22 一种基于金纳米颗粒和二碳化钛MXenes的双重催化鲁米诺电化学发光生物传感器 WO2020114124A1 (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112630279A (zh) * 2020-12-18 2021-04-09 常州大学 用于检测双氯酚酸的基于金纳米粒子的等离子共振增强型电化学发光传感器及制备方法

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109490284B (zh) * 2018-12-03 2020-06-19 青岛大学 一种基于金纳米颗粒和二碳化钛MXenes的双重催化鲁米诺电化学发光生物传感器
CN110320260B (zh) * 2019-07-27 2021-07-30 福建师范大学 基于MXenes和黑磷量子点增强的外泌体电致化学发光传感器
CN110988070B (zh) * 2019-12-20 2022-08-26 常州大学 一种电化学发光适配体传感器及检测氯霉素的方法
CN111551722A (zh) * 2020-05-27 2020-08-18 济南大学 一种识别和催化双功能化磁性海藻酸钠复合材料的制备及其应用
WO2024036560A1 (zh) * 2022-08-18 2024-02-22 中国科学院深圳先进技术研究院 一种修饰MXene的方法及其应用
CN117706088B (zh) * 2024-02-05 2024-05-03 首都医科大学附属北京康复医院 甲氧西林金黄色葡萄球菌的检测方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170294546A1 (en) * 2014-09-25 2017-10-12 Drexel University Physical Forms of MXene Materials Exhibiting Novel Electrical and Optical Characteristics
WO2018106186A1 (en) * 2016-12-06 2018-06-14 Singapore University Of Technology And Design Method of manufacturing capacitive deionization (cdi) device, cdi device and apparatus for treating water, electrode for capacitive deionization and method of forming same
CN108273058A (zh) * 2018-02-11 2018-07-13 中国人民解放军第二军医大学 一种肿瘤靶向治疗缓释制剂及其制备方法
CN108375564A (zh) * 2017-07-03 2018-08-07 天津大学 自支撑层状材料MXenes的制备方法及其作为拉曼基底的应用
CN108562573A (zh) * 2018-04-20 2018-09-21 青岛大学 一种基于二碳化三钛二维金属碳化物催化鲁米诺电化学发光探针的生物传感器及制备方法
CN109490284A (zh) * 2018-12-03 2019-03-19 青岛大学 一种基于金纳米颗粒和二碳化钛MXenes的双重催化鲁米诺电化学发光生物传感器

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2614905B2 (ja) * 1988-09-22 1997-05-28 帝人株式会社 免疫センサ
CN1737560A (zh) * 2005-09-02 2006-02-22 天津大学 静电纺丝制备的固定酶电极及其方法
CN101565469B (zh) * 2008-10-28 2011-05-11 中国海洋大学 酰胺接枝的海藻酸钠纳米材料及其制备方法与应用
CN102021226B (zh) * 2009-09-11 2014-04-23 中国科学技术大学 鲁米诺直接键合的纳米金核酸分析探针及其应用
CN102288656B (zh) * 2011-05-17 2014-05-28 上海师范大学 一种检测卵巢skov-3癌细胞的三明治型电化学传感器
CN105477678B (zh) * 2015-12-25 2019-01-29 四川大学 一种新型生物医用粘合剂及其制备方法
CN106589161A (zh) * 2016-12-21 2017-04-26 深圳先进技术研究院 改性海藻酸或其盐及制备方法、生物修复材料及支架
CN108872339B (zh) * 2018-04-20 2020-05-26 常州大学 一种用于电化学识别半胱氨酸对映体的分子印迹海藻酸钠/二氧化锰修饰电极的制备方法
CN108586664B (zh) * 2018-04-24 2019-11-12 华中科技大学 一种制备全水凝胶可拉伸超级电容器的方法及该电容器
CN108831760A (zh) * 2018-06-15 2018-11-16 武汉工程大学 一种N掺杂MXene材料及其制备方法和应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170294546A1 (en) * 2014-09-25 2017-10-12 Drexel University Physical Forms of MXene Materials Exhibiting Novel Electrical and Optical Characteristics
WO2018106186A1 (en) * 2016-12-06 2018-06-14 Singapore University Of Technology And Design Method of manufacturing capacitive deionization (cdi) device, cdi device and apparatus for treating water, electrode for capacitive deionization and method of forming same
CN108375564A (zh) * 2017-07-03 2018-08-07 天津大学 自支撑层状材料MXenes的制备方法及其作为拉曼基底的应用
CN108273058A (zh) * 2018-02-11 2018-07-13 中国人民解放军第二军医大学 一种肿瘤靶向治疗缓释制剂及其制备方法
CN108562573A (zh) * 2018-04-20 2018-09-21 青岛大学 一种基于二碳化三钛二维金属碳化物催化鲁米诺电化学发光探针的生物传感器及制备方法
CN109490284A (zh) * 2018-12-03 2019-03-19 青岛大学 一种基于金纳米颗粒和二碳化钛MXenes的双重催化鲁米诺电化学发光生物传感器

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112630279A (zh) * 2020-12-18 2021-04-09 常州大学 用于检测双氯酚酸的基于金纳米粒子的等离子共振增强型电化学发光传感器及制备方法
CN112630279B (zh) * 2020-12-18 2023-05-23 常州大学 用于检测双氯酚酸的基于金纳米粒子的等离子共振增强型电化学发光传感器及制备方法

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