Adapter functionalized magnetic blade spraying device

Abstract

The utility model discloses an adapter functionalized magnetic blade spraying device, which comprises a mass spectrometer (2) and a blade spraying ionization device which are connected through an alligator clip, the blade spraying ionization device comprises a blade (1) and a magnet (3) positioned below the blade (1), and the blade (1) comprises a detection solution. The detection solution comprises the aptamer functionalized magnetic nanoparticles and a sample. According to the device disclosed by the utility model, the aptamer functionalized magnetic nanoparticles, the blade spray ionization technology and the small portable mass spectrometer are ingeniously combined together. The aptamer functionalized magnetic nanoparticles can realize specific recognition of a target object, a nucleic acid dye mass spectrum label adsorbed on the aptamer improves the detection sensitivity, and a blade spray ionization technology and a small portable mass spectrometer can realize rapid, direct and real-time on-site analysis of the target object.

Description

Technical Field

[0001] The utility model relates to the field of inspection and testing, in particular to an aptamer-functionalized magnetic blade spray device. Background Art

[0002] Coated blade spray is an efficient in-situ ionization technology based on solid-phase microextraction. It selectively enriches analytes from complex matrices through the coating on the blade tip, and then uses a small amount of organic solvent for desorption and ionization. The immunomagnetic blade spray ionization technology replaces the traditional surface coating of the coated blade spray with immunomagnetic microspheres, and its immunological recognition combined with mass spectrometry analysis significantly improves the method selectivity. In theory, by replacing the monoclonal antibody on the surface of the magnetic beads, the immunological capture and analysis of various food contaminants can be achieved. Although immunological analysis technology has been continuously improved, the process of antibody development is complex and time-consuming. Therefore, it is particularly necessary to find alternative recognition molecules. Content of the Utility Model

[0003] The technical problem to be solved by the utility model is to provide an aptamer-functionalized magnetic blade spray device with a simple structure and convenient operation.

[0004] An aptamer-functionalized magnetic blade spray device includes a mass spectrometer and a blade spray ionization device connected by an alligator clip. The blade spray ionization device includes a blade and a magnet located below it, and a detection solution is included on the blade.

[0005] In the aptamer-functionalized magnetic blade spray device of the utility model, the detection solution includes aptamer-functionalized magnetic nanoparticles and a sample.

[0006] In the aptamer-functionalized magnetic blade spray device of the utility model, the alligator clip is made of copper. The aptamer-functionalized magnetic blade is fixed at a position 5 mm in front of the inlet of the mass spectrometer by using the alligator clip, and the rear end of the alligator clip is connected to the voltage of the mass spectrometer through a wire.

[0007] In the aptamer-functionalized magnetic blade spray device of the utility model, the mass spectrometer is a Miniβ small portable mass spectrometer.

[0008] In the aptamer-functionalized magnetic blade spray device of the utility model, the magnet is placed at the bottom of the blade tip.

[0009] The difference between the aptamer-functionalized magnetic blade spray device of the utility model and the prior art lies in:

[0010] The aptamer-functionalized magnetic blade spray device of the present utility model ingeniously combines aptamer-functionalized magnetic nanoparticles, blade spray ionization technology, and a small portable mass spectrometer. The aptamer-functionalized magnetic nanoparticles can achieve specific recognition of the target, and the nucleic acid dye mass spectrometry tags adsorbed on the aptamer improve the detection sensitivity, while the blade spray ionization technology and the small portable mass spectrometer can achieve rapid direct and real-time on-site analysis of the target.

[0011] In the present utility model, the combination of aptamer-functionalized magnetic nanoparticles and blade spray ionization technology is an efficient and powerful method that can give full play to their respective advantages, thus providing a more powerful tool for the analysis field.

[0012] The device of the present utility model uses a small portable mass spectrometer, eliminating the limitations of the size and weight of large mass spectrometers, and also solving the complexity and time-consuming nature of mass spectrometry methods to a certain extent; by using the device of the present utility model, online detection of different types of targets can be achieved by replacing nucleic acid aptamers.

[0013] The following further describes the aptamer-functionalized magnetic blade spray device of the present utility model with reference to the accompanying drawings. Description of the Drawings

[0014] Figure 1 It is a schematic structural diagram of the small portable mass spectrometry detection device for aptamer-functionalized magnetic blade spray in the present utility model;

[0015] Figure 2 It is the full-scan first-order mass spectrometry diagram of detecting cell membrane glycoprotein in blood samples using the device of the present utility model; (A) 0 ng / mL and (B) 50 ng / mL cell membrane glycoprotein; (C) Matrix-matched calibration curve (blue) and standard solution calibration curve (red) of cell membrane protein in the concentration range of 1 to 50 ng / mL;

[0016] All the English words that appear in the drawings of the present utility model are translated as follows:

[0017] Dye loading: Dye loading;

[0018] CD105 Adsorption: CD105 protein adsorption;

[0019] Dye desorption: Dye desorption;

[0020] Aptamer: Aptamer;

[0021] EBApt: Nucleic acid aptamer that binds to cell membrane glycoprotein;

[0022] CD105: Cell membrane glycoprotein;

[0023] CD26 / DPP4: Dipeptidyl peptidase 4;

[0024] PD-L1: Programmed death ligand 1;

[0025] HAS: Human serum albumin;

[0026] AFP: Alpha-fetoprotein;

[0027] Dye: Nucleic acid dye;

[0028] IS: Internal standard;

[0029] Magnet: Magnet;

[0030] EBApt-pMNPs: Aptamer-functionalized hydrophilic polymer magnetic nanoparticles;

[0031] AGMP: Aptamer-functionalized nucleic acid dye-loaded mass spectrometry probe;

[0032] pMNPs: Hydrophilic polymer magnetic nanoparticles;

[0033] Relative Abundance: Relative abundance;

[0034] Intensity ratio of m / z GelRed / IS: Mass spectrometry peak intensity ratio of GelRed dye and internal standard;

[0035] Standard solution: Standard solution;

[0036] Matrix-matched: Matrix-matched. Detailed implementation manners

[0037] I. Instruments and devices

[0038] Miniβ small portable mass spectrometer (55 cm × 24 cm × 31 cm, Qingpu Technology Co., Ltd., Beijing); Milli-Q ultrapure water purifier (Millipore Corporation, USA); Pipette tips used in the experiment (Bio-Rad Laboratories, Inc., USA).

[0039] The blade is made by cutting an ultra-thin rectangular iron sheet, and the ultra-thin rectangular iron sheet (length 20 × width 10 × thickness 0.3 mm, Youci Technology Co., Ltd., Shenzhen).

[0040] II. Materials and reagents

[0041] The cell membrane glycoproteins used in the experiment were purchased from Beijing Sino Biological Inc.; GelRed nucleic acid dye was purchased from Sigma-Aldrich (Beijing); crystal violet was from Beijing J&K Scientific Ltd.; human serum was purchased from Shanghai Chunshi Biotechnology Co., Ltd. (Shanghai); all solutions were prepared with pure water produced by a Milli Q pure water instrument from the United States.

[0042] III. Experimental Setup

[0043] As Figure 1 shown, an aptamer-functionalized magnetic blade spray device includes a mass spectrometer 2 and a blade spray ionization device connected by an alligator clip. The blade spray ionization device includes a blade 1 and a magnet 3 located below it. The detection solution is included on the blade 1. The detection solution includes aptamer-functionalized magnetic nanoparticles and a sample. The alligator clip is made of copper. The blade 1 is fixed at the front end of the sampling port of the mass spectrometer 2, 5 mm away, using an alligator clip. The rear end of the alligator clip is connected to the voltage of the mass spectrometer 2 through a wire. The mass spectrometer 2 is a Miniβ small portable mass spectrometer. The size of the blade 1 is 4 cm × 1.1 cm, and the tip angle is 60°; a magnet 3 is placed at the bottom of the tip of the blade 1.

[0044] IV. Experimental Method

[0045] The method for detecting cell membrane glycoproteins in human serum samples using the device of the present utility model includes the following steps:

[0046] (A) Add 100 μL of aptamer-functionalized magnetic nanoparticles to 1 mL of 10X GelRed aqueous solution and adsorb for 5 min; adsorb the magnetic nanoparticles with a magnet and wash 1 - 2 times with deionized water; add 100 μL of deionized water and vortex to mix evenly to obtain an aptamer-functionalized magnetic nanoparticle resuspension.

[0047] (B) Adjust the pH value of the human serum sample to 6.0 using acetic acid; add 10 μL of the aptamer-functionalized magnetic nanoparticle resuspension to 100 μL of the human serum sample and incubate at room temperature for 30 min; adsorb the magnetic nanoparticles with a magnet and wash 1 - 2 times with deionized water; add 20 μL of deionized water and vortex to mix evenly.

[0048] (C) Add the solution obtained in step (B) to the tip of the customized blade, place a magnet below it to fix, fix it to the front end of the sampling port of the mass spectrometer using a copper alligator clip, and connect the rear end of the alligator clip to the Miniβ small portable mass spectrometer through a wire; the size of the customized blade is 4 cm × 1.1 cm, and the tip angle is 60°.

[0049] (D) Use a clean tissue paper to absorb the solution at the tip of the blade; add 10 μL of methanol dropwise onto the surface of the magnetic beads and desorb for 1 min; add 10 μL of methanol solution containing 200 ng / mL crystal violet internal standard dropwise again; apply voltage for spray ionization.

[0050] The analyte is cell membrane glycoprotein, and the parameters for mass spectrometry detection and analysis are as follows: the spray voltage is 4.2 kV; the ionization mode is positive ion mode; the collision gas is air; the collision time is 500 ms; the average number of times is 2; the continuous number is 3; the injection volume is 40 ms; the mass-to-charge ratio of the injection low-mass end cut-off is m / z 50.

[0051] V. Results and Discussion

[0052] (1) Optimization of aptamer-functionalized magnetic nanoparticles

[0053] The CD105 aptamer End-A2-T was immobilized on hydrophilic polymer-modified magnetic nanoparticles to construct EBApt-pMNPs. By measuring the ultraviolet absorption value of the supernatant before and after the immobilization process, the immobilization amount of the aptamer on the surface of EBApt-pMNPs was determined. Higher aptamer levels were shown in the supernatants corresponding to EBApt-pMNPs-0 and EBApt-pMNPs-1, which was due to the too low usage amount of HEA during the synthesis process, thus limiting the polymerization degree on the material surface. With the increase in the amount of HEA used, the absorbance of the supernatants of EBApt-pMNPs-2 to EBApt-pMNPs-4 gradually decreased. Among them, the absorbance of EBApt-pMNPs-3 was the lowest, indicating that the largest amount of aptamer was immobilized on its surface. Through the standard curve of aptamer concentration - ultraviolet absorption value, the concentration of EBApt in the supernatant after the reaction was calculated to be 0.169 μM. This result shows that 536 μL of 4.831 μM EBApt was successfully immobilized on 5 mg of pMNPs, that is, the immobilization amount of EBApt was 0.518 μmol / g.

[0054] Synthesis of aptamer-functionalized hydrophilic polymer-modified magnetic nanoparticles:

[0055] 5.4 g of FeCl3·6H2O and 2 g of FeCl2·4H2O were dissolved in water. After adjusting the pH value to 12.0 with NH3·H2O, the mixture was stirred at 70 °C for 3 hours, washed with water and ethanol, and dried to obtain Fe3O4 powder. 200 mg of Fe3O4, 100 mL of ethanol, and 6 mL of 3-(trimethoxysilyl)propyl methacrylate (MPS) were added to a flask and stirred at room temperature for 24 hours. After washing with water and ethanol and drying, Fe3O4@MPS powder was obtained. 4-Vinyl-1,3-dioxolan-2-one (VEC) and 2-hydroxyethyl acrylate (HEA) were selected as functional monomers, and AIBN was used as an initiator. 85 mg of Fe3O4@MPS, 6 mg of initiator AIBN, 265 μL of VEC, and 48 μL of HEA were added to a three-necked round-bottom flask containing 10 mL of organic solvent 1,4-dioxane and stirred at 90 °C for 24 hours to obtain Fe3O4@MPS@p(VEC-HEA). Finally, 10 mg of Fe3O4@MPS@p(VEC-AA) was added to 570 μL of 10 μM EBApt (aptamer corresponding to CD105 protein) aqueous solution and stirred at room temperature for 24 hours to obtain EBApt-pMNPs.

[0056] (2) Determination of the fixed amount of nucleic acid dye

[0057] EBApt-pMNPs were incubated with a sufficient amount of nucleic acid dye (GelRed) to construct an aptamer-functionalized nucleic acid dye-loaded mass spectrometry probe (AGMP). The loading amount of GelRed dye on AGMP is the key to highly sensitive detection of CD105 protein. By plotting the standard curve of the peak absorption of GelRed at 280 nm in the concentration range of 0.1 - 10X, the adsorption amount of GelRed on AGMP was measured to be 1.86X. It should be noted that the UV absorption value of the magnetic polymer material without immobilized aptamer is not much different from that of the initial dye, indicating that the magnetic nanoparticles themselves do not adsorb dye molecules.

[0058] (3) Methodological investigation

[0059] Using 200 ng / mL crystal violet as an internal standard, a series of CD105 protein solutions with a concentration range of 1 to 50 ng / mL were prepared for serum sample analysis. By comparing the ratio of the mass spectrometry peak intensity of GelRed dye (m / z 412.22) and crystal violet (m / z 372.24) to the concentration of CD105 protein, a calibration curve was plotted ( Figure 2Red curve). The detection limit and quantification limit were calculated to be 0.2 ng / mL and 1.0 ng / mL respectively according to 3σ / S and 10σ / S (σ is the standard deviation of 6 blank samples, and S is the slope of the standard curve). To evaluate the matrix effect of this method, the differences in the calibration curve slopes between the matrix solution and the standard solution were compared within the same concentration range. As Figure 2 shown, compared with the calibration curve of the standard solution (red), the slope deviation of the matrix-matched calibration curve (blue) was 11.14%, indicating that this method can effectively reduce the influence of the matrix effect.

[0060] First, the ELISA method was used to determine the original content of CD105 in serum samples: a standard curve was established first, and the contents of CD105 in three serum samples were measured to be 12.1 ng / mL, 6.7 ng / mL, and 9.4 ng / mL respectively, and the results were of the same order of magnitude as those reported in the literature. Subsequently, different concentrations of CD105 (1 times the quantification limit, 10 times the quantification limit, and 20 times the quantification limit) were added to the serum samples and incubated with AGMP. The results were analyzed, and the spiked recoveries of the three samples were between 90.1% and 106.8%, and the relative standard deviations were between 3.6% and 5.1% (Table 2). In addition, compared with other reported CD105 detection methods, the method constructed by the present utility model showed a good linear relationship and comparable sensitivity (Table 1). These results demonstrated the feasibility of this method for the determination of CD105 in actual serum.

[0061] Table 1 Results of the evaluation of the detection limit, quantification limit, and linear relationship of the method using the device of the present utility model

[0062]

[0063] Table 2 Detection of spiked recoveries of AGMP for CD105 in human serum (n = 6)

[0064]

[0065] The embodiments described above are only descriptions of the preferred embodiments of the present utility model, and do not limit the scope of the present utility model. Without departing from the design spirit of the present utility model, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present utility model shall fall within the protection scope determined by the claims of the present utility model.

Claims

1. An aptamer-functionalized magnetic blade spraying device, characterized in that: It includes a mass spectrometer (2) connected by an alligator clip and a blade spray ionization device, and the blade spray ionization device includes a blade (1) and a magnet (3) located below it, and a detection solution is included on the blade (1).

2. The aptamer-functionalized magnetic blade spraying device according to claim 1, wherein: The detection solution includes aptamer-functionalized magnetic nanoparticles and a sample.

3. The aptamer-functionalized magnetic blade spraying device according to claim 1, characterized in that: The alligator clip is made of copper. The blade (1) is fixed at the front end of the inlet of the mass spectrometer (2) 5 mm away by using the alligator clip. The rear end of the alligator clip is connected to the voltage of the mass spectrometer (2) through a wire. The size of the blade (1) is 4 cm × 1.1 cm, and the tip angle is 60°.

4. The aptamer-functionalized magnetic blade spraying device according to claim 1, wherein: The mass spectrometer (2) is a Miniβ small portable mass spectrometer.

5. The aptamer-functionalized magnetic blade spraying device according to claim 1, wherein: The magnet (3) is placed at the bottom of the tip of the blade (1).

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