CN116930481A - Cross-molecule detection method for magnetic field driven micro-nano motor - Google Patents
Cross-molecule detection method for magnetic field driven micro-nano motor Download PDFInfo
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- CN116930481A CN116930481A CN202311170782.9A CN202311170782A CN116930481A CN 116930481 A CN116930481 A CN 116930481A CN 202311170782 A CN202311170782 A CN 202311170782A CN 116930481 A CN116930481 A CN 116930481A
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Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
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- 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
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- 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"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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Abstract
The invention relates to the technical field of biological materials. The invention provides a cross-molecule detection method for a magnetic field driven micro-nano motor. The method comprises the following steps: firstly synthesizing micro-nano motors with three sizes, and then respectively modifying different capturing units on the surfaces of the micro-nano motors to realize biological functionalization of the micro-nano motors so as to obtain the micro-nano motors with different biological activities. And separating the micro-nano motors with different biological activities through magnetophoresis separation, and finally combining with a corresponding detection technology to realize the integrated detection of the immune marker, the nucleic acid and the biological metabolite. According to the invention, the micro-nano motors with different biological activities are placed in the same reaction system, and the size separation principle under magnetophoresis movement is utilized by applying a magnetic field to the reaction system, so that the effective separation of the immune marker, the nucleic acid molecule and the biological metabolite is realized at the same time, and the cross-molecular detection of different substances in a detection sample is further realized by combining with a fluorescent detection probe.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to a cross-molecule detection method of a magnetic field driven micro-nano motor.
Background
Detection of different types of biomolecules, such as detection of immune markers, nucleic acid molecules, biological metabolites and the like, has important research significance in basic life science research, drug development, disease diagnosis and treatment and efficacy evaluation. For example, in clinical practice, a disease is often associated with multiple types of markers, and detection of multiple markers can provide a clinician with more comprehensive disease information, thereby helping them accurately identify patients with the disease and reducing misdiagnosis, missed diagnosis rates. However, due to the different physicochemical properties of different markers, it is difficult for a conventional laboratory or medical unit to perform detection of multiple markers simultaneously in the same system on the same platform to achieve detection across molecular classes.
Therefore, to meet the requirement of multi-element biomolecule cross-molecule detection, it is important to develop a method capable of detecting different biomolecules simultaneously in the same system.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a cross-molecule detection method for a magnetic field driven micro-nano motor.
A magnetic field driven micro-nano motor cross-molecule detection method comprises the following steps:
step one: respectively preparing magnetic rod-like micro-nano motors with different sizes;
step two: respectively coupling different biomolecules to the magnetic rod-shaped micro-nano motors with different sizes so as to obtain micro-nano motors with different biological activities;
the different biomolecules include: immunological markers, nucleic acid molecules and biochemical metabolites; the micro-nanomotor of different biological activities comprises: an immune marker micro-nanomotor, a nucleic acid molecule micro-nanomotor, a biochemical metabolite micro-nanomotor;
step three: adding the micro-nano motors with different biological activities into a biological sample to form a detection sample;
step four: applying a rotating magnetic field to the detection sample to enable the micro-nano motors with different biological activities to perform rotating stirring motion so as to accelerate the capture of the detection sample on biological molecules;
step five: applying a composite magnetic field to the detection sample subjected to the rotary stirring motion, and separating the micro-nano motors with different biological activities through the composite magnetic field, so as to respectively obtain micro-nano motors with corresponding biological activities;
step six: and detecting the micro-nano motor with corresponding biological activity by using a corresponding fluorescence detection probe so as to realize in-situ detection of the cross molecule.
Further, in the above method for detecting a cross molecule of a magnetic field driven micro-nano motor, the preparation of the magnetic rod-shaped micro-nano motor in the first step includes:
step 11: feCl is added 3 ·6H 2 Adding glycol into O, dispersing with ultrasound, adding ammonium acetate into the mixture, stirring and dissolving to obtain FeCl 3 A solution;
step 12: dissolving FeCl 3 Transferring the solution into a reaction kettle, performing high-temperature reaction on the solution 12 and h, and cooling the solution to room temperature to obtain Fe 3 O 4 A reaction product;
step 13: enriching the synthesized Fe with magnet 3 O 4 The reaction product is respectively washed and dried by ethanol and deionized water to obtain pure Fe 3 O 4 A reaction product;
step 14: will beThe pure Fe 3 O 4 Adding the reaction product into a mixed solution formed by deionized water and isopropanol, and carrying out ultrasonic treatment for 30 min;
step 15: applying a magnetic field of 1.6 mT to the mixed solution after ultrasonic treatment, adding ammonia water, and oscillating for 10 min;
step 16: TEOS is added to react at room temperature for 6 h, and bar-shaped Fe is obtained after the reaction is finished 3 O 4 @SiO 2 A product;
step 17: the rod-shaped Fe 3 O 4 @SiO 2 The product is enriched by a magnet, and is washed by ethanol and deionized water respectively, and finally Fe with a one-dimensional magnetic rod-shaped structure is obtained 3 O 4 @SiO 2 ；
Step 18: fe of the one-dimensional magnetic rod-like structure 3 O 4 @SiO 2 Ultrasonic dispersing in ethanol, adding APTES, stirring for 24 h to obtain aminated micro-nano motor Fe 3 O 4 @SiO 2 -NH 2 And washing the product with ethanol to finally obtain the magnetic rod-shaped micro-nano motor.
Further, according to the method for detecting the cross molecule of the magnetic field driven micro-nano motor, the preparation of the immune marker micro-nano motor comprises the following steps:
dissolving the magnetic rod-shaped micro-nano motor in DMF, adding succinic anhydride SAA and triethylamine TEA, stirring 24-h, and washing with ethanol for 3 times to obtain Fe 3 O 4 @SiO 2 -COOH, and storing it in plasma water by ultrasonic dispersion;
weighing the Fe 3 O 4 @SiO 2 -COOH, which is sonicated in ethanesulfonic acid MES buffer at ph=6.5, EDC and NHS are added and reacted at room temperature for 1 h to activate the carboxyl groups;
after the reaction was completed, the MES buffer was replaced with phosphate PBS buffer at ph=7.4 (137 mM NaCl,2.7 mM KCl,8 mM Na 2 HPO 4 And 2 mM KH 2 PO 4 Ph=7.4), and then antibody Ab was added, and reaction was performed at room temperature 4h;
replacing the phosphate PBS buffer with 2mL 1% bovine serum albumin BSA, reacting 1 h to block unreacted carboxyl sites;
after the end of the closure, the reaction product Fe was collected 3 O 4 @SiO 2 And Ab, finally obtaining the immune marker micro-nano motor.
Further, the method for detecting the cross-molecule of the magnetic field driven micro-nano motor comprises the following steps:
the magnetic rod-shaped micro-nano motor is weighed and added into PBS solution containing glutaraldehyde (glutaraldehyde 0.5 M,137 mM NaCl,2.7 mM KCl,8 mM Na) 2 HPO 4 And 2 mM KH 2 PO 4 pH 7.2-7.4), for 4h;
after the reaction is finished, phosphate PBS buffer solution is washed for three times, and avidin is added for reaction 4h;
after the reaction is finished, washing the mixture for three times by using a phosphate PBS buffer solution, and dispersing the mixture in the phosphate PBS buffer solution to obtain the avidin micro-nano motor;
taking the avidin-treated micro-nano motor, adding the DNA of the biotinylated nucleic acid capture probe, reacting at 37 ℃ for 2 h, and then washing with phosphate PBS buffer solution for 3 times to obtain Fe 3 O 4 @SiO 2 -DNA, i.e. the nucleic acid molecule micro-nanomotor.
Further, the method for detecting the cross-molecule of the magnetic field driven micro-nano motor comprises the following steps:
dissolving the magnetic rod-shaped micro-nano motor in DMF, adding succinic anhydride SAA and triethylamine TEA, stirring 24-h, and washing with ethanol for 3 times to obtain Fe 3 O 4 @SiO 2 -COOH, and storing it in plasma water by ultrasonic dispersion;
the Fe is 3 O 4 @SiO 2 After washing COOH 3 times with phosphate PBS buffer, it was added to a PBS solution containing NHS and EDC, and reacted for 15 min to activate carboxyl groups;
after the reaction is finished, phosphate PBS buffer solution is washed for 3 times, and aminated DNA single-chain aptamer is added for reaction 4h, thus obtaining Fe 3 O 4 @SiO 2 Apt, the biochemical metabolite micro-nanomotor.
Further, the method for detecting the cross-molecule of the magnetic field driven micro-nano motor, after the third step and before the fourth step, further comprises the following steps:
and applying an oscillating dispersion magnetic field to the detection sample to remove biomolecules which are not specifically bound on the surface of the micro-nano motor.
The beneficial effects are that: according to the invention, the magnetic rodlike micro-nano motors with different sizes are prepared, and corresponding different biological molecules are respectively coupled on the magnetic rodlike micro-nano motors with different sizes, so that the micro-nano motors with different biological activities are obtained, and finally, the micro-nano motors with different biological activities are placed in the same reaction system, and through a mode of applying a magnetic field to the reaction system, the effective separation of immune markers, nucleic acid molecules and biological metabolites is realized simultaneously by utilizing the size separation principle under magnetophoresis movement, and the detection of the cross molecules of different types of substances in a detection sample is further realized by combining with a fluorescent detection probe. Furthermore, in order to enable the effective separation of the immunological marker, the nucleic acid molecule and the biological metabolite, the present invention realizes the effective separation of the immunological marker, the nucleic acid molecule and the biological metabolite by preparing the magnetic rod-shaped micro-nano motor.
In addition, the present invention utilizes carboxylated Fe 3 O 4 @SiO 2 The micro-nano motor for capturing the immune marker is prepared by dehydrating condensation of the micro-nano motor and the antibody, and the capture of the immune marker by the micro-nano motor is realized based on the specific combination of antigen-antibody; the invention prepares a micro-nano motor for capturing nucleic acid molecules by utilizing the specific combination of avidin and a biotinylated nucleic acid capture probe, and realizes the capturing of the micro-nano motor to the nucleic acid molecules based on the principle of base complementation pairing; the invention utilizes carboxylated Fe 3 O 4 @SiO 2 The micro-nano motor for capturing biological metabolites is prepared by dehydration condensation of the micro-nano motor and the aminated nucleic acid aptamer, and the capturing of the biological metabolites by the micro-nano motor is realized based on the specific recognition of the aptamer-ligand.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In order to solve the problem that different biomolecules are difficult to detect simultaneously on the same platform in the prior art, the invention provides a cross-molecule detection method for a magnetic field driven micro-nano motor. Firstly, three micro-nano motors with different sizes are synthesized, and different capturing units (an immune marker capturing unit, a nucleic acid molecule capturing unit and a biological metabolite capturing unit) are further modified on the surfaces of the micro-nano motors respectively so as to realize biological functionalization of the micro-nano motors, thereby obtaining the labeled micro-nano motors for immune markers, nucleic acid molecules and biological metabolites. And separating the micro-nano motors with different biological activities into different detection grooves through magnetophoresis separation, and then combining with corresponding detection technologies to realize the integrated detection of the immune markers, the nucleic acid and the biological metabolites.
The cross-molecule detection method for the magnetic field driven micro-nano motor provided by the embodiment of the invention comprises the following steps:
step one: respectively preparing magnetic rod-like micro-nano motors with different sizes
(11) Fe synthesis by hydrothermal method 3 O 4 Nano microsphere: weighing a certain amount of FeCl 3 ·6H 2 O30 mL ethylene glycol was added and dispersed with ultrasound, then 1.925 g ammonium acetate was added thereto and dissolved with stirring. Transferring the dissolved solution into a reaction kettle, performing high-temperature reaction for 12-h, cooling to room temperature, enriching the synthesized reaction product by using a magnet, and washing with ethanol and deionized water for 3 times respectively. Subsequently, the product is dried in air to obtain Fe 3 O 4 The nanometer microsphere is dried and preserved at normal temperature.
(12) Preparation of Fe by sol-gel method 3 O 4 @SiO 2 Rod-like microNano motor: weigh the Fe stored in one step of 50 mg 3 O 4 The nanoparticle was added to a mixed solution of 40 mL deionized water and 100 mL isopropyl alcohol and sonicated for 30 min. Then a magnetic field of 1.6 and mT is applied to the reaction system, 500 mu L of ammonia water is added, and the mixture is vibrated for 10 min; then, 500. Mu.L of tetraethyl orthosilicate (TEOS) was added thereto, and the reaction was carried out at room temperature for 6 h. After the reaction is finished, the reaction products are respectively washed for 2 times by using ethanol and deionized water through magnet enrichment, and Fe is obtained by collecting 3 O 4 @SiO 2 One-dimensional magnetic rod-shaped structure is stored in ethanol for standby.
The invention is realized by controlling FeCl 3 ·6H 2 The addition amount of O can prepare Fe with different particle diameters 3 O 4 The nano microsphere adopts the addition amount of 0.5 g, 0.6 g and 0.7 g to prepare the Fe with three particle sizes 3 O 4 A nanoparticle; then Fe with three particle sizes 3 O 4 The nanometer microspheres are aminated respectively, so that three sizes of aminated micro-nano motor Fe are obtained 3 O 4 @SiO 2 -NH 2 。
Wherein the formation of the rod-like structure is due to Fe 3 O 4 Self-assembling the nano microsphere into a rod shape in an externally applied magnetic field, and condensing tetraethyl orthosilicate catalyzed and hydrolyzed by ammonia water on the surface of the rod-shaped structure to form SiO 2 A shell layer to cure and stabilize the structure.
(13) To achieve biological functionalization of the micro-nano motor, fe with a one-dimensional magnetic rod-like structure is needed to be prepared 3 O 4 @SiO 2 Surface amination:
respectively obtaining three rod-shaped micro-nano motors Fe with three sizes 3 O 4 @SiO 2 Ultrasonic dispersing in ethanol, adding 100 μl of 3-aminopropyl triethoxysilane (APTES), stirring for 24 h to obtain product Fe 3 O 4 @SiO 2 -NH 2 And the product was washed three times with ethanol; subsequently, fe 3 O 4 @SiO 2 -NH 2 Dispersed in Dimethylformamide (DM)F) In the process, the mixture is stored at 4 ℃ for standby.
Step two: and respectively coupling the magnetic rod-shaped micro-nano motors with different sizes with corresponding different biomolecules to obtain micro-nano motors with corresponding different biological activities, thereby realizing the biological functionalization of the micro-nano motors. The different biomolecules include: immunological markers, nucleic acid molecules and biochemical metabolites; the micro-nanomotor of different biological activities comprises: immune marker micro-nanomotor, nucleic acid molecule micro-nanomotor, biochemical metabolite micro-nanomotor.
Wherein, the preparation of the immune marker micro-nano motor comprises the following steps:
monoclonal antibodies for immunolabel capture were coupled to micro-nanomotor surfaces by covalent binding of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide to N-hydroxysuccinimide (EDC)/N-hydroxysuccinimide (NHS) by the principle of: by means of EDC supplied-NH 2 With Fe 3 O 4 @SiO 2 Dehydration condensation of-COOH to form an intermediate product which is catalyzed by NHS with-NH of the antibody 2 And (3) reacting, and coupling the antibody on the surface of the micro-nano motor. The method comprises the following specific steps:
first, 15mg of Fe 3 O 4 @SiO 2 -NH 2 Dissolving in 5mL DMF, adding 17 μl succinic anhydride (Succinic anhydride, SAA) and 17 mg Triethylamine (TEA), stirring 24 h, and washing with ethanol for 3 times to obtain Fe 3 O 4 @SiO 2 -COOH, stored with ultrasonic dispersion in plasma water. Subsequently, 1mgFe was weighed 3 O 4 @SiO 2 -COOH, sonicated in ethanesulfonic acid (MES) buffer at ph=6.5, and 1.5 mg EDC and 1.2mg nhs were added and reacted at room temperature for 1 h to activate carboxyl groups. After the reaction was completed, the MES buffer was replaced with phosphate PBS (phosphate buffered saline, PBS) buffer (137 mM NaCl,2.7 mM KCl,8 mM Na 2 HPO 4 And 2 mM KH 2 PO 4 ) An additional 0.3mg of antibody (Ab) was added and reacted at room temperature 4. 4 h. Next, phosphoric acid is addedThe salt PBS buffer was replaced with 2mL of 1% bovine serum albumin (bovine serum albumin, BSA) by mass fraction, and 1 h was reacted to block unreacted carboxyl sites. After the end of the closure, the reaction mass was collected and the Fe obtained 3 O 4 @SiO 2 -Ab was dispersed in 1% BSA solution and stored at 4 ℃ for later use.
In the embodiment of the invention, an NGAL antibody is selected as a target coupled with a magnetic rod-shaped micro-nano motor.
The preparation of the nucleic acid molecule micro-nano motor comprises the following steps:
the biotinylated nucleic acid capture probe is coupled to the surface of the avidin-labeled micro-nanomotor by utilizing the specific binding of biotin to avidin. The specific method comprises the following steps:
weighing 15mg of Fe obtained in the step (3) 3 O 4 @SiO 2 -NH 2 25mL of a PBS solution containing 5% glutaraldehyde (glutaraldehyde 0.5 M,137 mM NaCl,2.7 mM KCl,8 mM Na) was added 2 HPO 4 And 2 mM KH 2 PO 4 pH 7.2-7.4), for 4h; after the reaction is finished, phosphate PBS buffer solution is washed for three times, 300 mu L of avidin with the concentration of 1 mg/mL is added, and the reaction is carried out for 4h; and after the reaction is finished, washing the mixture for three times by using a phosphate PBS buffer solution, and dispersing the mixture in the phosphate PBS buffer solution to obtain the avidin micro-nano motor. 100. Mu.L of the above-mentioned 1 mg/mL avidity Fe was taken 3 O 4 @SiO 2 20. Mu.L of 50 nM biotinylated nucleic acid capture probe (DNA) was added and reacted at 37℃for 2 h, followed by 3 washes with phosphate PBS buffer to obtain Fe 3 O 4 @SiO 2 DNA, dispersed in PBS by ultrasound, stored at 4℃for further use.
In the embodiment of the invention, miR-188 complementary fragments are selected as targets for coupling the magnetic rod-shaped micro-nano motor.
The preparation of the biochemical metabolite micro-nano motor comprises the following steps:
15mg Fe 3 O 4 @SiO 2 -NH 2 Dissolving in 5mL DMF, adding 17 μl succinic anhydride (Succinic anhydride, SAA) and 17 mg Triethylamine (TEA), stirring 24 h, and washing with ethanol for 3 times to obtain Fe 3 O 4 @SiO 2 -COOH, stored with ultrasonic dispersion in plasma water.
10 mu L (5 mg/mL) of Fe was taken 3 O 4 @SiO 2 -COOH with PBS buffer at ph=6.2 (137 mM NaCl,2.7 mM KCl,8 mM Na 2 HPO 4 And 2 mM KH 2 PO 4 ) Washing 3 times, adding the mixture into 200 mu L of PBS solution (100 mM NHS,400 mM EDC,pH6.2) containing NHS and EDC, and reacting for 15 min to activate carboxyl; after the reaction, PBS was washed 3 times, and an aminated DNA single-stranded aptamer (aptamer) was added to react with 4. 4h to obtain Fe 3 O 4 @SiO 2 Apt, washed 3 times with deionized water and dispersed in deionized water, stored at-4 ℃ for further use.
In the embodiment of the invention, a Cys C aptamer is selected as a target for coupling a magnetic rod-shaped micro-nano motor.
Step three: adding the micro-nanomotors with different biological activities to a biological sample to form a detection sample.
(31) Sequentially adding micro-nano motors with different sizes, which are coupled with NGAL antibodies, miR-188 complementary fragments and Cys C aptamers, and serum samples into a reaction tank;
(32) Applying a rotating magnetic field to enable the micro-nano motor to perform rotating stirring movement, and accelerating capturing of NGAL, miR-188 and Cys C in a sample;
(33) Applying a gradient magnetic field to enable the micro-nano motor to enter the cleaning tank from the reaction tank;
(34) Applying an oscillating dispersion magnetic field for removing biomolecules non-specifically bound to the surface of the motor;
(35) Applying a gradient magnetic field (composite magnetic field) parallel to and perpendicular to the movement direction of the motor, enabling the motor to enter a separation tank from a cleaning tank by using the magnetic field parallel to the motor, and sorting the micro-nano motors capturing different types of substances into different detection tanks by using the magnetic field perpendicular to the motor based on the size separation principle under magnetophoresis movement;
(36) And adding corresponding fluorescent detection probes into the detection groove for detecting NGAL, miR-188 and Cys C in the serum sample.
The method provided by the invention has the following advantages:
(1) The invention utilizes a magnetic field to control the sorting of micro-nano motors with different sizes, and builds a platform for capturing immune markers, nucleic acid molecules and biological metabolites in a liquid detection sample; by coupling the traditional optical detection method, the cross-molecule detection of different types of biomarkers in a single sample is realized;
(2) The method can be used for preparing magnetic rod-shaped micro-nano motors with different sizes, and the main raw material Fe related by the method 3 O 4 The preparation method has the advantages of easy acquisition, low cost, high stability of the obtained product, easy modification, simple operation, good repeatability and easy realization of large-scale batch production;
(3) The method for preparing the micro-nano motor with different biological activities is simple to operate and has higher universality, and the capture probes aiming at different biological molecules can be prepared by replacing the types of immune markers, nucleic acid molecules and biological metabolites, so that the method has a huge application prospect in the aspect of detecting the multiple biological molecules in a biological sample;
(4) The micro-nano motor prepared by the invention is of a magnetic rod-shaped structure with controllable movement, can be applied to detection of substances at the organ level, guides directional movement by utilizing magnetic field guidance, and can accurately position the distribution conditions of different substances in a designated area by combining with a corresponding detection technology, thereby further realizing in-situ detection and dynamic monitoring of substances of cross-molecular type. In addition, the cluster motion of the micro-nano motor can be utilized to perform rapid separation, extraction and enrichment of the object to be detected, so that complicated operations such as centrifugal treatment and the like are omitted, interference of matrix components in a complex sample can be avoided, and the sensitivity and the specificity of detection are enhanced.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (6)
1. A magnetic field driven micro-nano motor cross-molecule detection method is characterized by comprising the following steps:
step one: respectively preparing magnetic rod-like micro-nano motors with different sizes;
step two: respectively coupling different biomolecules to the magnetic rod-shaped micro-nano motors with different sizes so as to obtain micro-nano motors with different biological activities;
the different biomolecules include: immunological markers, nucleic acid molecules and biochemical metabolites; the micro-nanomotor of different biological activities comprises: an immune marker micro-nanomotor, a nucleic acid molecule micro-nanomotor, a biochemical metabolite micro-nanomotor;
step three: adding the micro-nano motors with different biological activities into a biological sample to form a detection sample;
step four: applying a rotating magnetic field to the detection sample to enable the micro-nano motors with different biological activities to perform rotating stirring motion so as to accelerate capture of the micro-nano motors on biological molecules in the detection sample;
step five: applying a composite magnetic field to the detection sample subjected to the rotary stirring motion, and separating the micro-nano motors with different biological activities through the composite magnetic field, so as to respectively obtain micro-nano motors with corresponding biological activities;
step six: and detecting the micro-nano motor with corresponding biological activity by using a corresponding fluorescence detection probe so as to realize in-situ detection of the cross molecule.
2. The method for cross-molecule detection of a magnetic field driven micro-nano motor according to claim 1, wherein the preparing of the magnetic rod-shaped micro-nano motor in the step one comprises:
step 11: feCl is added 3 ·6H 2 O is added with glycol and dispersed by ultrasonic, then ammonium acetate is added and stirred for dissolutionObtaining FeCl 3 A solution;
step 12: dissolving FeCl 3 Transferring the solution into a reaction kettle, performing high-temperature reaction on the solution 12 and h, and cooling the solution to room temperature to obtain Fe 3 O 4 A reaction product;
step 13: enriching the synthesized Fe with magnet 3 O 4 The reaction product is respectively washed and dried by ethanol and deionized water to obtain pure Fe 3 O 4 A reaction product;
step 14: subjecting the purified Fe to 3 O 4 Adding the reaction product into a mixed solution formed by deionized water and isopropanol, and carrying out ultrasonic treatment for 30 min;
step 15: applying a magnetic field of 1.6 mT to the mixed solution after ultrasonic treatment, adding ammonia water, and oscillating for 10 min;
step 16: TEOS is added to react at room temperature for 6 h, and bar-shaped Fe is obtained after the reaction is finished 3 O 4 @SiO 2 A product;
step 17: the rod-shaped Fe 3 O 4 @SiO 2 The product is enriched by a magnet, and is washed by ethanol and deionized water respectively, and finally Fe with a one-dimensional magnetic rod-shaped structure is obtained 3 O 4 @SiO 2 ；
Step 18: fe of the one-dimensional magnetic rod-like structure 3 O 4 @SiO 2 Ultrasonic dispersing in ethanol, adding APTES, stirring for 24 h to obtain aminated micro-nano motor Fe 3 O 4 @SiO 2 -NH 2 And washing with ethanol to finally obtain the magnetic rod-shaped micro-nano motor.
3. The method for cross-molecule detection of a magnetic field driven micro-nanomotor of claim 2, wherein the preparation of the immunomarker micro-nanomotor comprises:
dissolving the magnetic rod-shaped micro-nano motor in DMF, adding succinic anhydride SAA and triethylamine TEA, stirring 24-h, and washing with ethanol for 3 times to obtain Fe 3 O 4 @SiO 2 -COOH and ultrasonically dispersing it in a plasmaPreserving in the seed water;
the Fe is 3 O 4 @SiO 2 -COOH, sonicated in ethanesulfonic acid MES buffer at ph=6.5, and EDC and NHS added to react at room temperature 1 h to activate the carboxyl group;
after the reaction is finished, replacing the MES buffer solution with a phosphate PBS buffer solution, adding an antibody Ab, and reacting at room temperature for 4h;
replacing the phosphate PBS buffer with 1% bovine serum albumin BSA by mass fraction, and reacting 1 h to block unreacted carboxyl sites;
after the end of the closure, the reaction product Fe was collected 3 O 4 @SiO 2 -Ab, as the reaction product Fe 3 O 4 @SiO 2 -Ab as the immune marker micro-nanomotor.
4. The method for cross-molecule detection of a magnetic field driven micro-nanomotor of claim 2, wherein the preparation of the nucleic acid molecule micro-nanomotor comprises:
adding the magnetic rod-shaped micro-nano motor into a PBS solution with mass fraction of 5% glutaraldehyde, and reacting 4. 4h;
after the reaction is finished, phosphate PBS buffer solution is washed for three times, and avidin is added for reaction 4h;
after the reaction is finished, washing the mixture for three times by using a phosphate PBS buffer solution, and dispersing the mixture in the phosphate PBS buffer solution to obtain the avidin micro-nano motor;
taking the avidin-treated micro-nano motor, adding the DNA of the biotinylated nucleic acid capture probe, reacting at 37 ℃ for 2 h, and then washing with phosphate PBS buffer solution for 3 times to obtain Fe 3 O 4 @SiO 2 -DNA, i.e. the nucleic acid molecule micro-nanomotor.
5. The method for cross-molecule detection of a magnetic field driven micro-nanomotor of claim 2, wherein the preparation of the biochemical metabolite micro-nanomotor comprises:
dissolving the magnetic rod-shaped micro-nano motor in DMF, and then adding succinic anhydride SAA and triethylamineTEA, stirring 24 and h, and washing with ethanol for 3 times to obtain Fe 3 O 4 @SiO 2 -COOH, and storing it in plasma water by ultrasonic dispersion;
the Fe is 3 O 4 @SiO 2 After washing COOH 3 times with phosphate PBS buffer, it was added to a PBS solution containing NHS and EDC, and reacted for 15 min to activate carboxyl groups;
after the reaction is finished, phosphate PBS buffer solution is washed for 3 times, and aminated DNA single-chain aptamer is added for reaction 4h, thus obtaining Fe 3 O 4 @SiO 2 Apt, the biochemical metabolite micro-nanomotor.
6. The method for cross-molecule detection of a magnetic field driven micro-nanomotor according to any one of claims 1 to 5, further comprising, after step three and before step four:
and applying an oscillating dispersion magnetic field to the detection sample to remove biomolecules which are not specifically bound on the surface of the micro-nano motor.
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