CN113527600A - Microsphere and preparation method and detection method thereof - Google Patents
Microsphere and preparation method and detection method thereof Download PDFInfo
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- CN113527600A CN113527600A CN202110658756.5A CN202110658756A CN113527600A CN 113527600 A CN113527600 A CN 113527600A CN 202110658756 A CN202110658756 A CN 202110658756A CN 113527600 A CN113527600 A CN 113527600A
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- 238000002360 preparation method Methods 0.000 title abstract description 15
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- MYRTYDVEIRVNKP-UHFFFAOYSA-N 1,2-Divinylbenzene Chemical compound C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 claims description 6
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 6
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- 239000000126 substance Substances 0.000 claims description 5
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 claims description 4
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 claims description 4
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- 229910021577 Iron(II) chloride Inorganic materials 0.000 claims description 4
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 4
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 claims description 4
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- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 claims description 4
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- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 claims description 4
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- 238000005070 sampling Methods 0.000 claims description 4
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 claims description 3
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 3
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims description 3
- 239000004342 Benzoyl peroxide Substances 0.000 claims description 3
- OMPJBNCRMGITSC-UHFFFAOYSA-N Benzoylperoxide Chemical compound C=1C=CC=CC=1C(=O)OOC(=O)C1=CC=CC=C1 OMPJBNCRMGITSC-UHFFFAOYSA-N 0.000 claims description 3
- 229910002567 K2S2O8 Inorganic materials 0.000 claims description 3
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims description 3
- 239000007825 activation reagent Substances 0.000 claims description 3
- 235000019400 benzoyl peroxide Nutrition 0.000 claims description 3
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 claims description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F292/00—Macromolecular compounds obtained by polymerising monomers on to inorganic materials
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/06—Making microcapsules or microballoons by phase separation
- B01J13/14—Polymerisation; cross-linking
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F8/00—Chemical modification by after-treatment
- C08F8/42—Introducing metal atoms or metal-containing groups
-
- 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
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
-
- 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
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
- G01N27/626—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using heat to ionise a gas
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- Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Electrochemistry (AREA)
- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Polymers & Plastics (AREA)
- Immunology (AREA)
- General Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Inorganic Chemistry (AREA)
- Materials Engineering (AREA)
- Dispersion Chemistry (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention provides a microsphere and a preparation method and a detection method thereof, wherein the microsphere comprises a spherical body; the surface of the body is modified with functional groups, and a chelate is coupled to part of the functional groups and chelates metal ions. The invention has the advantages of high detection accuracy, rapidness and the like.
Description
Technical Field
The invention relates to microspheres, in particular to microspheres and a preparation method and a detection method thereof.
Background
The magnetic polymer microsphere is a micron-sized magnetic sphere which is prepared by combining an organic polymer and an inorganic magnetic material by a certain method and has magnetism and a certain special structure. Because of the characteristics of unique magnetic responsiveness, large specific surface area, good biocompatibility, easy surface functionalization and the like, the Magnetic Resonance Imaging (MRI) probe has wide application in various fields of biomedicine, separation engineering and the like, such as Magnetic Resonance Imaging (MRI), cell marking and separation, nucleic acid and protein separation and purification and the like. The magnetic microspheres can realize high flux and rapid detection of the biomarkers when applied to the flow cytometry, and have very important application prospects in the field of biological detection.
In the traditional flow cytometry technology, magnetic microspheres are used as main detection carriers, the microspheres can be coded through fluorescent dyeing, then protein/nucleic acid probes and the microspheres are coupled to form fluorescent probes, and then the fluorescent probes and an object to be detected perform biological reaction, so that the aim of detecting antibodies/nucleic acids is fulfilled. The technology mainly uses various fluorescent groups as labels of objects to be detected, the detection means is a laser and a photomultiplier, in the detection process, the problem of fluorescence cross color easily occurs between different channels, additional fluorescence compensation adjustment is needed, the detection channel of the flow type fluorescence technology is limited, and when multi-parameter detection is carried out, the adjustment of fluorescence compensation becomes abnormally complex.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a microsphere.
The purpose of the invention is realized by the following technical scheme:
a microsphere comprising a spherical body; the surface of the body is modified with functional groups, and a chelate is coupled to part of the functional groups and chelates metal ions.
The invention also aims to provide a preparation method of the microsphere, and the aim of the invention is realized by the following technical scheme:
according to the preparation method of the microsphere, the preparation method of the microsphere comprises the following steps:
adding the spherical body with the surface modified with the functional group into a chemical activation reagent to activate the functional group;
adding a metal chelating agent, and reacting;
a metal salt is added and the chelating agent chelates the metal ions in the metal salt.
The invention also aims to apply the detection method of the microsphere, and the invention aims to be realized by the following technical scheme:
the detection method based on the mass spectrometry and the microspheres comprises the following steps:
providing a plurality of microspheres of the application, wherein the composition of metal ions chelated by each microsphere is different, probes on the surface of each microsphere are different, and the mass number of the metal ions corresponds to the mass number of the probes one by one;
the probes of the microspheres respectively perform specific reaction with various nucleic acids or antibodies in a sample to be detected;
the composition of metal ions on various microspheres is detected by using a mass spectrometry technology, so that the information of specific reaction between a sample and a nucleic acid probe or an antibody modified on the microspheres is obtained.
Compared with the prior art, the invention has the beneficial effects that:
1. the method for modifying lanthanide metal ions by the (magnetic) microspheres is simple and effective, and has strong operability;
ANTA and Iso-EDTA have very strong coordination ability, can be tightly chelated with metal ions, and have high binding efficiency;
2. the detection accuracy and the resolution are high;
the majority of the metals selected as the markers are rare metals, such as lanthanide metals, and the content of the metals in the cells is basically zero, so that the detection background is extremely low;
mass spectrometry technologies such as ICP-MS (inductively coupled plasma-mass spectrometry) are large in detection channel number, hundreds of different parameters can be detected at one time, interference between adjacent channels is small, the problem of fluorescence cross color during flow cytometry detection is solved, and additional adjustment of fluorescence compensation is not needed;
the mass spectrometry technology, such as ICP-TOF-MS technology, is used for detecting the content of metal ions with different mass quantities, so that the detection accuracy of the metal ions is obviously improved, namely the accuracy of nucleic acid detection is improved;
in debugging, the spatial positions of the vertical torch tube and the coil are accurately and synchronously adjusted to reach the optimal position under each working condition, and the most accurate detection data is obtained;
the fine adjustment of the torch tube in three dimensions is realized, the position precision and the repetition precision in three directions can reach 0.01mm, and the optimal position of the flame and the cone of the torch tube is realized;
the flight time mass spectrometer can realize second-order time focusing on wider ion initial position dispersion, and the mass resolution is obviously improved;
3. the detection sensitivity is high;
the technical requirement on high-voltage pulse can be reduced by adopting a double-pulse repulsion technology; the invention adopts a double-repulsion mode of positive pulse pushing (repulsion electrode) and negative pulse pulling (traction electrode), the requirement of high voltage can be reduced by half, so that the rising edge is steeper and the pulse waveform can be improved;
the first grid and the second grid with equal electric potential are added in the middle of the double-pulse repulsion, so that the electric field permeation effect of the acceleration region on the ion modulation region can be reduced;
the first grid mesh and the second grid mesh are directly grounded, no extra voltage is added, and the adjusting difficulty is small;
the wider modulation region can be realized, and the ion flux and the sensitivity are improved;
4. the reliability is good;
the torch tube is vertically arranged and keeps relative static with the coil, and the torch tube and the coil move synchronously, so that the torch tube is prevented from being burnt out due to the fact that the torch tube is close to the coil when moving, and the problem that a sampling cone is burnt and deformed is solved;
the rotation of the motor is reliably converted into the vertical movement of the conversion piece by utilizing the conversion module and the conversion piece, and the bearing piece and the conversion piece only move in the vertical direction by using a plurality of guide pieces, so that the inclination of the bearing piece is effectively prevented, and the positioning accuracy and reliability of the torch tube are ensured;
drawings
The disclosure of the present invention will become more readily understood with reference to the accompanying drawings. As is readily understood by those skilled in the art: these drawings are only for illustrating the technical solutions of the present invention and are not intended to limit the scope of the present invention. In the figure:
FIG. 1 is a flow chart of a method of making microspheres according to an embodiment of the invention;
FIG. 2 is a schematic diagram of the structure of a microsphere according to an embodiment of the invention;
FIG. 3 is a schematic representation of another structure of a microsphere according to an embodiment of the present invention;
FIG. 4 is a schematic diagram of a mass spectrometer according to an embodiment of the present invention;
FIG. 5 is a schematic structural diagram of a carrying unit according to an embodiment of the present invention;
FIG. 6 is a schematic diagram of a time-of-flight mass analyzer in accordance with an embodiment of the present invention;
fig. 7 is a schematic structural diagram of a time-of-flight mass analyzer according to an embodiment of the present invention.
Detailed Description
Fig. 1-7 and the following description depict alternative embodiments of the invention to teach those skilled in the art how to make and use the invention. Some conventional aspects have been simplified or omitted for the purpose of explaining the technical solution of the present invention. Those skilled in the art will appreciate that variations or substitutions from these embodiments will be within the scope of the invention. Those skilled in the art will appreciate that the features described below can be combined in various ways to form multiple variations of the invention. Thus, the present invention is not limited to the following alternative embodiments, but is only limited by the claims and their equivalents.
Example 1:
fig. 1 shows a flow chart of a preparation method of microspheres according to an embodiment of the present invention, and as shown in fig. 1, the preparation method of the microspheres includes:
adding the spherical body with the surface modified with the functional group into a chemical activation reagent to activate the functional group;
adding a metal chelating agent, and reacting;
a metal salt is added and the chelating agent chelates the metal ions in the metal salt.
The microsphere prepared by the preparation method comprises a spherical body, for example, the microsphere is made of polystyrene; the surface of the body is modified with functional groups, and a chelate is coupled to part of the functional groups and chelates metal ions.
To enhance the coupling effect, further, the functional group is an amino group or a carboxyl group, the chelate is ANTA or Iso-EDTA, and the metal ion is a lanthanide metal ion.
In the above preparation method, the spherical body with the surface modified with the functional group is prepared by the following steps:
FeCl2·4H2o and FeCl3·6H2Adding pure water and dispersing, and heating and stirring under the condition of inert gas; addition of NH3·H2O and oleic acid react; magnetically separating the product and washing with pure water until the pH value of the washing liquid is neutral to obtain Fe3O4A nanoparticle;
Fe3O4dissolving solid and SDS in pure water, dispersing, adding styrene, stirring under inert gas, adding K2S2O8Heating for reaction; adding acrylic acid for continuous reaction, and obtaining powder Fe after vacuum drying3O4Sample @ PS-COOH; or taking Fe3O4Dispersing in styrene, adding divinylbenzene, benzoyl peroxide and methacrylic acid to form magnetic fluid, and adding dropwise the magnetic fluid into an aqueous solution containing cetyl alcohol and sodium dodecyl sulfate; adding aqueous solution of acrylamide and potassium persulfate under the condition of inert gas, wherein the product is Fe3O4@PS-NH2。
The detection method based on the mass spectrometry and the microspheres of the embodiment of the invention comprises the following steps:
providing a plurality of microspheres prepared in the embodiment, wherein each microsphere has different compositions of chelated metal ions (such as different mass numbers or different metal ions) and different probes on the surface, and the mass numbers of the metal ions correspond to the probes one by one;
the probes of the microspheres respectively perform specific reaction with various nucleic acids or antibodies in a sample to be detected;
the composition of metal ions on various microspheres is detected by using a mass spectrometry technology, so that the information of specific reaction between a sample and a nucleic acid probe or an antibody modified on the microspheres is obtained.
In order to improve the detection accuracy, further, in the mass spectrometry, the specific ionization mode is as follows:
the motor rotates, and the conversion module converts the rotation of the motor into the linear movement of the sliding piece;
converting the linear movement of the sliding part into the vertical movement of the conversion part, so as to drive the bearing part connected with the conversion part to vertically move along a plurality of guide parts, and the bearing unit arranged on the bearing part vertically moves along with the bearing part; the torch tube is vertically arranged on the carrying unit, and the coil is fixed on the carrying unit and is kept static relative to the torch tube;
the position of the bearing unit is adjusted in a two-dimensional mode in the horizontal direction, the bearing unit is arranged on the two-dimensional adjusting unit, and the two-dimensional adjusting unit is arranged on the bearing piece;
the cells enter the torch tube one by one, and are excited into plasma through the coil, so that ions are formed;
the ions pass through a sampling cone and enter a mass spectrometry unit.
To improve the detection accuracy, further, in the mass spectrometric detection of single cells, a time-of-flight mass analyzer is used, comprising a repeller, a field-free flight zone comprising a first entrance grid and a detector; the time-of-flight mass analyser further comprises:
a first ion acceleration region is formed between the traction electrode and the first incident grid;
the device comprises a first grid and a second grid, wherein the potential difference between the first grid and the second grid is zero; a second ion acceleration area is formed between the repulsion electrode and the first grid, and between the second grid and the traction electrode; the ions sequentially pass through the first grid mesh, the second grid mesh, the traction electrode, the first incidence grid mesh and the field-free flight area and are received by the detector.
The time-of-flight mass analyser further comprises:
a reflective region including a first reflective field including a second incident grid and reflective electrodes and a second reflective field including the reflective electrodes and reflective plates; ions emerging from the field-free flight zone are reflected by the reflective zone and are then received by the detector.
In order to realize second-order focusing, the second ion acceleration region and the first and second reflection fields satisfy the following conditions:
E1、E3、E4、E5electric field intensity, z, of the second ion acceleration region, the first reflection field and the second reflection field, respectively0、dG、d2、d3、d4、d5The distance between the incident ions and the first grid, the distance between the first grid and the second grid, the distance between the second grid and the traction electrode, the distance between the traction electrode and the first incident grid, the distance between the second incident grid and the reflection electrode, and the distance between the reflection electrode and the reflection plate are respectively; l is the length of flight of the ions in the field-free flight region between the first entrance grid and the detector.
In order to realize second-order focusing, the distance between the first grid and the second grid and the field-free flight area satisfy that:
E1、E3electric field intensity, z, of the second ion acceleration region and the first ion acceleration region, respectively0、dG、d2、d3Respectively the distance between the incident ion and the first grid, the distance between the first grid and the second grid, the distance between the second grid and the traction electrode, and the distance between the traction electrode and the first incident grid; l is the length of flight of the ions in the field-free flight region between the first entrance grid and the detector.
Example 2:
the microsphere of embodiment 1, the preparation method thereof and the application example of the detection method are provided.
In this application example, the preparation method of the microsphere is as follows:
FeCl2·4H2o and FeCl3·6H2Adding ultrapure water, performing ultrasonic dispersion, and heating and stirring under the nitrogen condition; addition of NH3·H2O and oleic acid react; magnetically separating the product and washing with ultrapure water until the pH value in the washing liquid is neutral to obtain stable magnetic fluid Fe3O4A nanoparticle;
taking Fe3O4Dissolving solid and SDS in ultrapure water and ultrasonically dispersing, adding styrene into the mixed solution and dispersing, heating and stirring the suspension under the condition of nitrogen, adding K2S2O8Heating for reaction; adding acrylic acid for continuous reaction, washing a product with absolute ethyl alcohol and ultrapure water, and drying in vacuum to obtain powder Fe3O4The sample @ PS-COOH, i.e. the surface of the microsphere is modified with carboxyl(functional group);
taking Fe3O4Adding the powder of @ PS-COOH into solvent with chemical activating agent dissolved, ultrasonic dispersing, stirring to react and activate Fe3O4The carboxyl on the surface of the @ PS-COOH nanosphere is added with an ANTA solution of a metal chelating agent for continuous reaction; finally, LaCl is added3Salt solution reaction; after the reaction is finished, centrifugally washing to remove excessive metal salt, and vacuum drying the obtained precipitate to obtain magnetic microsphere powder Fe3O4@PS-Chelate-La3+The structure is shown in figure 2.
The detection method based on the mass spectrometry and the microspheres of the embodiment of the invention comprises the following steps:
providing a plurality of microspheres prepared in the embodiment, wherein each microsphere has different compositions of chelated metal ions (such as different mass numbers or different metal ions) and different probes on the surface, and the mass numbers of the metal ions correspond to the probes one by one;
the probes of the microspheres respectively perform specific reaction with a plurality of nucleic acids or antibodies in a sample to be detected, namely the probes correspond to the nucleic acids or the antibodies one by one, namely the nucleic acids or the antibodies are marked by using the composition of metal ions;
detecting the composition of metal ions on a plurality of microspheres by using a mass spectrometry technology (using ICP-TOF-MS), thereby obtaining the information of the specific reaction between a sample and a nucleic acid probe or an antibody modified on the microspheres
The specific structure of ICP-TOF-MS is as follows:
as shown in fig. 4, the torch tube 101 and the coil 102 are respectively disposed on different parts of the carrier unit, the torch tube 101 and the coil 102 remaining relatively stationary;
as shown in fig. 5, the carrying unit includes a fixing portion 301, a first mounting portion 302, a second mounting portion 303, and a connecting portion 304; the second mounting part 303 is horizontally fixed on the first adjusting unit 201, the fixing part 301 is fixed on the upper side of the second mounting part 303, the connecting part 304 is vertically arranged, the lower end is fixed on the second mounting part 303, and the upper end is fixed with the horizontally arranged first mounting part 302; the torch tube 101 is vertically arranged on the first mounting part 302, one end of the coil 102 is fixed on the fixing part 301, and the other end of the coil surrounds the torch tube 101;
as shown in fig. 4, the second adjusting unit is fixed on the chassis 100 and includes a motor 401, a screw 402, a nut, a guide rail 404, a slider 405, and a bearing, the motor 401 drives the horizontally arranged screw 402 to rotate, the nut is sleeved on the screw 402 by using a thread, the slider 405 is arranged on the guide rail 404 arranged in parallel with the screw 402, and the nut is connected with the slider 405, so that when the unit screw 402 rotates, the slider 405 is driven to move horizontally and linearly along the guide rail 404; the bearing is disposed at the top end of the slider 405; the four corners of the carrier 503 are respectively provided with through holes for allowing the vertically arranged guides 504 to pass through, and the difference between the inner diameter of the through holes and the outer diameter of the guides 504 is small, so that the carrier 503 can only move vertically along the guides 504; the conversion piece is provided with a vertical part 501 and an inclined part 502, the lower end of the vertical part 501 is connected with the bearing part 503, the upper end of the vertical part is connected with the inclined part 502, the inclined part 502 is propped by the bearing, and the sliding piece 405 moves horizontally and linearly on the lower side of the inclined part 502, so that the inclined part 502 only moves vertically;
the first adjusting unit 201 adopts an electric two-dimensional moving platform to realize two-dimensional adjustment in the horizontal direction, and is arranged on the bearing part 503;
fig. 6 shows a schematic structural diagram of a time-of-flight mass analyzer according to an embodiment of the present invention, as shown in fig. 5, the time-of-flight mass analyzer includes:
a repeller 11, a field-free flight zone 30 and a detector 51, said field-free flight zone 30 comprising a first entrance grid 31;
a first ion acceleration region is formed between the traction electrode 12 and the first incident grid 31;
a first grid 21 and a second grid 22, wherein the potential difference between the first grid 21 and the second grid 22 is zero; a second ion acceleration region is formed between the repulsion electrode 11 and the first grid 21, and between the second grid 22 and the traction electrode 12; the ions sequentially pass through the first grid 21, the second grid 22, the traction electrode 12, the first incidence grid 31 and the field-free flight area 30, and are received by the detector 51; the first grid 21 and the second grid 22 are grounded, so that the first grid 21 and the second grid 22 are ensured to be in equal potential;
a plurality of electrodes allowing ions to pass through are arranged between the traction electrode 12 and the first incidence grid 31, and voltage division is carried out on the plurality of electrodes by using a voltage division resistor; a plurality of electrodes allowing ions to pass through are arranged between the traction electrode 12 and the first incident grid 31, and the voltage of the plurality of electrodes is divided by using a voltage dividing resistor, so that the electric field intensity of the first ion acceleration area is uniform; the pull electrode 12 has slots allowing ions to pass through or has a grid structure allowing ions to pass through; the power supply applies a positive pulse voltage to the repeller 11 and a negative pulse voltage to the trailing electrode 12.
In order to realize second-order focusing, the distance between the first grid and the second grid and the field-free flight area satisfy that:
E1、E3electric field intensity, z, of the second ion acceleration region and the first ion acceleration region, respectively0、dG、d2、d3The distance between the incident ion and the first grid 21, the distance between the first grid 21 and the second grid 22, the distance between the second grid 21 and the traction electrode 12, and the distance between the traction electrode 12 and the first incident grid 31, respectively; l is the length of flight of the ions in the field-free flight region between the first entrance grid 31 and the detector 51.
The ionization mode of the single cell is as follows:
the motor rotates to drive the nut to carry the sliding piece 405 to move horizontally and linearly on the guide rail 404;
the sliding member 405 moves linearly horizontally under the inclined portion 502 of the converting member, thereby converting into a vertical movement of the converting member (the converting member only moves vertically), thereby driving the carrier 503 connected to the converting member to move vertically along the plurality of guides 504 (the carrier 503 only moves vertically), and the carrier unit disposed on the carrier 503 moves vertically along with the carrier 503; the torch tube 101 is vertically arranged on the first mounting portion 302 of the carrier unit, and the coil 102 is fixed on the fixing portion 301 of the carrier unit and is kept relatively stationary with respect to the torch tube 101;
two-dimensionally adjusting the position of the bearing unit in the horizontal direction by using a first adjusting unit 201, the bearing unit being disposed on the first adjusting unit 201, the first adjusting unit 201 being disposed on the bearing 503; it can be seen that in the three-dimensional adjustment of the carrier unit, the torch tube 101 and coil 102 remain relatively stationary;
the sample is excited into a plasma by the coil 102, forming ions, upon entering the torch 101;
the ions pass through a sampling cone 103 and enter the mass analysis unit.
Example 3:
the microsphere of embodiment 1, the preparation method thereof and the application example of the detection method are provided.
In this application example, the preparation method of the microsphere is as follows:
FeCl2·4H2o and FeCl3·6H2Adding ultrapure water, performing ultrasonic dispersion, and heating and stirring under the nitrogen condition; addition of NH3·H2O and oleic acid react; magnetically separating the product and washing with ultrapure water until the pH value in the washing liquid is neutral to obtain stable magnetic fluid Fe3O4A nanoparticle;
taking Fe3O4Dispersing in styrene, adding divinylbenzene, benzoyl peroxide and methacrylic acid to form magnetic fluid, and uniformly dropwise adding the magnetic fluid into an aqueous solution containing hexadecanol and sodium dodecyl sulfate; adding an aqueous solution of acrylamide and potassium persulfate under the condition of nitrogen, keeping the temperature at 70 ℃, stirring for reaction, and washing a product by water magnetism to obtain Fe3O4@PS-NH2Namely, the surface of the microsphere is modified with amino (functional group);
taking Fe3O4@PS-NH2Powder is added intoUltrasonically dispersing in solvent with chemical activating agent dissolved, and stirring for reaction to activate Fe3O4@PS-NH2And adding a metal chelating agent Iso-EDTA solution into the amino on the surface of the nano-ball to continue the reaction. Finally, TbCl is added3、TmCl3And HoCl3Lanthanide metal salt solution mixed according to a certain proportion (all lanthanide metal salts) is reacted; after the reaction is finished, centrifugal washing is carried out to remove excessive metal salt, and the obtained precipitate is dried in vacuum to obtain the magnetic microsphere Fe3O4@PS-EDTA-Tb3+/Tm3+/Ho3+The structure is shown in figure 3, thereby realizing the detection of nucleic acid or antibody.
In the detection method of the embodiment, in the time-of-flight mass analyzer, as shown in fig. 7, the first grid 21 and the second grid 22 are grounded, so that the first grid 21 and the second grid 22 are guaranteed to be equipotential; a plurality of electrodes allowing ions to pass through are arranged between the traction electrode 12 and the first incident grid 31, and the voltage of the plurality of electrodes is divided by using a voltage dividing resistor, so that the electric field intensity of the first ion acceleration area is uniform; the power supply applies positive pulse voltage to the repulsion electrode 11 and applies negative pulse voltage to the traction electrode 12;
the reflective region includes a first reflected field including the second incident grid 32 and the reflective electrode 41, and a second reflected field including the reflective electrode 41 and the reflective plate 42; ions exiting the field-free flight zone 30 are reflected by the reflecting zone and then received by the detector 51; the reflective electrode 41 has a slot hole for allowing ions to pass through, or has a grid structure for allowing ions to pass through;
arranging a plurality of electrodes allowing ions to pass through in the first ion acceleration area, the first reflection field and the second reflection field, and dividing the voltage of the plurality of electrodes by using a voltage dividing resistor so that the electric field intensity in the first ion acceleration area, the first reflection field and the second reflection field is uniform;
in order to realize second-order focusing, the second ion acceleration region and the first and second reflection fields satisfy the following conditions:
E1、E3、E4、E5electric field intensity, z, of the second ion acceleration region, the first reflection field and the second reflection field, respectively0、dG、d2、d3、d4、d5The distance between the incident ion and the first grid 21, the distance between the first grid 21 and the second grid 22, the distance between the second grid 22 and the traction electrode 12, the distance between the traction electrode 12 and the first incident grid 31, the distance between the second incident grid 32 and the reflective electrode 41, and the distance between the reflective electrode 41 and the reflective plate 42, respectively; l is the length of flight of the ions in the field-free flight region between the first entrance grid 31 and the detector 51.
In ICP, unlike example 2, is:
1. the second mounting part and the connecting part are not arranged, the fixing part is directly fixed on the first adjusting unit, and the first mounting part is horizontally fixed on the fixing part;
2. the screw rod and the guide rail are kept parallel and are obliquely arranged relative to the horizontal plane;
3. the conversion piece comprises a horizontal part and a vertical part, and the horizontal part is supported by the sliding piece; when the slide member, which is moved in a straight line in an inclined manner, moves on the lower side of the horizontal portion of the switching member, the switching member moves vertically therewith, and only moves vertically.
Claims (10)
1. A microsphere comprising a spherical body; the metal ion chelating agent is characterized in that a functional group is modified on the surface of the body, a chelate is coupled to a part of the functional group, and the chelate chelates metal ions.
2. The microsphere of claim 1, wherein the functional group is amino or carboxyl, the chelate is ANTA or Iso-EDTA, and the metal ion is a lanthanide metal ion.
3. The method for producing microspheres according to claim 1 or 2, which comprises:
adding the spherical body with the surface modified with the functional group into a chemical activation reagent to activate the functional group;
adding a metal chelating agent, and reacting;
a metal salt is added and the chelating agent chelates the metal ions in the metal salt.
4. The method for preparing microspheres according to claim 3, wherein the spherical body with the surface modified with functional groups is prepared by:
FeCl2·4H2o and FeCl3·6H2Adding pure water and dispersing, and heating and stirring under the condition of inert gas; addition of NH3·H2O and oleic acid react; magnetically separating the product and washing with pure water until the pH value of the washing liquid is neutral to obtain Fe3O4A nanoparticle;
Fe3O4dissolving solid and SDS in pure water, dispersing, adding styrene, stirring under inert gas, adding K2S2O8Heating for reaction; adding acrylic acid for continuous reaction, and obtaining powder Fe after vacuum drying3O4Sample @ PS-COOH; or taking Fe3O4Dispersing in styrene, adding divinylbenzene, benzoyl peroxide and methacrylic acid to form magnetic fluid, and adding dropwise the magnetic fluid into an aqueous solution containing cetyl alcohol and sodium dodecyl sulfate; adding aqueous solution of acrylamide and potassium persulfate under the condition of inert gas, wherein the product is Fe3O4@PS-NH2。
5. The detection method based on the mass spectrometry and the microspheres comprises the following steps:
providing a plurality of microspheres according to claim 1 or 2, each microsphere having a different composition of chelated metal ions and a different probe on the surface, the mass number of the metal ions corresponding to the probes one to one;
the probes of the microspheres respectively perform specific reaction with various nucleic acids or antibodies in a sample to be detected;
the composition of metal ions on various microspheres is detected by using a mass spectrometry technology, so that the information of specific reaction between a sample and a nucleic acid probe or an antibody modified on the microspheres is obtained.
6. The method of claim 5, wherein the ionization mode in mass spectrometry detection is as follows:
the motor rotates, and the conversion module converts the rotation of the motor into the linear movement of the sliding piece;
converting the linear movement of the sliding part into the vertical movement of the conversion part, so as to drive the bearing part connected with the conversion part to vertically move along a plurality of guide parts, and the bearing unit arranged on the bearing part vertically moves along with the bearing part; the torch tube is vertically arranged on the carrying unit, and the coil is fixed on the carrying unit and is kept static relative to the torch tube;
the position of the bearing unit is adjusted in a two-dimensional mode in the horizontal direction, the bearing unit is arranged on the two-dimensional adjusting unit, and the two-dimensional adjusting unit is arranged on the bearing piece;
the sample enters the torch tube and is excited into plasma through the coil, so that ions are formed;
the ions pass through a sampling cone and enter a mass spectrometry unit.
7. The method of claim 5, wherein a time-of-flight mass analyzer is used in the mass spectrometric detection, the time-of-flight mass analyzer comprising a repeller, a field-free flight zone, and a detector, the field-free flight zone comprising a first entrance grid; the time-of-flight mass analyser further comprises:
a first ion acceleration region is formed between the traction electrode and the first incident grid;
the device comprises a first grid and a second grid, wherein the potential difference between the first grid and the second grid is zero; a second ion acceleration area is formed between the repulsion electrode and the first grid, and between the second grid and the traction electrode; the ions sequentially pass through the first grid mesh, the second grid mesh, the traction electrode, the first incidence grid mesh and the field-free flight area and are received by the detector.
8. The method of mass spectrometry and microsphere based detection according to claim 7, wherein said time of flight mass analyzer further comprises:
a reflective region including a first reflective field including a second incident grid and reflective electrodes and a second reflective field including the reflective electrodes and reflective plates; ions emerging from the field-free flight zone are reflected by the reflective zone and are then received by the detector.
9. The mass spectrometry and microsphere based detection method of claim 8, wherein the second ion acceleration region and the first and second reflected fields satisfy:
E1、E3、E4、E5electric field intensity, z, of the second ion acceleration region, the first reflection field and the second reflection field, respectively0、dG、d2、d3、d4、d5Respectively the distance between the incident ion and the first grid, the distance between the first grid and the second grid, and the second gridAnd the distance between the traction electrodes, the distance between the traction electrodes and the first incident grid, the distance between the second incident grid and the reflecting electrodes, and the distance between the reflecting electrodes and the reflecting plates; l is the length of flight of the ions in the field-free flight region between the first entrance grid and the detector.
10. The mass spectrometry and microsphere based detection method of claim 7, wherein the distance between the first grid and the second grid and the field-free flight zone satisfy:
E1、E3electric field intensity, z, of the second ion acceleration region and the first ion acceleration region, respectively0、dG、d2、d3Respectively the distance between the incident ion and the first grid, the distance between the first grid and the second grid, the distance between the second grid and the traction electrode, and the distance between the traction electrode and the first incident grid; l is the length of flight of the ions in the field-free flight region between the first entrance grid and the detector.
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