CN112280827B - Preparation method of core-shell nucleic acid immobilized microspheres - Google Patents
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Abstract
The invention provides a preparation method of a core-shell nucleic acid immobilized microsphere, which can effectively immobilize nucleic acid molecules. The nucleic acid immobilized microspheres have good unicity and are beneficial to application in aspects of gene sequencing and the like. Connecting an initiator or a carbon-carbon double bond on the surface of the microsphere; then, the microspheres are subjected to polymerization reaction in a water phase, and polymer chains containing click chemical functional groups and azide groups are connected to the surfaces of the microspheres; then the nucleic acid molecule is immobilized through click chemistry. The method has simple steps and can be applied to common sequencers.
Description
Technical Field
The invention relates to a preparation method of a core-shell nucleic acid immobilized microsphere, which can effectively immobilize nucleic acid molecules and belongs to the field of gene sequencing.
Background
The bioactive load Microsphere (Microsphere) is spherical particle with the particle diameter of 50 nm-2 mm and is provided with reactive groups such as-NH 2, -COOH, -SH and the like. The microspheres have small size, so that the microspheres have obvious surface effects such as good affinity and biocompatibility of materials, easy absorption and migration in organisms and the like, and are widely applied to the fields of cytology, immunology, microbiology, molecular biology, clinical diagnosis and treatment, high-throughput gene detection and the like.
In the method of microsphere for high-throughput gene detection analysis of certain nucleic acid sequences, the size distribution of microspheres is about 1 μm, the microspheres are highly uniformly dispersed, and the loading of the microspheres on nucleic acid chains is relied on. The sequence of the loaded nucleic acid strand can then be determined by a number of different methods well known in the art.
In some of the microspheres used in nucleic acid sequencing methods, the procedure involves loading with a nucleic acid sample to be sequenced by rapidly and efficiently binding to the corresponding reactive groups of the microspheres by a number of methods well known in the art; nucleic acid-loaded microspheres are introduced into the flow cell channels and into the corresponding reaction micro-tunnels, and incubated for a fixed time to produce all downstream chemical processing steps that are consistently capable of supporting amplification and sequencing. Compared with other types of nucleic acid loading methods, such as chip coating loading coating, the nucleic acid loading microspheres have the advantages of better controllability, large nucleic acid loading capacity and the like.
The invention provides a preparation method of nucleic acid immobilized microspheres, which can effectively immobilize nucleic acid molecules. The nucleic acid immobilized microsphere has good unicity and is beneficial to the application in the aspects of gene sequencing and the like.
Disclosure of Invention
The invention provides a preparation method of core-shell nucleic acid immobilized microspheres, which is characterized by comprising the following steps,
(1) Connecting a first active group on the surface of the amino polystyrene microsphere through reaction, wherein the structure of the first active group is a group with carbon-carbon double bond at the alpha position of carbonyl group, or a group with halogen at the alpha position of carbonyl group, preferably bromine, or azo group;
(2) In an aqueous phase, reacting the microspheres connected with the first active groups obtained in the step (1) with an acrylamide compound to generate microspheres containing azide groups; wherein the acrylamide compound refers to that at least part of the acrylamide compound contains an azide group;
(3) And (3) reacting the microspheres containing the azide groups obtained in the step (2) with nucleic acid molecules with alkynyl to obtain the microspheres carrying the nucleic acid molecules.
According to a preferred embodiment, in step 2, the acrylamide compound refers to a mixture of acrylamide and acrylamide having an azide group.
According to a preferred embodiment, the acrylamide compound in the step 2 refers to a mixture of substituted acrylamide and acrylamide with an azide group.
According to a preferred embodiment, the step 1 of attaching the first active group on the surface of the aminostyrene microsphere through reaction means that the aminostyrene microsphere is reacted with 4,4 '-azobis (4-cyanovaleric acid), N-hydroxysuccinimide bromo-2-isobutyrate, acrylic acid to attach the active group, or substituted 4,4' -azobis (4-cyanovaleric acid), N-hydroxysuccinimide substituted bromo-2-isobutyrate, substituted acrylic acid to attach the active group.
According to a preferred embodiment, the group having a halogen in position α to the carbonyl group is a group having a bromine in position α to the carbonyl group.
The invention has the following remarkable advantages:
(1) The controllability of the microsphere is good, and comprises the regulation of the reactive group of the microsphere, the regulation of the size of the microsphere and the regulation of the amount of the nucleic acid loaded on the microsphere. Because the overall size of the shell polymer chains is small compared to the overall size of the microspheres, the size of the microspheres is largely determined by the use of commercially available amino PS microspheres, of which there are various alternatives.
(2) The mechanical strength of the microspheres is high. Because the core is PS microspheres, the mechanical strength of the microspheres is much higher than that of polyacrylamide microspheres commonly used in other sequencers.
(3) The microspheres are good in observability. Polyacrylamide microspheres commonly used by other sequencers are invisible in a bright field, and can be observed in a fluorescent field by connecting a fluorescent group on the surface. The PS core of the microsphere can be observed in a bright field.
Drawings
Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. In the drawings:
FIG. 1 is a micrograph of microspheres of example 1;
FIG. 2 is a micrograph of microspheres from example 2;
FIG. 3 micrograph of microspheres of example 3.
Detailed Description
Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure have been described, it should be understood that the present disclosure may be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.
The invention is further illustrated by the following examples in conjunction with the accompanying drawings. The specific embodiments of the present invention are further illustrative of the present invention and do not limit the scope of the present invention.
The invention provides a preparation method of nucleic acid immobilized microspheres, which comprises the following steps:
(1) Initiator or carbon-carbon double bond is connected on the surface of the purchased amino PS (polystyrene) microsphere.
(2) And (2) carrying out polymerization reaction on the product microspheres obtained in the step (1) in an aqueous phase to connect polymer chains containing click chemistry functional groups and azide groups on the surface.
(3) The polymer microspheres can effectively immobilize nucleic acid molecules through click chemistry.
The click chemical condition is that the molar ratio of Cu-THPTA to the azido functional group is 1-100.
Example 1
A core is a PS microsphere with the diameter of 1um, and a shell is a core-shell nucleic acid immobilized microsphere with a polyacrylamide-methacrylamide azide copolymer chain.
The method comprises the following specific steps:
1) A radical initiator ACVA (4, 4' -azobis (4-cyanovaleric acid)) is connected on the surface of a 1um amino PS microsphere of purchased Tianjin big goose.
A water/isopropanol solution of ACVA, an aqueous solution of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and an aqueous solution of NHS (N-hydroxysuccinimide) were prepared at concentrations of 25mg/ml,50mg/ml and 50mg/ml, respectively.
In a 2.5ml ep tube, 224. Mu.l ACVA, 92. Mu.l NHS, 154. Mu.l EDC, 800. Mu.l 1 μm PS-NH2 microspheres were added and the reaction was carried out under shaking at 1200rpm in a dark place (coated with aluminum foil) for 60 minutes.
Centrifuging for 2min at 15kG centrifugal force, decanting the supernatant, adding 1000. Mu.l of 0.2% tween20 water, shaking for dispersion, centrifuging for 1min at 15kG, decanting the supernatant, adding 1000. Mu.l of 0.2% tween20 water, shaking for dispersion; centrifuging at 15kG for 1min, decanting the supernatant, adding 1000. Mu.l of 0.2% tween20 water, and shaking for dispersion; centrifuge at 15kG for 1min, pour off the supernatant, add 1000. Mu.l of 0.2% tween20 water and disperse by shaking.
Placing into a shading bottle for later use, and refrigerating for storage.
2) Carrying out polymerization reaction on the product microspheres obtained in the step 1) to coat polyacrylamide-methacrylamide azide copolymer chains.
Preparing an AM (acrylamide) aqueous solution with the concentration of 50g/l; preparing an AM-N3 (methacrylamide azide monomer, shown as the following) aqueous solution with the concentration of 50g/l; an aqueous solution of AM-biotin (acrylamide biotin monomer, shown below) was prepared at a concentration of 20g/l. The addition of AM-biotin monomer is required for subsequent immobilization of the microspheres to a specific surface.
Methacrylamide azide monomer:
acrylamide biotin monomer:
in a 20ml glass tube, 568. Mu.l of AM solution was added, 192. Mu.l of AM-N3 solution was added, 50. Mu.l of AM-biotin solution was added, 1990. Mu.l of water was added, a magnetic rotor was added, stirring was carried out at 300rpm, and 200. Mu.l of the product microsphere PS-ACVA of step 1) was added.
Inserting into reaction solution of the test tube with a trachea with an inner diameter of about 1mm, approaching the bottom of the test tube, and introducing nitrogen for 10min.
The test tube is sealed by a rubber plug with a hole, and the trachea is arranged at the top of the test tube through the hole. The stirring was maintained at 300rpm under a nitrogen atmosphere, and the mixture was heated to 70 ℃ for 60min.
Heating to 90 ℃, and timing for 120min.
Cooling, packaging into 2 ep tubes of 2ml, centrifuging for 2min (15 kG), decanting the supernatant, adding 0.2% of tween water each 1ml, shaking for dispersion, centrifuging at 15kG for 1min, decanting the supernatant; adding tween water 1ml 0.2% each, shaking for dispersing, centrifuging at 15kG for 1min, and pouring off the supernatant; adding tween water 1ml 0.2% each, shaking for dispersing, centrifuging at 15kG for 1min, and pouring off the supernatant; 0.2% of tween water was added to each, and 0.5ml of tween water was added, dispersed by shaking, and combined and stored under refrigeration.
3) The product microspheres of 2) are surface-immobilized with DNA fragments using "click" chemistry.
Using Cu-THPTA and NaVc as catalysts to carry out 'click' chemical connection on the azide group on the surface of the product microsphere in the step 2) and the DNA fragment with terminal alkynyl and fluorescein groups.
FIG. 1 is a photograph taken under a microscope with microspheres immobilized in a microfluidic chip, the left side under a fluorescent field and the right side under an identical position bright field. The positions of the two microspheres are observed to be in one-to-one correspondence.
Example 2
A core is a PS microsphere with the diameter of 1um, and a shell is a core-shell nucleic acid immobilized microsphere with a polyacrylamide-methacrylamide azide copolymer chain.
The method comprises the following specific steps:
1) Connecting a SI-ATRP (surface initiated atom transfer radical polymerization) free radical initiator to the surface of a purchased 1um amino PS microsphere of the Swan: n-hydroxysuccinimide ester of bromo-2-isobutyric acid (abbreviated as NHS-alphaBr, shown below).
Bromo-2-isobutyric acid N-hydroxysuccinimide ester:
a water/isopropanol solution of NHS-alphaBr was prepared at a concentration of 25mg/ml.
212 microliter of NHS-alphaBr solution is added into a 2.5ml ep tube, 800 microliter of 1 micron PS-NH2 microspheres are added, and the oscillation reaction is carried out for 120 minutes at normal temperature and 1200rpm under the condition of shading (wrapping aluminum foil paper).
Centrifuging for 2min at 15kG centrifugal force, decanting the supernatant, adding 1000. Mu.l of 0.2% tween20 water, shaking for dispersion, centrifuging for 1min at 15kG, decanting the supernatant, adding 1000. Mu.l of 0.2% tween20 water, shaking for dispersion; centrifuging at 15kG for 1min, decanting the supernatant, adding 1000. Mu.l of 0.2% tween20 water, and shaking for dispersion; centrifuge at 15kG for 1min, pour off the supernatant, add 1000. Mu.l of 0.2% tween20 water and disperse by shaking.
Placing into a shading bottle for later use, and refrigerating for storage.
2) Carrying out SI-ATRP polymerization reaction on the product microspheres in the step 1), and coating polyacrylamide-methacrylamide azide copolymer chains.
Preparing an AM (acrylamide) aqueous solution with the concentration of 50g/l; preparing an AM-N3 (methacrylamide azide monomer) aqueous solution with the concentration of 50g/l; preparing an AM-biotin (acrylamide biotin monomer) aqueous solution with the concentration of 20g/l; preparing a CuBr-bipy (2, 2-bipyridine) solution, wherein the formula is CuBr: bipy: IPA =143:312:3120 obtaining a red-brown solution
In a 20ml glass tube, 568. Mu.l of AM solution was added, 192. Mu.l of AM-N3 solution was added, 50. Mu.l of AM-biotin solution was added, 1990. Mu.l of water was added, a magnetic rotor was added, stirring was carried out at 300rpm, and 200. Mu.l of the product microspheres PS-Br of step 1) were added.
Inserting into reaction solution of the test tube with a trachea with an inner diameter of about 1mm, approaching the bottom of the test tube, and introducing nitrogen for 10min.
2.5ul of CuBr-bipy solution was added.
The test tube is sealed by a rubber plug with a hole, and an air pipe is arranged at the top of the test tube through the hole. Stirring was maintained at 300rpm under a nitrogen atmosphere, and the mixture was heated to 90 ℃ for 180min.
After cooling, they were dispensed into 2ml ep tubes, centrifuged for 2min (15 kG), the supernatant was decanted, 0.2% of tween water was added to each tube at 1ml, dispersed by shaking, centrifuged at 15kG for 1min, and the supernatant was decanted; adding tween water 1ml 0.2% each, shaking for dispersing, centrifuging at 15kG for 1min, and pouring off the supernatant; adding tween water 1ml 0.2% each, shaking for dispersing, centrifuging at 15kG for 1min, and pouring off the supernatant; adding 0.2% tween water 0.5ml each, shaking for dispersion, mixing, and refrigerating for storage.
3) The product microspheres of 2) are surface-immobilized with DNA fragments using "click" chemistry.
Using Cu-THPTA and NaVc as catalysts to carry out 'click' chemical connection on the azide group on the surface of the product microsphere in the step 2) and the DNA fragment with terminal alkynyl and a fluorescein group.
FIG. 2 is a photograph taken under a microscope after microspheres are immobilized in a microfluidic chip, with the left side under a fluorescent field and the right side under an collocated bright field. The positions of the two microspheres are observed to be in one-to-one correspondence.
Example 3
The preparation method of the core-shell type nucleic acid immobilized microsphere with the core being the PS microsphere with the diameter of 1um and the shell being the polyacrylamide-methacrylamide azide copolymer chain.
The method comprises the following specific steps:
1) Acrylamide is connected to the surface of a purchased 1um amino PS microsphere of the Tianjin big goose.
An aqueous solution of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) was prepared at a concentration of 50mg/ml. .
685ul of a 0.2% aqueous solution of tween20 was added to a 2ml ep tube, 110 ul of EDC solution was added, 200 ul of 1 micron PS-NH2 microspheres and 2ul of acrylic acid were added, and the reaction was carried out with shaking at 1200rpm for 60 minutes under a dark condition (coated aluminum foil).
Centrifuging for 2min at 15kG centrifugal force, decanting the supernatant, adding 1000. Mu.l of 0.2% tween20 water, shaking for dispersion, centrifuging for 1min at 15kG, decanting the supernatant, adding 1000. Mu.l of 0.2% tween20 water, shaking for dispersion; centrifuging at 15kG for 1min, decanting the supernatant, adding 1000. Mu.l of 0.2% tween20 water, and dispersing by shaking; centrifuge at 15kG for 1min, decant the supernatant, add 500. Mu.l of 0.2% tween20 water, and disperse by shaking.
Placing into a shading bottle for later use, and refrigerating for storage.
2) Carrying out polymerization reaction on the product microspheres obtained in the step 1) to coat a polyacrylamide-methacrylamide azide copolymer chain.
Preparing an AM (acrylamide) aqueous solution with the concentration of 50g/l; preparing an AM-N3 (methacrylamide azide monomer, shown as the following) aqueous solution with the concentration of 50g/l; an aqueous solution of AM-biotin (acrylamide biotin monomer shown below) was prepared at a concentration of 20g/l. A water/isopropanol solution of ACVA was prepared at a concentration of 25g/l. The addition of AM-biotin monomer is required for subsequent immobilization of the microspheres to a specific surface.
Acrylamide biotin monomer:
in a 20ml glass tube, 568. Mu.l of AM solution was added, 192. Mu.l of AM-N3 solution was added, 50. Mu.l of AM-biotin solution was added, 1990. Mu.l of water was added, 40. Mu.l of ACVA solution was added, a magnetic rotor was added, stirring was carried out at 300rpm, and 200. Mu.l of the product microspheres PS-AM of step 1) were added.
Inserting into reaction solution of the test tube with a trachea with an inner diameter of about 1mm, approaching the bottom of the test tube, and introducing nitrogen for 10min.
The test tube is sealed by a rubber plug with a hole, and an air pipe is arranged at the top of the test tube through the hole. Stirring was maintained at 300rpm under a nitrogen atmosphere, and the mixture was heated to 70 ℃ for 60min.
Heating to 90 ℃, and timing for 120min.
After cooling, they were dispensed into 2ml ep tubes, centrifuged for 2min (15 kG), the supernatant was decanted, 0.2% of tween water was added to each tube at 1ml, dispersed by shaking, centrifuged at 15kG for 1min, and the supernatant was decanted; adding tween water 1ml 0.2% each, shaking for dispersing, centrifuging at 15kG for 1min, and pouring off the supernatant; adding tween water 1ml 0.2% each, shaking for dispersing, centrifuging at 15kG for 1min, and pouring off the supernatant; 0.2% of tween water was added to each, and 0.5ml of tween water was added, dispersed by shaking, and combined and stored under refrigeration.
3) The product microspheres of 2) are surface-immobilized with DNA fragments using "click" chemistry.
Using Cu-THPTA and NaVc as catalysts to carry out 'click' chemical connection on the azide group on the surface of the product microsphere in the step 2) and the DNA fragment with terminal alkynyl and fluorescein groups.
FIG. 3 is a photograph taken under a microscope after microspheres are immobilized in a microfluidic chip, with the left side under a fluorescent field and the right side under an collocated bright field. The positions of the two microspheres are observed to be in one-to-one correspondence.
While the invention has been described with reference to specific preferred embodiments, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the following claims. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (6)
1. A preparation method of core-shell nucleic acid immobilized microspheres is characterized by comprising the following steps:
step 1: connecting a first active group on the surface of the amino polystyrene microsphere through reaction, wherein the structure of the first active group is a group with a carbon-carbon double bond at a carbonyl alpha position; or a group having a halogen in the alpha-position to the carbonyl group, or an azo group;
step 2: in an aqueous phase, reacting the microspheres connected with the first active groups obtained in the step (1) with an acrylamide compound to generate microspheres containing azide groups; wherein, the acrylamide compound refers to that at least part of the acrylamide compound contains azide groups;
and step 3: and (3) reacting the microspheres containing the azide groups obtained in the step (2) with nucleic acid molecules with alkynyl to obtain the microspheres carrying the nucleic acid molecules.
2. The method according to claim 1, wherein in step 2, the acrylamide compound is a mixture of acrylamide and acrylamide having an azide group.
3. The method of claim 1, wherein the acrylamide compound in the step 2 is a mixture of substituted acrylamide and acrylamide having an azide group.
4. The method of claim 1, wherein the step 1 of attaching the first reactive group to the surface of the aminostyrene microsphere by reaction is carried out by reaction of the aminostyrene microsphere with 4,4 '-azobis (4-cyanovaleric acid), N-hydroxysuccinimide ester of bromo-2-isobutyric acid, acrylic acid or with substituted 4,4' -azobis (4-cyanovaleric acid), N-hydroxysuccinimide ester of bromo-2-isobutyric acid, substituted acrylic acid.
5. The method of claim 1, wherein the group having a halogen alpha to the carbonyl group is a group having a bromine alpha to the carbonyl group.
6. The method of claim 1, wherein the azide group on at least a portion of the acrylamide compound is a polymer chain having a click chemistry functional group on at least a portion of the acrylamide compound, wherein the polymer chain is a polyacrylamide chain and the functional group is an azide group.
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