CN109490523A - Method for the nano material of label, nucleic acid probe and nucleic acid and nano material coupling - Google Patents
Method for the nano material of label, nucleic acid probe and nucleic acid and nano material coupling Download PDFInfo
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- CN109490523A CN109490523A CN201811230629.XA CN201811230629A CN109490523A CN 109490523 A CN109490523 A CN 109490523A CN 201811230629 A CN201811230629 A CN 201811230629A CN 109490523 A CN109490523 A CN 109490523A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—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
- G01N33/54346—Nanoparticles
Abstract
The invention discloses a kind of methods for the nano material of label, nucleic acid probe and nucleic acid and nano material coupling.Wherein, which includes: nano material, has tracer characteristic and acetylenic click chemistry derivatives group, is coupled at nano-material surface, and acetylenic click chemistry derivatives group includes clicking group.It applies the technical scheme of the present invention, using Surface-modification of Nanoparticles click chemistry coupling group, the end modified click chemistry group matched with it of nucleic acid, to realize nucleic acid and the quick special coupling of nano material.
Description
Technical field
The present invention relates to field of nano biotechnology, visit in particular to a kind of for the nano material of label, nucleic acid
The method of needle and nucleic acid and nano material coupling.
Background technique
Traditional biological labled material is usually small molecule compound, such as various fluorescent chemicals or colored dyes.It is suitable
The general partial size of nano material for biomarker and immunodiagnosis is in 1~200nm spherical shape or almost spherical, as a kind of novel
Marker material, it is microcosmic bigger volume, obtain higher detection signal, improve detection sensitivity, while can be reduced detection
The use of material.Common nano material for biological detection includes that quantum dot, magnetic microsphere, color latex microballoon, fluorescence are micro-
Ball, colloidal gold, colloidal-carbon etc..
It is a kind of nano-luminescent material that quantum dot, which is also known as semiconductor nano, how spherical in shape, and radius is less than or close to exciton
Bohr radius, for diameter generally in 1~12nm, core material is generally IV, II-VI, IV-VI or III-V element forms, such as
CdSe/ZnS, CdTe/ZnS, CdSe/ZnSe or CdTe/ZnSe etc..Quantum dot has excellent fluorescence special as fluorescent material
Property: possess narrow and symmetrical fluorescence emission spectrum, launch wavelength is related with material and partial size, adjustable realization multicolor emission,
Have extensively and continuous absorption spectrum and its Stokes shift is larger, it is same that the above characteristic makes quantum dot be easily achieved multi-wavelength
The multi-color marking function of Shi Jifa.In addition, quantum dot has photostability high, and resistance to photobleaching is glimmering relative to organic fluorescent substance
The good characteristics such as light efficiency height, and with the development of quantum dot surface aglucon chemical modification technology, the biocompatibility of quantum dot
And markup can get a promotion, so that related quantum dot biomarker is further opened up in the application of scientific research, detection field
Exhibition.
Magnetic microsphere is to wrap up magnetic particle using materials such as high molecular material such as polystyrene, cellulose etc. or silica
And emulsion dispersion is the magnetic Nano material for being suitable for size.Magnetic microsphere marks the biomolecule combination magnetic flux inspection of specific function
Measurement equipment can be realized simultaneously the purpose of the magnetic force enrichment and highly sensitive detection of sample, it is low but big to be highly suitable for detectable concentration
The sample of sample size.
Color latex microballoon is the nanometer material using the materials such as high molecular material or silica package coloured dye molecule
Material, partial size generally in 50nm~200nm, can form high-visible immunoblotting, due to single microballoon under visible light conditions
It comprising multiple dye molecules, therefore detects signal and significantly increases, there is higher sensitivity.
Fluorescent microsphere is divided into fluorescein microballoon, time-resolved fluorescence microballoon, quantum dot fluorescence microballoon etc., uses macromolecule material
The materials such as material or silica are wrapped up, and realize that the fluorescence signal of the bigger range of linearity detects by photoluminescence.Compared to biography
The fluorescent microsphere of the fluorescein preparation of system, time-resolved fluorescence microballoon are prepared using rare earth material, have extremely long fluorescence lifetime,
It realizes time resolution detection, background fluorescence in biological sample can be effectively reduced and interfere, improve detection sensitivity.Quantum dot fluorescence is micro-
Ball is then also able to achieve height by high extinction coefficient and fluorescence efficiency, stable fluorescence property and biggish Stokes shift
Sensitivity technique, while the unique multiple wave length simultaneously exciting discovery of quanta point material is as, it can be achieved that multi objective detects simultaneously.
Colloidal gold and colloidal-carbon are stable gold and carbon colloid nano particle respectively, a certain size colloid gold particle is red
For color to partially blue, colloidal-carbon is black particle, can be with quick adsorption albumen or other compounds, and then further functionalization,
Realize multipath coupling.Compared to colloidal gold, colloidal-carbon synthetic method cost is lower, and environmental-friendly, is that high efficiency of new generation is low
Cost labelled reagent.
Nano material will solve the efficient of nano material and nucleic acid molecules in the application in molecular Biological Detection field at first
Coupling, and the conformation and complementation Annealing Property of nucleic acid molecules cannot be influenced.Traditional chemical conjugation methods utilize nucleic acid end
The amino or sulfydryl of modification are connect with the active group of nano-material surface aglucon.This coupling mode low efficiency, differences between batches
It is larger, and it is easy occur the problems such as nano-grain aggregation in coupling process.
Summary of the invention
The present invention is intended to provide a kind of side for the nano material of label, nucleic acid probe and nucleic acid and nano material coupling
Method, to realize efficient, the special coupling of nucleic acid molecules and nano material.
To achieve the goals above, according to an aspect of the invention, there is provided a kind of nano material for label.It should
Nano material for label includes: nano material, has tracer characteristic and acetylenic click chemistry derivatives group, coupling
In nano-material surface, acetylenic click chemistry derivatives group includes clicking group.
Further, the coupling residue and click base being coupled in acetylenic click chemistry derivatives group with nano-material surface
Also there is spacerarm, spacerarm is carbochain, PEG, copolymer or polypeptide between group.
Further, the length of spacerarm is 4~50 C-C keys, preferably 8~15 C-C keys；Preferably, PEG is
PEG2~20.
Further, nano material be selected from by quantum dot, magnetic microsphere, color latex microballoon, fluorescent microsphere, colloidal gold and
One of group of colloidal-carbon composition is a variety of.
Further, acetylenic click chemistry derivatives group includes straight chain alkynes click chemistry derivatives group and hexichol basic ring
Octyne click chemistry derivatives group is derived from acetylenic click chemistry derivatives group with the coupling residue of nano-material surface
Coupling group, coupling group select free sulfhydryl group, polynary sulfydryl, nitrilotriacetic acid base, iminodiacetic acid, poly imidazoles, poly group
One of group of propylhomoserin, amino, carboxyl, hydrazides, n-hydroxysuccinimide and maleimide composition is a variety of.
According to another aspect of the present invention, a kind of nucleic acid probe is provided.The nucleic acid probe includes: above-mentioned for marking
Nano material, nano material surface coupling have acetylenic click chemistry derivatives group；And nucleic acid molecules, nucleic acid molecules are
The nucleic acid molecules of azido group activation, nucleic acid molecules are connect by azido group with acetylenic click chemistry derivatives group.
Further, nucleic acid molecules include azido group, intervening sequence area and functional sequence area, and intervening sequence area is located at folded
Between nitrogen groups and functional sequence area, intervening sequence is poly thymidine or poly adenine.
A kind of method according to the present invention in one aspect, providing nucleic acid and nano material coupling.This method include with
Lower step: the surface group of acetylenic click chemistry derivative and nano material is coupled by S1, and generating has click chemistry even
Join active nano material, acetylenic click chemistry derivative includes coupling group and click group；S2, by azido group activation
Nucleic acid molecules mix in buffer with the nano material with click chemistry coupling activity, realize nucleic acid molecules and nano material
Coupling.
Further, nano material be selected from by quantum dot, magnetic microsphere, color latex microballoon, fluorescent microsphere, colloidal gold and
One of group of colloidal-carbon composition is a variety of.
Further, acetylenic click chemistry derivative includes straight chain alkynes click chemistry derivative and/or diphenyl cyclooctyne
Click chemistry derivative, it is preferable that coupling group selects free sulfhydryl group, polynary sulfydryl, nitrilotriacetic acid base, iminodiacetic acid, more
One of polyimidazole, polyhistidine, amino, carboxyl, hydrazides, n-hydroxysuccinimide and group of maleimide composition
Or it is a variety of.
Further, the coupling group of acetylenic click chemistry derivative and clicking has between group between pre-set space distance
Every arm, spacerarm is selected from one of carbochain, PEG, copolymer or polypeptide or a variety of.
Further, the length of spacerarm is 4~50 C-C keys, preferably 8~15 C-C keys, more preferably 10 C-
C key.
Further, the nucleic acid molecules of azido group activation and the mixing of the nano material with click chemistry coupling activity
Molar ratio when coupling is 1:1~1000:1.
Further, the molar ratio of acetylenic click chemistry derivative and nano material is 10:1~100:1 in S1.
Further, the reaction temperature in S2 is less than or equal to 40 degrees Celsius, and the pH of buffer is 7~8.
It applies the technical scheme of the present invention, using Surface-modification of Nanoparticles click chemistry coupling group, nucleic acid end is repaired
The click chemistry group matched with it is adornd, to realize nucleic acid and the quick special coupling of nano material.
Detailed description of the invention
The accompanying drawings constituting a part of this application is used to provide further understanding of the present invention, and of the invention shows
Examples and descriptions thereof are used to explain the present invention for meaning property, does not constitute improper limitations of the present invention.In the accompanying drawings:
The ultraviolet light irradiation of quantum dot microsphere (paper slip) that Fig. 1 shows embodiment 13 excites figure of taking pictures；
Fig. 2 shows the PCR purified product amount comparison diagrams for calculating T/C and the measurement of minim DNA analyzer of embodiment 13；
Colloidal-carbon (paper slip) daylight light irradiation that Fig. 3 shows embodiment 14 is taken pictures figure；
Fig. 4 shows the magnetic particle transmitting peak area of embodiment 15 to bacterial concentration figure；And
Fig. 5 shows the ultraviolet light irradiation excitation figure of paper slip in embodiment 16.
Specific embodiment
It should be noted that in the absence of conflict, the features in the embodiments and the embodiments of the present application can phase
Mutually combination.Below in conjunction with embodiment, the present invention will be described in detail.
For relevant issues present in background technique, the present invention is directed to realize nucleic acid molecules and nano material it is efficient,
Special coupling, is applied to detection field relevant to nucleic acid or aptamers.To realize the purpose, the present invention provides one kind to be applicable in
In the nano material for label that the nucleic acid molecules with chemical synthesis with azido group are coupled, while optimizing coupling buffering
Liquid condition, coupling time and environmental condition can be used for developing series detection reagent relevant to nucleic acid or aptamers.
Wherein, which includes nano material, and is coupled at the acetylenic point of nano-material surface
Chemical derivative group is hit, acetylenic click chemistry derivatives group includes clicking group.A kind of typical implementation according to the present invention
Mode also has interval in acetylenic click chemistry derivatives group between the click group of nano-material surface coupling residue
Arm, spacerarm be carbochain, PEG, copolymer or polypeptide, it is preferred that the length of spacerarm be 4~50 C-C keys, more preferably 8
~15 C-C keys.Wherein, the space length between spacerarm offer coupling residue and click group is to reduce steric hindrance, point
Hitting group can be used for that click-reaction occurs with the nucleic acid molecules that nitrine is modified.In acetylenic click chemistry derivatives group with nanometer
Material surface coupling residue is derived from coupling group, and coupling group can be sulfydryl, polynary sulfydryl, nitrilotriacetic acid base (NTA), Asia
Aminodiacetic acid (IDA), poly imidazoles, polyhistidine, amino, carboxyl, hydrazides, n-hydroxysuccinimide (NHS), Malaysia
Acid imide etc..
The spacerarm of acetylenic click chemistry derivative is smaller on coupling efficiency influence, but since biological detection probe generally exists
It is used in aqueous environment, needs to select the higher structure of hydrophily, therefore the general PEG or hydrophilic more for selecting low molecular weight
Peptide, for length in 4~50 C-C keys, too long spacerarm causes compound viscosity to increase, and synthesizes purity reduction, is unfavorable for idol
Joining homogeneity, too short spacerarm causes there are steric hindrance, and the skin effect of nano material will affect the conformation of nucleic acid molecules,
To hinder nucleic acid molecules to function.Preferably, the spacerarm of 8~15 C-C keys or so to specific project detection effect compared with
It is good.Click group is the acetylenics click chemistry derivatives such as straight-chain alkynyl, diphenyl cyclooctyne (DBCO), can be driven by ring strain
It is efficiently coupled with the nucleic acid of nitrine modification.
Nano material in the present invention includes water-soluble quantum dot, magnetic microsphere, quantum dot fluorescence microballoon, colloidal-carbon, coloured silk
Color latex beads, fluorescent microsphere, colloidal gold.
The above-mentioned nano material for having click coupling activity can be prepared by following method:
(1) it clicks coupling quantum dot: (1) using and contain sulfydryl, polynary sulfydryl, nitrilotriacetic acid base (NTA), iminodiacetic acid (salt)
Sour (IDA), poly imidazoles, polyhistidine coupling group click chemistry derivative mixed with oil-soluble quantum dot, addition water
Phase solution is switched to by the methods of stirring, ultrasound directly coordination with the water-soluble quantum dot for clicking coupling activity, or (2)
It is mixed with water-soluble quantum dot, ligand displacement is carried out by the methods of stirring, ultrasound and carries out clicking reagent activation；Or (3) make
With the quantum dot of 1- ethyl-(3- dimethylaminopropyl) carbodiimide (EDC) activated water-soluble carboxyl ligand, then uses and contain ammonia
The click chemistry derivatives reaction of base carries out clicking reagent activation；Or (4) use carboxylic click chemistry derivative and 1-
After ethyl-(3- dimethylaminopropyl) carbodiimide (EDC) reaction activation, and use the quantum dot of water soluble amino ligand anti-
It answers, carries out clicking reagent activation；Or (5) using ligand containing terminal aldehyde groups or with the sodium periodate oxidation ligand of dihydric alcohol containing end
Quantum dot and the click chemistry derivatives reaction containing hydrazides, carry out click reagent activation；Or (6) use and contain N- hydroxyl
The click chemistry derivative of succinimide (NHS) is reacted with the quantum dot containing water soluble amino ligand, carries out click reagent
Activation；Or (6) are anti-using the click chemistry derivative containing maleimide and the quantum dot containing water-soluble thiol ligand
It answers, carries out clicking reagent activation.
(2) materials such as macromolecule or silica package can be used in magnetic microsphere, fluorescent microsphere, color latex microballoon
Water-soluble magnetic particle, coloured dye molecule, fluorescein, time-resolved fluorescence rare earth material, quantum dot, pass through bifunctional reagent
Or different copolymers, realize that surface has carboxyl, amino, aldehyde radical, sulfydryl isoreactivity group.Carboxyl magnetic microsphere can be by making
It is anti-with the click chemistry derivative containing amino with 1- ethyl-(3- dimethylaminopropyl) carbodiimide (EDC) reaction activation
It answers, carries out clicking reagent activation；Amino-magnetic microballoon can by with 1- ethyl-(3- dimethylaminopropyl) carbodiimide
(EDC) the carboxyl click chemistry derivative activated or the click chemistry derivatives reaction containing n-hydroxysuccinimide (NHS),
It carries out clicking reagent activation；Aldehyde radical magnetic microsphere can be clicked with the click chemistry derivatives reaction containing hydrazides group
Reagent activation；Sulfydryl magnetic microsphere it is living can to carry out click reagent with the click chemistry derivatives reaction containing maleimide
Change.
(3) colloidal gold is directly covalently attached activation using the click chemistry derivative containing sulfydryl, polynary sulfydryl, or uses
Click chemistry derivative activating ammonia based high molecular compound package colloid gold particle containing n-hydroxysuccinimide (NHS) is realized
Click reagent activation.
(4) colloidal-carbon utilizes the click chemistry derivative activating ammonia based high molecular for containing n-hydroxysuccinimide (NHS)
Closing object includes that colloidal-carbon carries out clicking reagent activation.
A kind of nano material typical embodiment according to the present invention, provides a kind of nucleic acid probe.The nucleic acid probe includes
The surface coupling of the above-mentioned nano material for label, the nano material has acetylenic click chemistry derivatives group；And nucleic acid
Molecule, nucleic acid molecules are the nucleic acid molecules of azido group activation, and nucleic acid molecules are clicked by the azido group and the acetylenic
The connection of chemical derivative group.
Acetylenic click chemistry derivative has the spacerarm for providing coupling group and clicking the space length between group,
Every carbochain, PEG, copolymer or the polypeptide that arm includes different length, has certain hydrophily, be able to maintain nano material water-soluble
Stablize in liquid, while maintain a certain distance the nucleic acid molecules of coupling spatially with nano material, reduces winding problem.It is excellent
Choosing, the length of spacerarm is 4~50 C-C keys, and preferably 8~15 C-C keys enable coupled product effectively to participate in nucleic acid miscellaneous
The reaction such as friendship.
In practical applications, quantum dot, magnetic microsphere, color latex microballoon, fluorescent microsphere, colloid may be selected in nano material
One of group of gold and colloidal-carbon composition is a variety of.Micron-sized microballoon is also included in nano material by the application.
Fields, the amplifying nucleic acids such as the nucleic acid probe in the present invention can be used for hybridizing, aptamer sensor can be deoxidation core
Ribosomal ribonucleic acid sequence (DNA) is also possible to RNA sequence (RNA).
In addition, in practical applications, interval sequence can be inserted between click chemistry modification group and functional sequence region
Column, conformation needed for making nucleic acid sequence that can form function.When designing nucleic acid molecules, generally in azido group and functional sequence area
Poly thymidine (polyT) is added or poly adenine (polyA) is used as intervening sequence, can be 0~15 base.It is required that
Intervening sequence itself does not influence the hybridization function of functional sequence, and unexpected complementary annealing effect does not occur.If sample is special,
It can also be added without intervening sequence, only with acetylenic click chemistry derivative spacerarm scheme.
A kind of typical embodiment according to the present invention provides the method for a kind of nucleic acid and nano material coupling.This method
The following steps are included: S1, acetylenic click chemistry derivative and nano-material surface group are coupled, generating has clickization
Learn the nano material of coupling activity, acetylenic click chemistry derivative includes coupling group and clicks group, coupling group and nanometer
Material connection is clicked group and is connect with nucleic acid molecules；The nucleic acid molecules of azido group activation are coupled by S2 with having click chemistry
Active nano material mixes in buffer realizes nucleic acid and the fast coupling of nano material.
Cyclenes and azido compound reaction by ring strain driving, i.e. ring strain cause nitrine-alkynes cycloaddition (SPAAC) and keep away
Exempt to be catalyzed using univalent copper ion, has been coupled in nucleic acid and applies upper great potential.It applies the technical scheme of the present invention, utilizes nanometer
Material surface modify click chemistry coupling group, nucleic acid it is end modified its pairing click chemistry group, thus realize nucleic acid with
The quick special coupling of nano material.
Alkynes-nitrine click coupling reaction is mild, is not likely to produce coagulation, coupled product homogeneity is higher.Reaction is usually in room
Temperature or 37 DEG C or so progress, pH value of solution condition is more wide in range, and it is steady that the general range for selecting pH 7~8 is conducive to large biological molecule
It is fixed.Reaction time be usually 24 hours can with fully reacting, ratio appropriate and under the conditions of, reaction efficiency can achieve 90%
More than.Nucleic acid molecules and the coupling Mole Ratio of nano material can change in 1~1000 range.It is different depending on the application, it is used for
Highly sensitive Tracing detection, conjugation ratios can optimize in 1~10 range, for cell dyeing or film hybridization generally 50~200
Range optimization.According to nano material property, need that the effective coupling group number of nano-material surface and granular size is combined to select
Suitable ratio.
In practical applications, quantum dot, magnetic microsphere, color latex microballoon, fluorescent microsphere, colloid may be selected in nano material
One of group of gold and colloidal-carbon composition is a variety of.
Click group is the acetylenics click chemistry derivatives such as straight-chain alkynyl, diphenyl cyclooctyne (DBCO), can pass through ring
The nucleic acid that power drive is modified with nitrine is efficiently coupled.Acetylenic click chemistry derivative can have different coupling groups, coupling
Group selects free sulfhydryl group, polynary sulfydryl, nitrilotriacetic acid base, iminodiacetic acid, poly imidazoles, polyhistidine, amino, carboxylic
One of group of base, hydrazides, n-hydroxysuccinimide and maleimide composition is a variety of.
Acetylenic click chemistry derivative has the spacerarm for providing coupling group and clicking the space length between group,
Every carbochain, PEG, copolymer or the polypeptide that arm includes different length, has certain hydrophily, be able to maintain nano material water-soluble
Stablize in liquid, while maintain a certain distance the nucleic acid molecules of coupling spatially with nano material, reduces winding problem.It is excellent
Choosing, the length of spacerarm is 4~50 C-C keys, and preferably 8~15 C-C keys enable coupled product effectively to participate in nucleic acid miscellaneous
The experiment such as friendship.
After above-mentioned technical solution, the beneficial effect that the present invention obtains is, due to the nucleic acid probe of Azide modification
Commercially available product, therefore the activation nano material described through the invention, can be rapidly completed nucleic acid coupling reaction, be different core
Acid or aptamers detection project provide a variety of detection probes, to realize highly sensitive detection.
Beneficial effects of the present invention are further illustrated below in conjunction with embodiment.
Examples 1 to 8 is the preparation of the nano material (also referred to as activation nano material) for label.
Embodiment 1`
Sulfydryl and polynary sulfydryl and the direct covalent coupling of colloidal gold:
The colloidal gold of every milliliter of optical concentration OD=1 be added 100~1000nmol sulfydryl-PEG4- diphenyl cyclooctyne or
The mixing of sulfydryl-PEG5- alkynyl, after being stored at room temperature 1 hour, addition 500mM pH8 buffer solution of sodium phosphate to final concentration of 10mM,
Magneton stirring room temperature reaction 1 hour, then 8000g was centrifuged 10 minutes, and precipitating 10mM pH8 buffer solution of sodium phosphate is redissolved to 1
Milliliter is completed to click reagent activation.Colloidal gold by clicking activation can pass through Huisgen cycloaddition or nitrine-alkynes cycloaddition
It is coupled the nucleic acid molecules modified containing azido group.
Embodiment 2
Nitrilotriacetic acid base (NTA), iminodiacetic acid (IDA), poly imidazoles or polyhistidine and quantum dot surface gold
Belong to ion coordination coupling.10~100nmol is added in every milliliter of 1 μm of ol/L water solubility thioacetic acid ligand quantum dot solution
The mixing of NTA-PEG4- diphenyl cyclooctyne, boiling water bath 15 minutes, is then stored at room temperature 1 hour, and 40000g is centrifuged 20 minutes, sinks
Shallow lake pure water, which is redissolved to 1 milliliter of completion, clicks reagent activation.Quantum dot by clicking activation can pass through nitrine-alkynes cycloaddition
It is coupled the nucleic acid molecules modified containing azido group.
Embodiment 3
Amino can be with the carboxyl microballoon through EDC (carbon imidodicarbonic diamide) or EDC/NHS (n-hydroxysuccinimide) activation
(fluorescent microsphere, magnetic microsphere, color micro-sphere etc.) coupling.After carboxyl microballoon 20mM pH6MES solution centrifuge washing, redissolution,
It is activated, is stored at room temperature 30 minutes with the EDC or EDC+NHS of 100~500 μ g/mg microballoons, then 8000g is centrifuged 10 minutes, precipitating
It is redissolved with 20mM pH6MES solution to initial volume, the amino-PEG4- diphenyl cyclooctyne of 10~100 times of molar ratios is added,
It is stored at room temperature reaction 1 hour after mixing, final concentration 10mM glycine is then added and mixes, continues room temperature reaction 1 hour, 8000g
Centrifugation 10 minutes, precipitating 10mM pH8 sodium phosphate buffer, which is redissolved to initial volume, to be completed to click reagent activation.By clicking
The microballoon of activation can be coupled the nucleic acid molecules modified containing azido group by nitrine-alkynes cycloaddition.
Embodiment 4
Carboxyl using EDC or EDC/NHS activation after, then with amino microballoon (fluorescent microsphere, magnetic microsphere, color micro-sphere etc.)
Coupling.Carboxyl-PEG4- diphenyl cyclooctyne is had with EDC or the EDC+NHS activation of 1~5 times of molar ratio, room temperature is quiet after mixing
Reaction 15 minutes is set, then by click reagent: microspheres quality ratio=1:200~1000 is added amino microballoon and continues room temperature reaction 1
Hour, 8000g is centrifuged 10 minutes, and precipitating, which is redissolved with 8 sodium phosphate buffer of 10mM pH to microballoon initial volume, to be completed to click examination
Agent activation.Microballoon by clicking activation can be coupled the nucleic acid molecules modified containing azido group by nitrine-alkynes cycloaddition.
Embodiment 5
N-hydroxysuccinimide (NHS) can be directly even with amino microballoon (fluorescent microsphere, magnetic microsphere, color micro-sphere etc.)
Connection.It is mixed with amino microballoon by 10~100 times of molar ratios with NHS-PEG4- diphenyl cyclooctyne, in 10mM pH7.4 sodium phosphate
37 degree of incubations in buffer, stand 1 hour, and 8000g is centrifuged 10 minutes, and precipitating 10mM pH8 sodium phosphate buffer is redissolved to micro-
Ball initial volume is completed to click reagent activation.Microballoon by clicking activation can be by nitrine-alkynes cycloaddition coupling containing folded
The nucleic acid molecules of nitrogen groups modification.
Embodiment 6
Hydrazides and aldehyde radical microballoon (fluorescent microsphere, magnetic microsphere, color micro-sphere etc.) are coupled.Hydrazide group-PEG4- hexichol basic ring
Octyne is mixed with aldehyde radical microballoon by 10~100 times of molar ratios, and 37 degree of incubations in 10mM pH 6MES buffer stand 3 hours,
8000g centrifugation 10 minutes, precipitating 8 sodium phosphate buffer of 10mM pH redissolve living to microballoon initial volume completion click reagent
Change.Microballoon by clicking activation can be coupled the nucleic acid molecules modified containing azido group by nitrine-alkynes cycloaddition.
Embodiment 7
Maleimide and sulfydryl microballoon (fluorescent microsphere, magnetic microsphere, color micro-sphere etc.) are coupled.Maleimide-
PEG4- diphenyl cyclooctyne is mixed with sulfydryl microballoon by 10~100 times of molar ratios, in 7.4 sodium phosphate buffer of 10mM pH
37 degree of incubations, stand 1 hour, and 8000g is centrifuged 10 minutes, and precipitating 8 sodium phosphate buffer of 10mM pH redissolves original to microballoon
Volume is completed to click reagent activation.Microballoon by clicking activation can contain azido group by nitrine-alkynes cycloaddition coupling
The nucleic acid molecules of modification.
Embodiment 8
NHS activates casein and is coupled colloidal-carbon.5~20 times moles are pressed with NHS-PEG4- diphenyl cyclooctyne and casein
Than mixing, 37 degree of incubations in 10mM pH7.4 sodium phosphate buffer stand 1 hour, and 50KDa super filter tube 8000g is centrifuged 15 points
Clock, fluid infusion dilution are centrifuged 3 times again, remove extra click reagent.Casein after modification in 1mg/100mg colloidal-carbon ratio with
Colloidal-carbon mixes, and room temperature is adsorbed 5 hours in 7.4 sodium phosphate buffer of 10mM pH, and 5000g is centrifuged 15 minutes, and precipitating uses 10mM
8 sodium phosphate buffer of pH, which is redissolved to colloidal-carbon initial volume, to be completed to click reagent activation.Colloidal-carbon by clicking activation can be with
The nucleic acid molecules modified containing azido group are coupled by nitrine-alkynes cycloaddition.
Embodiment 9~12 is the preparation of nucleic acid probe
Embodiment 9
It carries out clicking reagent activation by the specific method of embodiment 3 using the carboxyl quantum dot fluorescence microballoon of 100nm partial size,
With the 5 ' N3- of the characteristic area staphylococcus aureus MRSA gene probe with the modification of the nitrine of 5 '-polyA and 5 ' of 10 times of molar ratios
AAAAAAAAGTTGTAGTTGTCGGGTTTGG-3 ' (SEQ ID NO:1) mixing, after 37 degree are reacted 24 hours, 5000g centrifugation 15
Minute, it discards supernatant, precipitates after centrifugation 2 times and redissolved with 5mM pH7 sodium phosphate buffer to initial volume, can be used to detect.
Embodiment 10
It carries out clicking reagent activation according to the specific method of embodiment 8 using colloidal-carbon, the colloidal-carbon after activation, which uses, to be divided
Light photometric determination 600nm absorbs (OD600), and colloidal-carbon is diluted to OD600=10 with 7 sodium phosphate buffer of 5mM pH,
The characteristic area staphylococcus aureus MRSA with the modification of the nitrine of 5 '-polyA and 5 ' of 1nmol is added in 1mL colloidal carbon solution
5 ' N3-AAAAAAAAGTTGTAGTTGTCGGGTTTGG-3 ' of gene probe (SEQ ID NO:1), after 37 degree are reacted 24 hours,
8000g is centrifuged 15 minutes, is discarded supernatant, and precipitating is redissolved with 7 sodium phosphate buffer of 5mM pH to initial volume after centrifugation 2 times, i.e.,
It can be used for detecting.
Embodiment 11
It carries out clicking reagent activation by the specific method of embodiment 5 using the amino-magnetic particle of 400nm partial size, 1mg is living
The staphylococcus aureus MRSA feature that 1nmol has the modification of the nitrine of 5 '-polyA and 5 ' is added in magnetic particle solution after change
5 ' N3-AAAAAAAAGTTGTAGTTGTCGGGTTTGG-3 ' of area's gene probe (SEQ ID NO:1), after 37 DEG C are reacted 24 hours,
By strong magnet adsorption and purification, precipitates after purifying 2 times and redissolved with 5mM pH7 sodium phosphate buffer to initial volume, be can be used to
Detection.
Embodiment 12
It carries out clicking reagent activation by the specific method of embodiment 2 using CdSe/ZnS quantum dot, the amount after 1nmol activation
50nmol is added in son point solution to visit with the staphylococcus aureus MRSA characteristic area gene of the nitrine of 5 '-polyA and 5 ' modification
5 ' N3-AAAAAAAAGTTGTAGTTGTCGGGTTTGG-3 ' of needle (SEQ ID NO:1), 37 DEG C reaction 24 hours after, 40000g from
The heart purifies 15 minutes, discards supernatant, and precipitating is redissolved with 5mM pH7 sodium phosphate buffer to initial volume after centrifugation 2 times, i.e., available
In detection.
Embodiment 13~16 is the detection application of nucleic acid probe
Embodiment 13
Quantum dot microsphere (paper slip)
The 5 μ L of quantum dot microsphere mark gene probe prepared using the specific method of embodiment 9,10 μM of downstream detector primers
5 ' Biotin-C6-CTTCCACATACCATCTTCTTTAAC-3 ' (SEQ ID NO:2) 5 μ L, Taq archaeal dna polymerase 1 μ L, Taq
53 μ L, 10mM MgSO of μ L, 50%DMSO of polymerase buffer43 μ L contain various concentration staphylococcus aureus MRSA heat
5 μ L of bacterium solution is cracked, pure water complements to 50 μ L.Using PCR thermal cycler according to 95 degree 5 minutes, 95 degree 30 seconds+55 degree 30 seconds+72
30 seconds 15 circulations of degree, 72 degree of 5 minutes programs carry out amplification reaction, and sample is detected using chromatograph test strip after reaction, test paper
Item is coated with 1mg/mL Streptavidin (TL) and 1mg/mL mouse by the PVC backer board with viscose glue and the middle part being adhered to
The nitrocellulose filter of IgG (CL), through 10mM PBS+5mg/mL BSA+1%Tween-20+0.01 μM sheep anti-Mouse labelled amount
Son point microspheres solution impregnates the fiberglass packing being dried and absorbent filter composition, and 5 μ of PCR product is directly put on glass pad
L is analysed using 70 μ L 10mM PB extension liquid layer, is taken pictures (Fig. 1) after 15 minutes with the ultraviolet light irradiation excitation of 365nm, and use fluorescence
Chart scanner measures the fluorescence signal value and their ratio T/C of TL and CL, and calculating T/C and the PCR that minim DNA analyzer measures are pure
Change product amount and compare (Fig. 2), it is seen that the two has good linear dependence.
Embodiment 14
Colloidal-carbon (paper slip)
The 5 μ L of colloidal-carbon mark gene probe prepared using the specific method of embodiment 10,10 μM of downstream detector primers 5 '
1 μ L, Taq of Biotin-C6-CTTCCACATACCATCTTCTTTAAC-3 ' (SEQ ID NO:2) 5 μ L, Taq archaeal dna polymerase are poly-
53 μ L, 10mM MgSO4 of μ L, 50%DMSO of synthase buffer, 3 μ L, contains various concentration staphylococcus aureus MRSA hot tearing
5 μ L of bacterium solution is solved, pure water complements to 50 μ L.Using PCR thermal cycler according to 95 degree 5 minutes, 95 degree 30 seconds+55 degree 30 seconds+72 spend
30 seconds 15 circulations, 72 degree of 5 minutes programs carry out amplification reaction, and sample is detected using chromatograph test strip after reaction, test strips
Nitrocellulose filter, the warp of 1mg/mL Streptavidin are coated with by the PVC backer board with viscose glue and the middle part being adhered to
10mM PBS+5mg/mL BSA+1%Tween-20 impregnates dry fiberglass packing and absorbent filter composition, on glass pad
5 μ L of PCR product is directly put, is analysed using 70 μ L 10mM PB extension liquid layer, is taken pictures (Fig. 3) after 15 minutes with daylight light irradiation, it can
See that paper slip outlet color depth and bacterial concentration have good correlation.
Embodiment 15
Magnetic particle
The 5 μ L of magnetic microsphere mark gene probe prepared using the specific method of embodiment 11, the specific side of embodiment 12
5 μ L, Taq archaeal dna polymerase of quantum dot-labeled gene probe, the 1 μ L of method preparation, 53 μ of μ L, 50%DMSO of Taq polymerase buffer
3 μ L of L, 10mM MgSO4, containing 10 μ L of various concentration staphylococcus aureus MRSA thermal cracking bacterium solution, pure water complements to 50 μ L.
Using PCR thermal cycler according to 95 DEG C 5 minutes, 95 DEG C 30 seconds+55 DEG C 30 seconds+72 DEG C 30 seconds carry out 20 circulation, 72 DEG C 5 minutes
Program carry out amplification reaction, after reaction sample strong magnet adsorb magnetic microsphere, TBST wash 2 times, with Fluorescence Spectrometer 365nm
Illumination excitation, scans the transmitting peak area (fluorescence intensity) at 560~680nm, emits peak area to bacterial concentration (CFU/mL)
It maps (Fig. 4), it is seen that fluorescence intensity has the good correlation (580-650nm of Fluorescence Spectrometer test QD610 with bacterial concentration
The integrated peak areas at place).
Embodiment 16
In every milliliter of 1 μm of ol/L water solubility thioacetic acid ligand 610nm emissive quantum dots solution be added 10~
The mixing of 100nmolNTA-PEG4- diphenyl cyclooctyne, boiling water bath 15 minutes, is then stored at room temperature 1 hour, 40000g centrifugation 20
Minute, precipitating pure water redissolves the click activation quantum dot being prepared to 1 milliliter containing spacerarm.
10~100nmol is added in every milliliter of 1 μm of ol/L water solubility thioacetic acid ligand 610nm emissive quantum dots solution
The mixing of NTA- diphenyl cyclooctyne, boiling water bath 15 minutes, is then stored at room temperature 1 hour, and 40000g is centrifuged 20 minutes, precipitates with pure
Water redissolves the click activation quantum dot being prepared to 1 milliliter without spacerarm.
It clicks for two kinds after 1nmol activation and 50nmol is added in activation quantum dot solution with the golden yellow of 5 ' nitrine modification
5 ' N3-GTTGTAGTTGTCGGGTTTGG-3 ' (SEQ ID NO:3) of the characteristic area color staphylococcus MRSA gene probe, 37 DEG C anti-
It after answering 24 hours, 40000g centrifugal purification 15 minutes, discards supernatant, precipitating is multiple with 5mM pH7 sodium phosphate buffer after centrifugation 2 times
It is molten to initial volume, the sub- point of two amounts-nucleic acid conjugated probes containing spacerarm and without containing spacerarm are prepared.
Using the complementary probe 5 '-CCAAACCCGACAACTACAAC-3 ' (SEQ ID NO:4) of gradient dilution in two nitre
5 sample spots of each point on acid cellulose film, the DNA content of each sample spot is about 10ng, 1ng, 0.1ng, 0.01ng,
0.001ng (contains 5% milk powder, 2 μ g/ml salmons with confining liquid after 56 DEG C of processing in dry drying 2 hours 30 minutes and 37 DEG C
Smart DNA, 8*SSC, 2.8%Triton X-100,0.2%SDS, 30% formamide) it is closed 1 hour at 37 DEG C.The sub- point-of two amounts
Nucleic acid conjugated probes are diluted to 0.1 μM using confining liquid respectively, are configured to hybridization solution.The nitrocellulose filter closed is taken out,
It is put into containing in the sub- point of two amounts-nucleic acid conjugated probes hybridization solution, 37 DEG C hybridize 2 hours, discard hybridization solution.Use film washing liquid
(containing 2*SSC, 0.2%SDS) washing 2 times is taken pictures (Fig. 5) with the ultraviolet light irradiation excitation of 365nm, and observation is it can be found that contain interval
Quantum dot-nucleic acid probe of arm has small-signal, and 1ng, 0.1ng sample spot signal in the sample spot of 0.01 box 0.001ng
Higher than the quantum dot-nucleic acid probe for being free of spacerarm, detection effect is more excellent.
It can be seen from the above description that the above embodiments of the present invention realized the following chievements: the present invention adopts
It is coupled nitrine modification of nucleic acids with the nano material that acetylenic clicks reagent activation, is coordinated using the suitable click reagent containing spacerarm
Efficient, stable, easy purification coupling is realized in coupling, and highly sensitive molecular diagnosis detection may be implemented.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Sequence table
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Claims (15)
1. a kind of nano material for label characterized by comprising
Nano material has tracer characteristic；And
Acetylenic click chemistry derivatives group is coupled at the nano-material surface, the acetylenic click chemistry derivatives group
Including clicking group.
2. nano material according to claim 1, which is characterized in that in the acetylenic click chemistry derivatives group with institute
State nano-material surface coupling coupling residue and the clicks group between also have spacerarm, the spacerarm be carbochain,
PEG, copolymer or polypeptide.
3. nano material according to claim 2, which is characterized in that the length of the spacerarm is 4~50 C-C keys,
Preferably 8~15 C-C keys；
Preferably, the PEG is PEG2~20.
4. nano material according to claim 1, which is characterized in that the nano material is selected from micro- by quantum dot, magnetism
One of group of ball, color latex microballoon, fluorescent microsphere, colloidal gold and colloidal-carbon composition is a variety of.
5. nano material according to claim 1, which is characterized in that the acetylenic click chemistry derivatives group includes straight
Alkyne click chemistry derivatives group and diphenyl cyclooctyne click chemistry derivatives group, the acetylenic click chemistry derivative
It is derived from coupling group with the nano-material surface coupling residue in group, the coupling group selects free sulfhydryl group, polynary mercapto
Base, nitrilotriacetic acid base, iminodiacetic acid, poly imidazoles, polyhistidine, amino, carboxyl, hydrazides, N- hydroxysuccinimidyl acyl are sub-
One of group of amine and maleimide composition is a variety of.
6. a kind of nucleic acid probe characterized by comprising
The surface coupling of the nano material for label as described in any one of claims 1 to 5, the nano material has alkynes
Class click chemistry derivatives group；And
Nucleic acid molecules, the nucleic acid molecules are the nucleic acid molecules of azido group activation, and the nucleic acid molecules pass through the azido
Group connect with the acetylenic click chemistry derivatives group.
7. nucleic acid probe according to claim 6, which is characterized in that the nucleic acid molecules include azido group, interval sequence
Area and functional sequence area are arranged, the intervening sequence area is between the azido group and functional sequence area, the intervening sequence
For poly thymidine or poly adenine.
8. a kind of method of nucleic acid and nano material coupling, which comprises the following steps:
The surface group of acetylenic click chemistry derivative and nano material is coupled by S1, and generating has click chemistry coupling
Active nano material, the acetylenic click chemistry derivative include coupling group and click group；
S2, by the nucleic acid molecules of azido group activation and the nano material with click chemistry coupling activity in buffer
Mixing, realizes the coupling of the nucleic acid molecules Yu the nano material.
9. according to the method described in claim 8, it is characterized in that, the nano material is selected from by quantum dot, magnetic microsphere, coloured silk
One of group of color latex beads, fluorescent microsphere, colloidal gold and colloidal-carbon composition is a variety of.
10. according to the method described in claim 8, it is characterized in that, the acetylenic click chemistry derivative includes straight chain alkynes point
Hit chemical derivative and/or diphenyl cyclooctyne click chemistry derivative, it is preferable that the coupling group selects free sulfhydryl group, more
First sulfydryl, nitrilotriacetic acid base, iminodiacetic acid, poly imidazoles, polyhistidine, amino, carboxyl, hydrazides, N- hydroxysuccinimidyl
One of group of acid imide and maleimide composition is a variety of.
11. according to the method described in claim 8, it is characterized in that, the coupled base of the acetylenic click chemistry derivative
There is the spacerarm of pre-set space distance between group and the click group, the spacerarm is selected from carbochain, PEG, copolymer or more
One of peptide is a variety of.
12. according to the method for claim 11, which is characterized in that the length of the spacerarm is 4~50 C-C keys, excellent
It is selected as 8~15 C-C keys, more preferably 10 C-C keys.
13. according to the method described in claim 8, it is characterized in that, the nucleic acid molecules and the tool of azido group activation
Having the molar ratio when mixing coupling for clicking the active nano material of chemical coupling is 1:1~1000:1.
14. according to the method described in claim 8, it is characterized in that, acetylenic click chemistry derivative described in the S1 and institute
The molar ratio for stating nano material is 10:1~100:1.
15. according to the method described in claim 8, it is characterized in that, reaction temperature in the S2 is less than or equal to 40 degrees Celsius,
The pH of the buffer is 7~8.
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Application publication date: 20190319 |