CN110108876A - A method of based on carcinomebryonic antigen in DNA carbon dots-silicon nano hydrogel material fluorescence detection serum - Google Patents
A method of based on carcinomebryonic antigen in DNA carbon dots-silicon nano hydrogel material fluorescence detection serum Download PDFInfo
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- 229910052799 carbon Inorganic materials 0.000 title claims abstract description 57
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 title claims abstract description 50
- 229910052710 silicon Inorganic materials 0.000 title claims abstract description 47
- 239000010703 silicon Substances 0.000 title claims abstract description 47
- 239000000017 hydrogel Substances 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 28
- 210000002966 serum Anatomy 0.000 title claims abstract description 27
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The application provides a kind of method based on carcinomebryonic antigen in DNA carbon dots-silicon nano hydrogel material fluorescence detection serum, this method includes the preparation of DNA carbon dots-silicon nano-hydrogel and DNA carbon dots-silicon nano-hydrogel fluorescence detection carcinomebryonic antigen, and the preparation of DNA carbon dots-silicon nano-hydrogel includes the preparation of fluorescent carbon point, the preparation of SiNPs-CD-DNA, DNA and the combination of methacrylic acid, the synthesis of polymethylacrylic acid (MAA)-DNA compound, the synthesis of DNA carbon dots-silicon nano-hydrogel.Reaction condition needed for DNA carbon dots-silicon nano-hydrogel of the application building is easy, build time is short, and the CEA in serum is detected using it, have the advantages that detection range is wide, detection sensitivity is high, detecting and selecting property is good, more accurate detection it can go out the CEA content in serum sample, the early diagnosis for tumour patient lesion provides help.
Description
Technical field
The present invention relates to carcinomebryonic antigen detection methods in serum, more particularly to one kind to be based on DNA carbon dots-silicon nano-hydrogel
The method of carcinomebryonic antigen in material fluorescence detection serum.
Background technique
Broad-spectrum tumor marker of the carcinomebryonic antigen (CEA) as clinical diagnosis cancer, with colon and rectum carcinoma, breast cancer,
The malignant tumours such as lung cancer are closely related.It determines its content in serum, has to early diagnosis, the therapeutic evaluation of malignant tumour
Very important meaning.
Currently, clinical generally using the content of CEA in enzyme-linked immunization (ELISA) detection serum.ELISA refers to antigen
Or antibody is fixed on solid carrier polyethylene microballoon, is identified and is reacted according to enzyme linked immunological, qualitative or quantitative detection target
Substance.This method accuracy is high, can detect the tumor markers of a certain concentration range.It is divided into according to the difference of immunologic process dual anti-
Body sandwich method, indirect method, competition law, prize law, surveys IgM antibody method at double site one-step method.But since operating process is complicated, hand
The disadvantages of section is cumbersome, detection limit is higher, the method that people have devised carcinomebryonic antigen in many novel detection serum again, such as electrification
Method and electrogenerated chemiluminescence method.Wei et al. utilizes Fe3O4@MnO2CEA in@Pt nano-complex Electrochemical Detection serum contains
Amount, detection are limited up to 0.16pg/mL.Guo et al. is examined using ruthenium silica core core-shell nanoparticles with electrogenerated chemiluminescence method
The CEA content in serum is surveyed, detection limit is up to 1.52 × 10-6ng/mL.Although both methods greatly improves CEA detection
Sensitivity, but have that the building time is long, stability is poor, urgently to be resolved the problems such as poor accuracy in complex system.
Summary of the invention
The application provides to solve above-mentioned technical problem.
The application is adopted the technical scheme that: one kind being based on DNA carbon dots-silicon nano hydrogel material fluorescence detection serum
The method of middle carcinomebryonic antigen, which comprises the following steps:
(1) DNA carbon dots-silicon nano-hydrogel preparation
(1) preparation of fluorescent carbon point
S1,0.6g urea and 0.3g citric acid are weighed in beaker, 10mL deionized water is added to be completely dissolved powder,
800W microwave heating 4min is put into 60 DEG C of the dry 1h of vacuum oven.Weigh dry powder quality.Black powder is put into 40mL
In ionized water, 3000rpm is centrifuged 20min, removes filter residue, collects supernatant, i.e. carbon dots stoste.
(2) preparation of SiNPs-CD-DNA
S2, the above-mentioned reaction clear liquid of 20 μ L is taken, is diluted to 500 μ L, be centrifuged 20min in 12500rpm, collect supernatant, i.e. carbon
Point base fluid is spare.
S3, in above-mentioned carbon dots base fluid, be added 20 μ L 10-5Initiation DNA chain and 2mg EDC (1- (the 3- dimethylamino third of M
Base) -3- ethyl-carbodiimide hydrochloride), room temperature activation 15min is after blending instrument mixes to get activating solution one；Meanwhile taking 5.5mg
NHS (n-hydroxysuccinimide) is added in the silica nanoparticle solution of surface amino groups modification, and blending instrument mixes rear chamber
Temperature activation 15min mixes two parts of solution to get activating solution two, after mixing reaction 2h in shaking table under room temperature, is put into 4
The reaction was continued in DEG C refrigerator 12h is to get nano SiO 2 particle-carbon dots-initiation DNA chain compound (SiNPs-CD-DNA).
(3) combination of DNA and methacrylic acid
S4,20 μ L 10 are taken-5DNA (the H comprising CEA aptamer structure of MA) mixed with the NHS solution of 10 μ L 0.55mg/mL
It closes, mixes post activation 30min to get HAActivating solution.Meanwhile by 17 μ L 2 × 10-10The methacrylic acid solution of M and 10 μ L
The EDC solution of 0.2mg/mL mixes, and reaction activation 30min is after mixing to get methacrylic acid activating solution one.Under room temperature,
By HAActivating solution and methacrylic acid activating solution one mix at room temperature, are put into shaking table, after reacting 2h at room temperature, are put into
The reaction was continued under 4 DEG C of environment of refrigerator 12h forms methacrylic acid-DNA1Compound (PA)。
S5,20 μ L 10 are taken-5DNA (the H of MB) mixed with the NHS of 10 μ L 0.55mg/mL, reaction activation 30min after mixing,
Both H is obtainedBActivating solution.Meanwhile by 17 μ L 2 × 10-8The methacrylic acid of M is mixed with the EDC solution of 10 μ L 0.2mg/mL, is mixed
Reaction activation 30min afterwards, both obtains methacrylic acid activating solution two.Under room temperature, by HBActivating solution and metering system acid activation
Liquid two mixes, and is put into shaking table, and after reacting 2h at room temperature, the 12h that is put under 4 DEG C of environment of refrigerator that the reaction was continued forms metering system
Acid-DNA2Compound (PB)。
(4) synthesis of polymethylacrylic acid-DNA compound
S6, the methacrylic acid-DNA for obtaining step (3)1Compound (PA) and methacrylic acid-DNA2Compound (PB)
In be separately added into 70 μ L concentration be 1% photoinitiator 12959,57 μ L HEPES buffer solutions are added, by methacrylic acid-
DNA1Compound (PA) and methacrylic acid-DNA2Compound (PB) two parts of solution are put into illumination reaction 19min under ultraviolet lamp, shape
At polymethylacrylic acid-DNA compound.
(5) DNA carbon dots-silicon nano-hydrogel synthesis
S7, the polymethylacrylic acid-DNA for obtaining step (4)1Compound (PA) and polymethylacrylic acid-DNA2Compound
(PB) two parts of solution are put into after heating 5min in 95 DEG C of water-baths, are transferred to rapidly in 0 DEG C of ice water, make solution temperature cool down rapidly to
With.
Nano SiO 2 particle-carbon dots-initiation DNA chain of S8, above-mentioned two parts of solution after cooling and step (2) generation
Compound (SiNPs-CD-DNA) solution mixes well, and 117 μ L HEPES buffer solutions are added, react 12h, i.e. shape in room temperature shaker
At DNA carbon dots-silicon nano-hydrogel.
(2) fluorescence intensity of CEA detects in serum
The serum containing CEA is added in the DNA carbon dots-silicon nano-hydrogel generated to step (5), is then placed in shaking table reaction
2-3h finally measures its fluorescence intensity with molecular fluorescence instrument, obtains fluorescence intensity level.
Further, in the combination of step (2) nano SiO 2 particle, carbon dots and initiation DNA chain, cause DNA
Chain-ordering is TTT TTT AGC TGA ACG ATA CCA, the terminal modified carboxyl of the initiation DNA chain 5 '.
Further, in the combination of step (3) DNA and methacrylic acid, the DNA (H with CEA aptamer structureA)
Structure sequence is TTT TTT CCA CGA TAC CAG CTT ATT CAA TTC GTG GGA TGT CTG GTA TCG TTC
AGC T, the DNA (H of the aptamer structure containing CEAA) the terminal modified amino of chain 3 ', 5 ' terminal modified methylene blues.
Further, in the combination of step (3) DNA and methacrylic acid, the DNA (HB) structure sequence be CCA
CGA ATT GAA CAA GAG CTG AAC GAT ACC ACT ATT GCT TAT TCA ATA TTT TT, the DNA
(HB) 5 ' terminal modified amino.
Further, in the step (4) and step (5), used HEPES buffer solution PH is 7.2, and is contained
0.15mol/L MgCl2With 0.15mol/L NaCl.
Further, in the step (5), the wavelength of ultraviolet lamp used is 365nm.
The application has the advantages and positive effects of:
1, using aptamer technology, a kind of means of new identification target is created, keep its biological safety high, it is special
Opposite sex identification is strong, and usage range is wide.Aptamer has many advantages, such as that generated time is short, and synthesis cost is low, stores convenient transportation, changes
It has been apt to the defect that traditional Ag-Ab identification technology build time is long, constructions cost is high, storage is difficult, has been more favorable for being commercialized
It promotes.
2, using carbon dots as fluorescence signal molecule, fluorescent quenching is made by the methylene blue of nucleic acid chain end, compared to biography
The electrochemistry and electrogenerated chemiluminescence method of CEA is determined in unified test, and fluorescence method is with good stability.
3, carbon dots-silicon nano-hydrogel composite construction is successfully realized in the application of biochemical analysis field, passes through precision
Structure design, in conjunction with the verifying of transmission electron microscope, it is ensured that it has good particle diameter distribution, is conducive to further realize industry
Change large-scale production.
4, there is stronger bio-toxicity for metal nano material, constructs main member using carbon, element silicon as life
The advantage of element, improves biocompatibility and the safety of carrier material, while constructing non pinetallic nano particle and can be greatly reduced
Material constructions cost.
It 5, is raw material using biological endogenous property molecular dna, preparation, which may be implemented, has good biocompatibility and biology drop
The fluorescence probe of solution property.Target substance detection of the Application of micron in complex sample, more fitting actual clinical tumour
Non-destructive testing in diagnosis needs.
6, relative to other complex detection means, item is reacted needed for the DNA carbon dots-silicon nano-hydrogel constructed in this case
Part is easy, and build time is short, low for equipment requirements, is conducive to actual production.
7, relative to clinically general detection method, this case detect serum using DNA carbon dots-silicon nano-hydrogel at present
In CEA, have the advantages that detection range is wide, detection sensitivity is high, detecting and selecting property is good, more accurate detection can go out serum
CEA content in sample, the early diagnosis for tumour patient lesion provide help.
The technical issues of in addition to the application described above solution, constitutes the technical characteristic of technical solution and by these
Except advantage brought by the technical characteristic of technical solution, wrapped in other technologies problem that the application can solve, technical solution
Advantage brought by the other technical characteristics contained and these technical characteristics, makees further details of hereinafter in conjunction with attached drawing
Explanation.
Detailed description of the invention
Fig. 1 is DNA carbon dots provided by the present application-silicon nano-hydrogel detection CEA schematic diagram；
Fig. 2 is DNA carbon dots in embodiment 1 provided by the present application-silicon nano-hydrogel electrophoresis phenogram；
Fig. 3 is the electron microscope of 1 nanostructure of embodiment provided by the present application；
Fig. 4 is DNA carbon dots provided by the present application-silicon nano-hydrogel detection CEA fluorescence kinetic profiles figure；
Fig. 5 is DNA carbon dots provided by the present application-silicon nano-hydrogel detection CEA working curve diagram；
Fig. 6 is bovine serum albumin(BSA) provided by the embodiments of the present application (BSA), fibrin ferment (thrombin), alpha-fetoprotein
(AFP) and carcinomebryonic antigen (CEA) fluorescence signal comparison diagram.
Specific embodiment
The application is described in further detail with reference to the accompanying drawings and examples.It is understood that this place is retouched
The specific embodiment stated is used only for explaining related invention, rather than the restriction to the invention.It also should be noted that in order to
Convenient for description, part relevant to invention is illustrated only in attached drawing.
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.The application is described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.
The preparation of embodiment 1:DNA carbon dots-silicon nano-hydrogel
(1) preparation of fluorescent carbon point
S1,0.6g urea and 0.3g citric acid are weighed in beaker, 10mL deionized water is added to be completely dissolved powder,
800W microwave heating 4min is put into 60 DEG C of the dry 1h of vacuum oven.Weigh dry powder quality.Black powder is put into 40mL
In ionized water, 3000rpm is centrifuged 20min, removes filter residue, takes supernatant, i.e. carbon dots stoste.
(2) preparation of SiNPs-CD-DNA
S2, carbon dots stoste made from 20 μ L steps (1) is taken, is diluted to 500 μ L, be centrifuged 20min in 12500rpm, carbon is made
Point base fluid is spare.
S3, in above-mentioned carbon dots base fluid, be added 20 μ L 10-5The initiation DNA chain and 2mg of the terminal modified carboxylic group of M 5 '
EDC, room temperature activation 15min is after blending instrument mixes to get activating solution one；Meanwhile taking 5.5mg NHS that surface amino groups modification is added
In silica nanoparticle solution, room temperature activation 15min mixes two parts of solution to get activating solution two after blending instrument mixes,
Under room temperature after shaking table mixes reaction 2h, the 12h that is put into 4 DEG C of refrigerators that the reaction was continued, both nano SiO 2 particle-carbon
Point-initiation DNA chain compound (SiNPs-CD-DNA).
(3) combination of DNA and methacrylic acid
S4,20 μ L 10 are taken-5DNA (the H comprising CEA aptamer structure of MA) mixed with the NHS solution of 10 μ L 0.55mg/mL
It closes, reaction activation 30min, both obtains H after mixingAActivating solution.Meanwhile by 17 L2 × 10 μ-10The methacrylic acid solution of M and 10 μ L
The EDC solution of 0.2mg/mL mixes, and reaction activation 30min, both obtains methacrylic acid activating solution one after mixing.Under room temperature,
By HAActivating solution and methacrylic acid activating solution one mix at room temperature, are put into shaking table and are put into 4 DEG C of rings of refrigerator after reaction activates 2h
The reaction was continued under border 12h forms methacrylic acid-DNA1Compound (PA)。
S5,20 μ L 10 are taken-5DNA (the H of MB) mixed with the NHS solution of 10 μ L 0.55mg/mL, post activation 30min is mixed,
Both H is obtainedBActivating solution.Meanwhile by 17 μ L 2 × 10-8The methacrylic acid long night of M mixes with the EDC solution of 10 μ L 0.2mg/mL,
Post activation 30min is mixed, methacrylic acid activating solution two is both obtained.Under room temperature, by HBActivating solution and metering system acid activation
Liquid two mixes, and is put into shaking table, and after reaction activation 2h, the 12h that is put into 4 DEG C of environment of refrigerator that the reaction was continued forms methacrylic acid-
DNA2Compound (PB)。
(4) synthesis of polymethylacrylic acid-DNA compound
S6, the methacrylic acid-DNA for obtaining step (3)1Compound (PA) and methacrylic acid-DNA2Compound (PB)
In be separately added into 70 μ L concentration be 1% photoinitiator 12959, add 57 μ L pH be 7.2 HEPES buffer solution, the buffering
Liquid contains MgCl2And NaCl, wherein MgCl2Concentration is 0.15M, concentration 0.15M, by methacrylic acid-DNA1Compound (PA)
With methacrylic acid-DNA2Compound (PB) two parts of solution are put into illumination 19min under 365nm ultraviolet lamp.Form polymethyl
Acid-DNA compound.
(5) DNA carbon dots-silicon nano-hydrogel synthesis
S7, the polymethylacrylic acid-DNA for obtaining step (4)1Compound (PA) and polymethylacrylic acid-DNA2Compound
(PB) after two parts of solution are put into 95 DEG C of heating water bath 5min, are transferred to rapidly in 0 DEG C of ice water, solution temperature is made to cool down rapidly for use.
Nano SiO 2 particle-carbon dots-initiation DNA that S8, above-mentioned two parts of solution after cooling and step (2) generate
Chain cpd (SiNPs-CD-DNA) solution mixes well, and the HEPES buffer solution that 117 μ L pH are 7.2 is added, which contains
There is MgCl2And NaCl, wherein MgCl2Concentration is 0.15M, concentration 0.15M, reacts 12h in room temperature shaker.Form DNA carbon
Point-silicon nano-hydrogel.
(6) DNA carbon dots-silicon nano-hydrogel carries out the verifying of electrophoresis characterization and preparation of surface nucleic acid property reasonable in design
The Electronic Speculum of material shape characterizes.
S9, electrophoresis characterization verifying
Ago-Gel is prepared, respectively to initiation DNA chain, the step in the SiNPs-CD-DNA compound of step (2) synthesis
Suddenly the P of (3) synthesisA, step (3) synthesis PB, cause chain DNA and PAHybridize the initiation chain DNA-P formedAStructure, step (5)
Initiation chain DNA-n (the P formed in synthesisA-PB) composite structure progress gel electrophoresis characterization.As a result as shown in Fig. 2, swimming lane 1 is
Cause chain DNA, swimming lane 2 is PA, swimming lane 3 is PB, swimming lane 4 is to cause chain DNA-PAStructure, swimming lane 5 are to cause chain DNA-n (PA-PB)
Composite structure.
As shown in Figure 2,1 bright band position of swimming lane is minimum, and swimming lane 2 and 3 bright band position of swimming lane are closer to, 4 bright band position of swimming lane
It sets and is higher than swimming lane 3,5 bright band position of swimming lane is much higher than swimming lane 1-4；Due to the position of bright band and nucleic acid base number in electrophoretic image and
Space structure is related, in the present embodiment, the corresponding base number for causing chain DNA of swimming lane 1 be it is the smallest, band is in most lower
End, the P after polymerizationA、PBMolecular weight, which is all larger than, causes chain DNA, and the pillar location of swimming lane 2 and swimming lane 3 is above swimming lane 1, due to
It is identical reaction condition and similar product structure, therefore the two is closer to, swimming lane 4 is to cause chain DNA-PAStructure, by
In cause chain DNA in conjunction with and so that its molecular weight is greater than simple PA, therefore band is higher than swimming lane 3, swimming lane 5 is corresponding to be caused
Chain DNA-n (PA-PB) composite structure, pillar location is much higher than swimming lane 1-4 in the swimming lane, thus shows DNA-n (PA-PB) multiple
Close the molecular weight that object structural molecule amount is much larger than swimming lane 1-4 tie substance, it was demonstrated that PAWith PBChain type hybrid design obtained smoothly
Initiation, realize (PA-PB) hybridization growth, form n (PA-PB) stereochemical structure.
In the present embodiment, (PA-PB) to promote nano SiO 2 particle-carbon dots-initiation DNA chain multiple for chain type hybridization reaction
Close object surface n (PA-PB) 3D stereochemical structure formation, n (PA-PB) loose porous structure facilitates knowledge of the aptamer to CEA
The other and protection to internal fluorescent effect part fluorescent carbon point structure, improves the stabilization applied in the complex environments such as serum
Property.
S10, Electronic Speculum characterization
To carbon dots structure, silicon nano-particle structures, carbon dots-silicon nanostructure and DNA carbon dots-silicon nano-hydrogel structure into
The detection of row Electronic Speculum, investigates particle size and particle size uniformity, and result is as shown in figure 3, figure a is carbon dots structure chart, and partial size is about
For 10nm or so, lattice distance is 0.54nm；Figure b is nano silicon particles figure, and partial size is 50nm or so；Figure c is carbon dots-silicon nanometer
Structure chart, partial size are 62nm or so；Figure d is DNA carbon dots-silicon nano-hydrogel figure, and partial size is about 74nm or so.It can by Fig. 3
Know, compare carbon dots structure, silicon nano-particle structures, carbon dots-silicon nanostructure, DNA carbon dots-silicon nano-hydrogel present by
In the reasonable change of size that layer-by-layer modification generates, and there is uniform size composition, which is convenient for quantitative measurement target.
In the present embodiment, cause DNA chain sequence, the DNA (H with CEA aptamer structureA) structure sequence and DNA (HB) structure
Sequence is as follows:
Embodiment 2:DNA carbon dots-silicon nano-hydrogel detects various concentration CEA
1, the SiNPs-CD-MA-DNA carrier solution for taking 100 μ L embodiments 1 to prepare respectively divides in 18 centrifuge tubes
It Bian Hao not be a to r.
It 2, to above-mentioned number is respectively that 20 μ L concentration are added into r centrifuge tube is 0ng/mL, 4x10 to a-6ng/mL、7x10- 6ng/mL、1x10-5ng/mL、4x10-5ng/mL、7x10-5ng/mL、1x10-4ng/mL、4x10-4ng/mL、7x10-4ng/mL、
1x10-3ng/mL、4x10-3ng/mL、7x10-3ng/mL、1x10-2ng/mL、4x10-2ng/mL、7x10-2ng/mL、0.1ng/mL、
The determinand CEA standard solution of 0.4ng/mL, 1.0ng/mL.
3, the above-mentioned centrifuge tube sequentially added after various concentration CEA titer is placed in oscillating reactions 3h in 37 DEG C of shaking tables.
4, the time take outs of the shaker above-mentioned centrifuge tube after, and 50 μ L reaction solutions is taken to be placed in fluorescence from each centrifuge tube
In spectrophotometer cell, under the maximum excitation wavelength excitation of 405nm, the emission spectrum in the section 430-600nm is acquired.
5, repeat the above steps 1-4, more than five times, fluorescence kinetic profiles figure is drawn, according to fluorescence kinetic profiles figure
Determine best launch wavelength, each centrifuge tube reaction solution in step 3 when measuring repetition experiment every time under best launch wavelength one by one,
Fluorescent value is obtained, the average value of the best transmitted wave strong point fluorescence intensity level of each centrifuge tube reaction solution of step 3 is taken, draws detection
The working curve diagram of CEA.
Fig. 4 is fluorescence kinetic profiles figure, and best launch wavelength is 515nm known to fluorescence kinetic profiles figure, and Fig. 5 is
According to the working curve diagram for the detection CEA that the fluorescence intensity under the best launch wavelength of 515nm is drawn.As shown in Figure 5 in 515nm
Place, fluorescence intensity are linearly increased with the concentration increase of CEA, and the range of linearity is 4.0 × 10-6ng/mL-1.0×10-2ng/
ML, linear equation IF=4372.11lgCCEA+270.10(R2=0.9952), detection is limited to 1.48 × 10-6Ng/mL, the reality
It tests the result shows that compared to existing CEA detection method, DNA carbon dots-silicon nano-hydrogel is wider to CEA detection range, detection is sensitive
Du Genggao.
Embodiment 3: pure to ox blood based on DNA carbon dots-silicon nano hydrogel material fluorescence detection carcinomebryonic antigen method
Albumen (BSA), fibrin ferment (thrombin), alpha-fetoprotein (AFP) and the comparison of carcinomebryonic antigen (CEA) fluorescence signal
For the DNA carbon dots-silicon nano-hydrogel for taking 100 μ L embodiments 1 to prepare respectively in 6 test tubes, one of them is sky
White control, taking 4 test tubes to be separately added into 20 μ L concentration is 10-6Bovine serum albumin(BSA) (BSA), the fibrin ferment of ng/mL
(thrombin), 20 μ L bovine serum albumin(BSA)s are added in alpha-fetoprotein (AFP) and carcinomebryonic antigen (CEA) in last branch test tube
(BSA), fibrin ferment (thrombin), alpha-fetoprotein (AFP) and carcinomebryonic antigen (CEA) concentration are 10-6The mixing of ng/mL is molten
Liquid, and it is put into shaking table reaction 3h, 6 parts of solution fluorescence intensity then are measured with molecular fluorescence instrument, repeatedly experiment takes its fluorescence
The average value of intensity carries out subsequent data analysis.
As a result as shown in figure 5, blank group fluorescent value is very low in figure, 10 are added-6Ng/mL bovine serum albumin(BSA) (BSA), blood coagulation
The fluorescent value of the experimental group of enzyme (thrombin), alpha-fetoprotein (AFP) is also far below 10-6The fluorescence of ng/mL carcinomebryonic antigen (CEA)
Value；Bovine serum albumin(BSA) (BSA), fibrin ferment (thrombin), alpha-fetoprotein (AFP) and carcinomebryonic antigen (CEA) concentration are 10- 6The fluorescent value of the mixed solution of ng/mL and 10-6The fluorescent value difference of ng/mL carcinomebryonic antigen (CEA) is less.The present embodiment experiment
As a result illustrate that this method has good selectivity, suitable for detecting the levels of CEA in complex sample system.
Embodiments herein is described in detail above, but the content is only the preferred embodiment of the application,
It should not be construed as limiting the practical range of the application.All the changes and improvements made by all application ranges according to the application
Deng should still belong within the patent covering scope of the application.
Sequence table
<110>Qingdao University of Science and Technology
<120>a kind of method based on carcinomebryonic antigen in DNA carbon dots-silicon nano hydrogel material fluorescence detection serum
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>5 ' terminal modified carboxyls
<400> 1
ttttttagct gaacgatacc a 21
<210> 2
<211> 55
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>3 ' terminal modified amino, 5 ' terminal modified methylene blues
<400> 2
ttttttccac gataccagct tattcaattc gtgggatgtc tggtatcgtt cagct 55
<210> 3
<211> 53
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>5 ' terminal modified amino
<400> 3
ccacgaattg aacaagagct gaacgatacc actattgctt attcaatatt ttt 53
Claims (6)
1. a kind of method based on carcinomebryonic antigen in DNA carbon dots-silicon nano hydrogel material fluorescence detection serum, feature exist
In, comprising the following steps:
(1) DNA carbon dots-silicon nano-hydrogel preparation
(1) preparation of fluorescent carbon point
S1,0.6g urea and 0.3g citric acid are weighed in beaker, 10mL deionized water is added to be completely dissolved powder, it is micro- in 800W
Wave heating 4min is put into 60 DEG C of the dry 1h of vacuum oven.Weigh dry powder quality.Black powder is put into 40mL deionized water
In, 3000rpm is centrifuged 20min, removes filter residue, collects supernatant, i.e. carbon dots stoste；
(2) preparation of SiNPs-CD-DNA
S2, the above-mentioned reaction clear liquid of 20 μ L is taken, is diluted to 500 μ L, be centrifuged 20min in 12500rpm, collect supernatant, i.e. carbon dots base
Liquid, it is spare；
S3, in above-mentioned carbon dots base fluid, be added 20 μ L 10-5Initiation DNA chain and 2mg the EDC (1- (3- dimethylamino-propyl)-of M
3- ethyl-carbodiimide hydrochloride), room temperature activation 15min is after blending instrument mixes to get activating solution one；Meanwhile taking 5.5mg NHS
(n-hydroxysuccinimide) is added in the silica nanoparticle solution of surface amino groups modification, and room temperature is living after blending instrument mixes
Change 15min to get activating solution two, two parts of solution are mixed, after mixing reaction 2h in shaking table under room temperature, is put into 4 DEG C of ice
The reaction was continued in case 12h is to get nano SiO 2 particle-carbon dots-initiation DNA chain compound (SiNPs-CD-DNA)；
(3) combination of DNA and methacrylic acid
S4,20 μ L 10 are taken-5DNA (the H comprising CEA aptamer structure of MA) mixed with the NHS solution of 10 μ L 0.55mg/mL, it mixes
Even post activation 30min is to get HAActivating solution.Meanwhile by 17 μ L 2 × 10-10The methacrylic acid solution of M and 10 μ L 0.2mg/
The EDC solution of mL mixes, and reaction activation 30min is after mixing to get methacrylic acid activating solution one.Under room temperature, by HAIt is living
Change liquid and methacrylic acid activating solution one mixes at room temperature, be put into shaking table, after reacting 2h at room temperature, is put into refrigerator 4
The reaction was continued under DEG C environment 12h, forms methacrylic acid-DNA1Compound (PA)；
S5,20 μ L 10 are taken-5DNA (the H of MB) mixed with the NHS of 10 μ L 0.55mg/mL, reaction activation 30min after mixing, both
HBActivating solution.Meanwhile by 17 μ L 2 × 10-8The methacrylic acid of M is mixed with the EDC solution of 10 μ L 0.2mg/mL, anti-after mixing
30min should be activated, methacrylic acid activating solution two is both obtained.Under room temperature, by HBActivating solution and methacrylic acid activating solution two
Mixing, is put into shaking table, and after reacting 2h at room temperature, the 12h that is put under 4 DEG C of environment of refrigerator that the reaction was continued forms methacrylic acid-
DNA2Compound (PB)；
(4) synthesis of polymethylacrylic acid-DNA compound
S6, the methacrylic acid-DNA for obtaining step (3)1Compound (PA) and methacrylic acid-DNA2Compound (PB) in point
The photoinitiator 12959 that 70 μ L concentration are 1% is not added, 57 μ L HEPES buffer solutions are added, by methacrylic acid-DNA1It is multiple
Close object (PA) and methacrylic acid-DNA2Compound (PB) two parts of solution are put into illumination reaction 19min under ultraviolet lamp, form poly- first
Base acrylic acid-DNA compound；
(5) DNA carbon dots-silicon nano-hydrogel synthesis
S7, the polymethylacrylic acid-DNA for obtaining step (4)1Compound (PA) and polymethylacrylic acid-DNA2Compound (PB)
Two parts of solution are put into 95 DEG C of water-baths after heating 5min, are transferred to rapidly in 0 DEG C of ice water, solution temperature is made to cool down rapidly for use；
Nano SiO 2 particle-carbon dots-initiation DNA chain that S8, above-mentioned two parts of solution after cooling and step (2) generate is compound
Object (SiNPs-CD-DNA) solution mixes well, and 117 μ L HEPES buffer solutions are added, reacts 12h in room temperature shaker, that is, is formed
DNA carbon dots-silicon nano-hydrogel；
(2) fluorescence intensity of CEA detects in serum
The serum containing CEA is added in the DNA carbon dots-silicon nano-hydrogel generated to step (5), is then placed in shaking table reaction 2-
3h finally measures its fluorescence intensity with molecular fluorescence instrument, obtains fluorescence intensity level.
2. as described in claim 1 a kind of based on carcinomebryonic antigen in DNA carbon dots-silicon nano hydrogel material fluorescence detection serum
Method, which is characterized in that step (2) nano SiO 2 particle, carbon dots and cause DNA chain combination in, cause DNA
Chain-ordering is TTT TTT AGC TGA ACG ATA CCA, the terminal modified carboxyl of the initiation DNA chain 5 '.
3. as claimed in claim 2 a kind of based on carcinomebryonic antigen in DNA carbon dots-silicon nano hydrogel material fluorescence detection serum
Method, which is characterized in that in the combination of step (3) DNA and methacrylic acid, with CEA aptamer structure DNA (HA)
Structure sequence is TTT TTT CCA CGA TAC CAG CTT ATT CAA TTC GTG GGA TGT CTG GTA TCG TTC
AGC T, the DNA (H of the aptamer structure containing CEAA) the terminal modified amino of chain 3 ', 5 ' terminal modified methylene blues.
4. as claimed in claim 3 a kind of based on carcinomebryonic antigen in DNA carbon dots-silicon nano hydrogel material fluorescence detection serum
Method, which is characterized in that in the combination of step (3) DNA and methacrylic acid, the DNA (HB) structure sequence be CCA
CGA ATT GAA CAA GAG CTG AAC GAT ACC ACT ATT GCT TAT TCA ATA TTT TT, the DNA
(HB) 5 ' terminal modified amino.
5. as claimed in claim 4 a kind of based on carcinomebryonic antigen in DNA carbon dots-silicon nano hydrogel material fluorescence detection serum
Method, which is characterized in that in the step (4) and step (5), used HEPES buffer solution PH is 7.2, and is contained
0.15mol/L MgCl2With 0.15mol/L NaCl.
6. as claimed in claim 5 a kind of based on carcinomebryonic antigen in DNA carbon dots-silicon nano hydrogel material fluorescence detection serum
Method, which is characterized in that in the step (5), the wavelength of ultraviolet lamp used is 365nm.
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