CN107142311A - A kind of method that chemiluminescence detects DNA - Google Patents
A kind of method that chemiluminescence detects DNA Download PDFInfo
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- CN107142311A CN107142311A CN201710371612.5A CN201710371612A CN107142311A CN 107142311 A CN107142311 A CN 107142311A CN 201710371612 A CN201710371612 A CN 201710371612A CN 107142311 A CN107142311 A CN 107142311A
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- 238000000034 method Methods 0.000 title claims abstract description 28
- 239000003298 DNA probe Substances 0.000 claims abstract description 23
- 239000011324 bead Substances 0.000 claims abstract description 23
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000000523 sample Substances 0.000 claims abstract description 21
- 108020003215 DNA Probes Proteins 0.000 claims abstract description 20
- 239000002245 particle Substances 0.000 claims abstract description 9
- 239000000126 substance Substances 0.000 claims abstract description 6
- 239000000084 colloidal system Substances 0.000 claims abstract description 5
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 claims abstract description 5
- 238000009396 hybridization Methods 0.000 claims abstract description 3
- 108020004414 DNA Proteins 0.000 claims description 70
- 239000000243 solution Substances 0.000 claims description 37
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 17
- 238000001514 detection method Methods 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 8
- DQPBABKTKYNPMH-UHFFFAOYSA-N amino hydrogen sulfate Chemical compound NOS(O)(=O)=O DQPBABKTKYNPMH-UHFFFAOYSA-N 0.000 claims description 7
- 230000000694 effects Effects 0.000 claims description 7
- SJUCACGNNJFHLB-UHFFFAOYSA-N O=C1N[ClH](=O)NC2=C1NC(=O)N2 Chemical compound O=C1N[ClH](=O)NC2=C1NC(=O)N2 SJUCACGNNJFHLB-UHFFFAOYSA-N 0.000 claims description 6
- 238000009835 boiling Methods 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 6
- 230000009514 concussion Effects 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 6
- 230000004048 modification Effects 0.000 claims description 6
- 238000012986 modification Methods 0.000 claims description 6
- 230000008836 DNA modification Effects 0.000 claims description 5
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 claims description 4
- 238000002474 experimental method Methods 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 239000003292 glue Substances 0.000 claims description 3
- 238000007654 immersion Methods 0.000 claims description 3
- 238000003760 magnetic stirring Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- NICDRCVJGXLKSF-UHFFFAOYSA-N nitric acid;trihydrochloride Chemical compound Cl.Cl.Cl.O[N+]([O-])=O NICDRCVJGXLKSF-UHFFFAOYSA-N 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000002955 isolation Methods 0.000 claims description 2
- 238000007422 luminescence assay Methods 0.000 claims description 2
- 238000004020 luminiscence type Methods 0.000 claims description 2
- 238000004140 cleaning Methods 0.000 claims 1
- 238000002845 discoloration Methods 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 2
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 abstract 2
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 abstract 1
- 238000005516 engineering process Methods 0.000 description 5
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- RYYXDZDBXNUPOG-UHFFFAOYSA-N 4,5,6,7-tetrahydro-1,3-benzothiazole-2,6-diamine;dihydrochloride Chemical compound Cl.Cl.C1C(N)CCC2=C1SC(N)=N2 RYYXDZDBXNUPOG-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 125000000143 2-carboxyethyl group Chemical group [H]OC(=O)C([H])([H])C([H])([H])* 0.000 description 1
- 101100262441 Caenorhabditis elegans rfl-1 gene Proteins 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 229910004042 HAuCl4 Inorganic materials 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical class ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 239000012327 Ruthenium complex Substances 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- BRSVJNYNWNMJKC-UHFFFAOYSA-N [Cl].[Au] Chemical compound [Cl].[Au] BRSVJNYNWNMJKC-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000002038 chemiluminescence detection Methods 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 229910001429 cobalt ion Inorganic materials 0.000 description 1
- XLJKHNWPARRRJB-UHFFFAOYSA-N cobalt(2+) Chemical compound [Co+2] XLJKHNWPARRRJB-UHFFFAOYSA-N 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 238000007306 functionalization reaction Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- RJHLTVSLYWWTEF-UHFFFAOYSA-K gold trichloride Chemical class Cl[Au](Cl)Cl RJHLTVSLYWWTEF-UHFFFAOYSA-K 0.000 description 1
- 229910021505 gold(III) hydroxide Inorganic materials 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 150000004714 phosphonium salts Chemical class 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract
The invention belongs to analytical chemistry field, and in particular to a kind of method that chemiluminescence detects DNA, and its principle is to reduce gold chloride with luminol, obtains luminol colloid golden nanometer particle LumAuNPs, modifies LumAuNPs with DNA probe, obtains chemiluminescence probe；Then the capture dna of hairpin structure is fixed by carrier of magnetic bead, in the presence of object DNA, the hairpin structure of capture dna is opened, and under the hybridization of the DNA probe on chemiluminescence probe, and magnetic bead surfaces are connected to by being catalyzed hair fastener self-assembling technique by chemiluminescence probe；After Magneto separate, azanol O sulfonic acid is added, using LumAuNPs-azanol O sulfonic acid as chemical luminous system, chemical luminescent detecting is carried out, the measure of target dna is realized according to the chemiluminescence of generation.Method has the high advantage of simple, sensitivity.
Description
Technical field
The invention belongs to analytical chemistry field, and in particular to a kind of method that chemiluminescence detects DNA.
Background technology
Luminol is conventional chemical illuminating reagent, is widely used in various chemiluminescence analysis detections.However, Rumi
Promise chemiluminescence produces higher background signal, therefore its application is subject to certain restrictions.In recent years, the collaurum of functionalization by
The favor of researcher, can use the method synthesis collaurum of ruthenium complex, luminol and its derivative reduction gold chloride
(Cui H,Wang W,Duan C F,et al.Synthesis,characterization,and
electrochemiluminescence of luminol-reduced gold nanoparticles and their
application in a hydrogen peroxide sensor[J].Chemistry,2007,13(24):6975-6984；
Gao W,Qi W,Lai J,et al.Thiourea dioxide as a unique eco-friendly coreactant
for luminol chemiluminescence in the sensitive detection of luminol,thiourea
dioxide and cobalt ions[J].Chemical Communications,2014,51(9):1620-1623.), its
In, the collaurum referred to as LumAuNPs that gold chloride is obtained is reduced using luminol, stronger chemistry is obtained in the presence of hydrogen peroxide
Luminous signal.It can react due to dioxygen water unstable and with many metal ions, so its selectivity is bad.In view of existing
The deficiency of technology, the present invention realizes DNA measure using LumAuNPs-hydroxylamine-o-sulfonic acid chemiluminescence detection system, has
There is the features such as sensitivity is high, method is simple.
The content of the invention
It is contemplated that inventing a kind of method for the measure DNA that method is simple, sensitivity is high.
In view of the deficiencies in the prior art, it is an object of the invention to provide a kind of method that chemiluminescence detects DNA.
Realizing the object of the invention technical scheme is：
A kind of method that chemiluminescence detects DNA, its principle is to reduce gold chloride with luminol, obtains luminol glue
Body golden nanometer particle LumAuNPs, modifies LumAuNPs with DNA probe, obtains chemiluminescence probe；Then consolidate by carrier of magnetic bead
Determine the capture dna of hairpin structure, in the presence of object DNA, the hairpin structure of capture dna is opened, and visited in chemiluminescence
Under the hybridization of DNA probe on pin, magnetic bead surfaces are connected to by being catalyzed hair fastener self-assembling technique by chemiluminescence probe；
After Magneto separate, hydroxylamine-o-sulfonic acid is added, using LumAuNPs-hydroxylamine-o-sulfonic acid as chemical luminous system, chemistry is carried out
Luminescence assays, the measure of target dna is realized according to the chemiluminescence of generation.
The present invention is realized by following measures：A kind of method that chemiluminescence detects DNA, it is characterized in that bag
Include following steps：
(1) preparation of luminol colloid golden nanometer particle；
(2) capture dna modifies the preparation of magnetic bead；
(3) DNA probe modifies LumAuNPs preparation；
(4) detection of target dna.
It is preferred that, the preparation of described luminol colloid golden nanometer particle comprises the following steps：
Before experiment starts, by glass apparatus HNO used3/HCl(3:1, v/v) after chloroazotic acid immersion 24h, with secondary
Distilled water flushing, is put into oven for drying.The chlorauric acid solution plus deionized water for taking a certain amount of 1% are diluted to 0.02% chlorine gold
Acid solution is placed in three-necked flask, is heated to reflux boiling under magnetic stirring；After solution boiling after, rapidly join 0.01mL~
5mL 0.01M luminol solution, continues heating and boils, the color of solution is changed into black from light yellow, eventually becomes claret,
Stop heating after 40min, and be cooled to room temperature under continued mixing, obtain luminol colloid golden nanometer particle, i.e. LumAuNPs will
Obtained LumAuNPs is transferred in brown, wide-mouth bottle, is saved backup at 4 DEG C.
It is preferred that, the preparation of described capture dna modification magnetic bead comprises the following steps：
Take the μ L carboxylated magnetic bead solution of 10 μ L~100 to be put into 1.5mL centrifuge tubes, be 0.1M with the μ L concentration of 10 μ L~200
Imidazole buffer is washed three times, is subsequently dispersed the 0.1M imidazole buffers that 0.01mL~2mL contains 0.1M EDC and 0.05M NHS
In liquid, under the conditions of 37 DEG C, oscillating reactions 30min；Then it is 5.0 × 10 that the μ L concentration of 10 μ L~200 is added into centrifuge tube- 8M capture dnas, the shaken overnight under the conditions of 37 DEG C obtains capture dna modification magnetic bead, then again with 2.0mL 0.1M PBS bufferings
Solution is cleaned three times, is finally distributed in 2.0mL PBS cushioning liquid, 4 DEG C of preservations.
It is preferred that, described DNA probe modification LumAuNPs preparation comprises the following steps：
It is 1.0 × 10 that the μ L of 1 μ L~20 TCEP is added into the μ L concentration of 10 μ L~200-6In M DNA probe solution, 37 DEG C are shaken
Activation 1 hour is swung, then takes 100 μ L~LumAuNPs synthetic 1000 μ L to be added in the solution, is shaken under the conditions of 37 DEG C
Overnight, 10 μ L~200 μ L NaCl containing 0.3M pH 8.2 10mM Tris-HCl buffer solutions are then added；Continue to shake 48h
Afterwards, it is after centrifugation 30min under conditions of 12000rpm, red precipitate is clear with 1mL pH 7.4 0.1M PBS cushioning liquid
Wash, centrifuge again, so in triplicate, obtain DNA probe modification LumAuNPs, i.e. chemiluminescence probe.The chemistry finally obtained
Luminescence probe is distributed in 1000 μ L pH 7.4 0.1M PBS cushioning liquid, and 4 DEG C save backup.
It is preferred that, the detection of described target dna comprises the following steps：
Take the μ L capture dnas of 10 μ L~200 modification magnetic bead solution to be placed in centrifuge tube, then take the μ L of 10 μ L~100 to contain target
DNA solution is added in this centrifuge tube, concussion reaction 40min under the conditions of 37 DEG C, then adds the μ L probes of 10 μ L~100
DNA modification LumAuNPs solution, concussion reaction 40min under the conditions of 37 DEG C, passes through the effect of target dna and capture dna, probe
DNA and target dna and capture dna effect, chemiluminescence probe are connected to magnetic bead surfaces；After Magneto separate, by magnetic point
In the 0.1M PBS cushioning liquid that 50 μ L pH 7.4 are dispersed in from thing, hydroxylamine-o-sulfonic acid solution is then added, chemistry is produced
It is luminous, it is quantitative according to chemiluminescence intensity, realize the measure of target dna.
Described DNA sequence dna is：
Capture dna：5`-ATATACGCCATGTAGCATTCGGT TAGG CGTAT ATT TG C TT-NH2-3`；
Target dna：5`-AATATACGCCTAACCG-3`；
DNA probe：5`-SH-TATA CGCCTAACCGAATGCT TACCACGCGTAT AG C A T CCGA-3`
The present invention have studied the relation between various concentrations target dna and chemiluminescence intensity, obtain detection target dna
Standard curve, the range of linearity and linear equation.
The advantage and effect of invention
When the concentration of target dna is between 80pM to 10nM, with the change of target DNA concentration, chemiluminescence intensity
There is significant change.The nonlinear equation for calculating detection target dna is y=3663.39648+332.34823x (y：Chemistry
Luminous intensity；x：The logarithm of target DNA concentration, unit is M), linearly dependent coefficient is 0.9987, and detection is limited to 30pM (3 σ)
(Fig. 2).The precision of the assay method is calculated by carrying out 11 parallel determinations to concentration for 500pM target dna,
Relative standard deviation is 3.6%, shows the assay method of the present invention and has preferable reappearance.
In addition, utilizing LumAuNPs-H2O2For detection architecture, when other steps are identical, in the present inventive method to target
DNA is detected that the detection of measure is limited to 500pM.Show the method that a kind of chemiluminescence proposed by the present invention detects DNA
With high sensitivity.
Brief description of the drawings
Fig. 1 detects the principle schematic of target dna.
The concentration of Fig. 2 target dnas and chemiluminescence intensity graph of a relation.
Embodiment
The operating method of the present invention will be further illustrated in following example, but does not constitute the further limitation to invention.
Example 1：A kind of method that chemiluminescence detects DNA
1. experimental section
1.1 instruments and reagent
1.1.1 instrument and equipment
DHG air dry ovens (kind will experimental instruments and equipment limited, Shanghai)；AR224CN type Ao Haosi assay balances (Qingdao
Neutralize Hengxin Electronic Co., Ltd., Qingdao)；THZ type constant temperature oscillations case (good Asource industry Science and Technology Ltd., Beijing)；RFL-1 types
Ultraweak chemiluminescence detector (Rui Mai Analytical Instrument Co., Ltd, Xi'an)；Anke-TGL-16C flies father-in-law's board supercentrifuge
(peace booth scientific instrument factory, Shanghai).
1.1.2 reagent
1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC), N- hydroxysuccinimides (NHS), chlorine
Auric acid (HAuCl4) bought from Sigma companies；Particle diameter is 0.5 μm, concentration for 10mg/mL carboxyl magnetic bead from Tianjin times Si Lese
Buy spectral technology development centre；Luminol (Luminol), hydroxylamine-o-sulfonic acid (HOSA) and TCEP (three (2- carboxyethyls) phosphonium salts acid
Salt) buy in Aladdin companies；0.01M luminol 0.1M NaOH dissolve, and are stored in brown bottle in 4 DEG C of refrigerators；
Take 1g gold chlorides plus 100mL water to be made into 1% chlorauric acid solution, preserved with brown bottle, diluted using preceding with redistilled water.
PBS cushioning liquid is 0.10M, and pH 7.4, its compound method is to weigh 0.1g KH2PO4、4.0g NaCl、1.45g
Na2HPO4·12H2O and 0.1g KCl are dissolved in 1L water, are produced.
The sequence of the used single stranded DNA of this experiment and hairpin dna (Sheng Gong bioengineering Co., Ltd synthesizes by Shanghai)
It is as follows：
Capture dna：5`-ATATACGCCATGTAGCATTCGGT TAGG CGTAT ATT TG C TT-NH2-3`；
Target dna：5`-AATATACGCCTAACCG-3`；
DNA probe：5`-SH-TATA CGCCTAACCGAATGCT TACCACGCGTAT AG C A T CCGA-3`.
The DNA of hairpin structure is reused after carrying out incubation processing.
1.2 LumAuNPs synthesis
Before experiment starts, glass apparatus used uses HNO3/HCl(3:1, v/v) after chloroazotic acid immersion 24h, with secondary
Distilled water flushing, is put into oven for drying.100 μ L 1% chlorauric acid solution plus deionized water is taken to be diluted to 50mL's 0.02%
Chlorauric acid solution is placed in three-necked flask, magneton is added in three-necked flask, and put it into magnetic stirring apparatus, magnetic agitation
Under be heated to reflux boiling.After after solution boiling, 1mL 0.01M luminol solution is rapidly joined, continues heating and boils 40min,
The color of solution is changed into black from light yellow, eventually becomes and stops heating and being cooled under continued mixing after claret, 40min
Room temperature.Obtained LumAuNPs is transferred in brown, wide-mouth bottle, saved backup at 4 DEG C.
1.3 capture dnas modify the preparation of magnetic bead
Take 50 μ L carboxylated magnetic bead solution to be put into 1.5mL centrifuge tubes, be that 0.1M imidazole buffers are washed with 100 μ L concentration
Three times, it is subsequently dispersed in the 0.1M imidazole buffers that 1mL contains 0.1M EDC and 0.05M NHS, under the conditions of 37 DEG C, vibration
React 30min；Then it is 5.0 × 10 that 100 μ L concentration are added in centrifuge tube-8M capture dnas, the shaken overnight under the conditions of 37 DEG C,
The magnetic bead of capture dna modification is obtained, is then cleaned three times with 2.0mL 0.1M PBS cushioning liquid again, is finally distributed to 2.0mL
In PBS cushioning liquid, 4 DEG C of preservations.
1.4 DNA probes modify LumAuNPs preparation
It is 1.0 × 10 that 5 μ L TCEP is added into 100 μ L concentration-6In M DNA probe solution, 37 DEG C of vibrations are activated 1 hour,
The LumAuNPs for taking 600 μ L synthetic again is added in the solution, is shaken and is stayed overnight under the conditions of 37 DEG C, is then added 50 μ L and is contained
0.3M NaCl pH 8.2 10mM Tris-HCl buffer solutions；Continue to shake after 48h, centrifuged under conditions of 12000rpm
After 30min, red precipitate is cleaned with 1mL pH 7.4 0.1M PBS cushioning liquid, centrifuges, so in triplicate, obtains again
DNA probe modifies LumAuNPs, i.e. chemiluminescence probe.The chemiluminescence probe finally obtained is distributed to 1000 μ L pH 7.4
0.1M PBS cushioning liquid in, 4 DEG C save backup.
The detection of 1.5 target dnas
Take 50 μ L capture dnas to modify magnetic bead solution to be placed in centrifuge tube, then take solution of the 50 μ L containing target dna to add again
Into centrifuge tube, then 37 DEG C of concussion reaction 40min are added under the conditions of 50 μ L DNA probes modification LumAuNPs solution, 37 DEG C
Concussion reaction 40min, effect and DNA probe and the effect of target dna and capture dna by target dna and capture dna,
Chemiluminescence probe is connected to magnetic bead surfaces；After Magneto separate, Magnetic Isolation thing is dispersed in 50 μ L pH 7.4 0.1M
In PBS cushioning liquid, hydroxylamine-o-sulfonic acid solution is then added, chemiluminescence is produced.Sent out according to concentration of standard solution and chemistry
Luminous intensity relation is mapped to obtain standard curve.
Example 2：Sample analysis
Sample solution containing target dna is tested by step 1.5 method in example 1, according to chemiluminescence intensity and
Step 1.5 gained standard curve can obtain target dna content in example 1.
Target dna content is determined according to the method for invention, and method commented using standard addition method
Valency, it is 96.00-102.2% that sample, which determines the rate of recovery, and measurement result is shown in Table 1, and method of the invention has in target dna detection
There is the characteristics of precision is high.
The sample analysis measurement result of table 1.
| Numbering | Contenta,b | Standard entertion amount | Measured amount | The rate of recovery |
| 1 | 1.22 | 1.00 | 2.18 | 96.0% |
| 2 | 3.59 | 5.00 | 8.67 | 103.4% |
| 3 | 4.70 | 5.00 | 9.81 | 102.2% |
a7 measurement results
bUnit：nM.
SEQUENCE LISTING
<110>Qingdao University of Science and Technology
<120>A kind of method that chemiluminescence detects DNA
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 40
<212> DNA
<213>Artificial sequence
<400> 1
ATATACGCCA TGTAGCATTC GGTTAGGCGT ATATTTGCTT 40
<210> 2
<211> 16
<212> DNA
<213>Artificial series
<400> 2
AATATACGCC TAACCG 16
<210> 3
<211> 41
<212> DNA
<213>Artificial sequence
<400> 1
TATACGCCTA ACCGAATGCT TACCACGCGT ATAGCATCCG A 41
Claims (3)
1. a kind of method that chemiluminescence detects DNA, it is characterised in that gold chloride is reduced with luminol, luminol glue is obtained
Body golden nanometer particle LumAuNPs, modifies LumAuNPs with DNA probe, obtains chemiluminescence probe；Then consolidate by carrier of magnetic bead
Determine the capture dna of hairpin structure, in the presence of object DNA, the hairpin structure of capture dna is opened, and visited in chemiluminescence
Under the hybridization of DNA probe on pin, magnetic bead surfaces are connected to by being catalyzed hair fastener self-assembling technique by chemiluminescence probe；
After Magneto separate, hydroxylamine-o-sulfonic acid is added, using LumAuNPs-hydroxylamine-o-sulfonic acid as chemical luminous system, chemistry is carried out
Luminescence assays, the measure of target dna is realized according to the chemiluminescence of generation, is concretely comprised the following steps：
(1) preparation of luminol colloid golden nanometer particle：Before experiment starts, by glass apparatus HNO used3/HCl(3:1,v/
V) after chloroazotic acid immersion 24h, rinsed with redistilled water, be put into oven for drying；A certain amount of 1% chlorauric acid solution is taken to add
The chlorauric acid solution that ionized water is diluted to 0.02% is placed in three-necked flask, is heated to reflux boiling under magnetic stirring；Treat molten
After liquid boiling, 0.01mL~5mL 0.01M luminol solution is rapidly joined, continues heating and boils, the color of solution is by pale yellow
Discoloration is black, eventually becomes and stops heating after claret, 40min, and is cooled to room temperature under continued mixing, obtains luminol glue
Body golden nanometer particle, i.e. LumAuNPs, obtained LumAuNPs is transferred in brown, wide-mouth bottle, saved backup at 4 DEG C；
(2) capture dna modifies the preparation of magnetic bead：Take the μ L carboxylated magnetic bead solution of 10 μ L~100 to be put into 1.5mL centrifuge tubes, use
The μ L concentration of 10 μ L~200 is that 0.1M imidazole buffers are washed three times, be subsequently dispersed 0.01mL~2mL contain 0.1M EDC and
In 0.05M NHS 0.1M imidazole buffers, under the conditions of 37 DEG C, oscillating reactions 30min；Then 10 are added into centrifuge tube
The μ L concentration of μ L~200 is 5.0 × 10-8M capture dnas, the shaken overnight under the conditions of 37 DEG C obtains capture dna modification magnetic bead, then
Cleaned three times, be finally distributed in 2.0mL PBS cushioning liquid with 2.0mL 0.1M PBS cushioning liquid again, 4 DEG C of preservations；
(3) DNA probe modifies LumAuNPs preparation：By the μ L of 1 μ L~20 TCEP be added to the μ L concentration of 10 μ L~200 be 1.0 ×
10-6In M DNA probe solution, 37 DEG C of vibrations are activated 1 hour, then take 100 μ L~LumAuNPs synthetic 1000 μ L to be added to
In the solution, shake and stay overnight under the conditions of 37 DEG C, then add 10 μ L~200 μ L NaCl containing 0.3M (pH 8.2) 10mM
Tris-HCl buffer solutions；Continue to shake after 48h, after centrifugation 30min under conditions of 12000rpm, by red precipitate 1mL (pH
7.4) 0.1M PBS cushioning liquid cleaning, is centrifuged again, so in triplicate, obtains DNA probe modification LumAuNPs, i.e. chemistry
Luminescence probe；The chemiluminescence probe finally obtained is distributed in 1000 μ L pH 7.4 0.1M PBS cushioning liquid, 4 DEG C of guarantors
Deposit standby；
(4) detection of target dna：Take the μ L capture dnas of 10 μ L~200 modification magnetic bead solution to be placed in centrifuge tube, then take 10 μ L
~100 solution of the μ L containing target dna are added in this centrifuge tube, and then concussion reaction 40min under the conditions of 37 DEG C adds 10 μ L
~100 μ L DNA probes modify LumAuNPs solution, concussion reaction 40min under the conditions of 37 DEG C, pass through target dna and capture dna
The effect of effect, DNA probe and target dna and capture dna, chemiluminescence probe is connected to magnetic bead surfaces；After Magneto separate,
Magnetic Isolation thing is dispersed in 50 μ L (pH 7.4) 0.1M PBS cushioning liquid, hydroxylamine-o-sulfonic acid solution is then added, produced
It is biochemical luminous, it is quantitative according to chemiluminescence intensity, realize the measure of target dna.
2. the method that a kind of chemiluminescence according to claim 1 detects DNA, it is characterised in that described DNA sequence dna is such as
Under：
Described DNA sequence dna is：Capture dna：5`-ATATACGCCATGTAGCATTCGGT TAGG CGTAT ATT TG C
TT-NH2-3`；
Target dna：5`-AATATACGCCTAACCG-3`；
DNA probe：5`-SH-TATA CGCCTAACCGAATGCT TACCACGCGTAT AG C A T CCGA-3` .
3. detection DNA according to claim 1 method, it is characterised in that it is limited that described DNA gives birth to work bioengineering by Shanghai
Company synthesizes.
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