CN110441525A - Protein chemistry luminescence imaging analysis method based on DNA microarray - Google Patents
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- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 50
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 50
- 238000000018 DNA microarray Methods 0.000 title claims abstract description 36
- 238000004458 analytical method Methods 0.000 title claims abstract description 21
- 238000003384 imaging method Methods 0.000 title claims abstract description 12
- 238000004020 luminiscence type Methods 0.000 title claims abstract description 12
- 238000006243 chemical reaction Methods 0.000 claims abstract description 23
- 238000001514 detection method Methods 0.000 claims abstract description 14
- 230000006287 biotinylation Effects 0.000 claims abstract description 10
- 238000007413 biotinylation Methods 0.000 claims abstract description 9
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims abstract description 8
- 239000000758 substrate Substances 0.000 claims abstract description 8
- 150000001875 compounds Chemical class 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims abstract description 6
- 239000011259 mixed solution Substances 0.000 claims abstract description 5
- 238000010191 image analysis Methods 0.000 claims abstract description 4
- 239000011521 glass Substances 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 12
- 238000009396 hybridization Methods 0.000 claims description 10
- 239000000523 sample Substances 0.000 claims description 9
- 238000011010 flushing procedure Methods 0.000 claims description 4
- 108090001008 Avidin Proteins 0.000 claims description 3
- 238000013461 design Methods 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000012488 sample solution Substances 0.000 claims description 3
- 238000011088 calibration curve Methods 0.000 claims description 2
- 230000002209 hydrophobic effect Effects 0.000 claims description 2
- 239000000243 solution Substances 0.000 claims description 2
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 claims 1
- 230000008901 benefit Effects 0.000 abstract description 4
- 230000004907 flux Effects 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 29
- 238000003018 immunoassay Methods 0.000 description 12
- 238000002493 microarray Methods 0.000 description 7
- 230000000875 corresponding effect Effects 0.000 description 5
- 238000001035 drying Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000028993 immune response Effects 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 238000011896 sensitive detection Methods 0.000 description 3
- 235000011330 Armoracia rusticana Nutrition 0.000 description 2
- 240000003291 Armoracia rusticana Species 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000003498 protein array Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 108020005031 Concatenated DNA Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 230000003172 anti-dna Effects 0.000 description 1
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- 229960002685 biotin Drugs 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
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- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
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- 239000011159 matrix material Substances 0.000 description 1
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
- G01N21/763—Bioluminescence
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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
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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Abstract
The protein chemistry luminescence imaging analysis method based on DNA microarray that the present invention relates to a kind of.Its corresponding capture hairpin dna and antibody-DNA are designed based on different target proteins.Different capture hairpin dnas is distinguished on point sample to glass substrate by auto sample applicator, constructs DNA microarray.It is detection liquid with the mixed solution of the antibody-DNA for different target protein, in the presence of target protein, target protein can be by its corresponding a pair of antibody-DNA sandwich identification simultaneously, so that this progress ortho position close to each other to the DNA on antibody-DNA hybridizes to form compound, and then hybridize with corresponding capture hairpin dna, open its hairpin structure.The capture hairpin dna of opening can cause biotinylation hairpin dna (H1 and H2) and cross chain reaction occurs, and long-chain strand of dna is generated on DNA microarray point and joins object.Finally by biotin-avidin specific reaction, horseradish peroxidase is fixed on DNA microarray, is catalyzed H2O2Luminol reaction, generates chemiluminescence signal, is scanned by CCD, realize the image analysis of target protein.The advantages such as the protein analysis method has multicomponent, flux is high, highly sensitive, universality is good, there is good clinical value.
Description
One, technical field
The present invention is a kind of protein chemistry luminescence imaging analysis method based on DNA microarray, for plurality of target
While protein, high throughput, Sensitive Detection.By constructing DNA microarray, the sandwich of antibody-DNA and target protein is utilized
Immune response, generates vucubak effectl alpha effect and promotes DNA hybridization, further opens the capture hairpin dna on DNA microarray and causes hybridization
Chain reaction is reacted by biotin-avidin, so that a large amount of horseradish peroxidases combine on DNA microarray, generates chemistry
Luminous signal amplification, realize multiple proteins while, highly sensitive image detection.
Two, background technique
Specific immunity association reaction that immunoassay is to rely between Ag-Ab and realize to target antigen detection
Analysis method, have superior specificity, in conjunction with colorimetric, fluorescence, electrochemistry, chemiluminescence etc. efficiently quick detection means,
The panimmunities such as ELISA, fluoroimmunoassay, electrochemical immunoanalytical and chemiluminescence immune assay have been developed
Analysis method.According to the difference of operating procedure, immunoassay can be divided into homogeneous immunoassay and out-phase immunoassay, Qian Zheyin again
It operates relatively easy, without multi-step incubation and cleaning, thus is widely used.Directly mixing relative to homogeneous immunoassay is anti-
It answers, though out-phase immunoassay operates cumbersome due to multistep is cleaned and separated, usually can obtain higher sensitivity, thus exempting from
It is equally occupied an important position in epidemic disease analysis.In addition, the content for efficiently measuring multiple components in complex sample simultaneously has become the modern times
Analysis detection there is an urgent need to and homogeneous immunoassay usually requires to consume a large amount of detection sample and analytical reagent is just able to achieve
This target, out-phase immunoassay then have natural advantage in this aspect.
Chemiluminescence immune assay, the i.e. signal using chemiluminescence as immune response are read, back simple with equipment
The advantages that scape interference is small, detection sensitivity is high.In conjunction with spatial discrimination mode, chemiluminescence immune assay is in multi-component immunity analytical
Field obtains extensive concern, this mode is multiple by independently occurring in parallel in the different spaces region of immune response carrier
Immune response, then each regional signal is acquired simultaneously or when similar by detector, it is detected while realization to multiple target proteins.
Thus develop the protein array technology come, be of great significance in proteomics, however, protein array is due to direct
Use protein as fixed matrix, larger challenge is deposited in terms of cost of manufacture, technical requirements.For protein,
DNA then has outstanding advantage because of synthetic technology maturation, environmental resistance degree height, sequence polymorphism, in genomics research and quotient
Industry is significant using upper all achievements.Protein will greatly be made up by carrying out protein correlative study as technology platform using DNA microarray
Suffered limitation in array development.
Ortho position inductive effect is reacted by sandwich immunoassay, promote to be coupled at a pair of of DNA on antibody because distance furthers,
Local concentration increases and partial hybridization occurs, and then triggers concatenated dna assembling.This method is will to be immunoreacted that be converted into DNA anti-
Should after carry out signal detection, the various DNA signal amplification techniques of grafting can be facilitated, realize the Sensitive Detection to target protein.
Three, summary of the invention
The contents of the present invention are: based on immune vucubak effectl alpha effect and DNA assembling design, target egg occurring on DNA microarray
White matter induces formula cross chain reaction, has developed a kind of protein chemistry luminescence imaging analysis method based on DNA microarray.
Protein chemistry luminescence imaging analysis method proposed by the present invention based on DNA microarray is by following technical side
Case is realized:
Its corresponding capture hairpin dna and antibody-DNA are designed according to different target proteins first；Secondly according to empty
Between differentiate mode, the sensing hole array of pad pasting building 4 × 12 on aldehyde radical sheet glass, then in each sensing bore midpoint amino
The capture hairpin dna of modification constructs the DNA microarray of 3 × 3 formulas, is reacted based on amino with aldehyde radical, captures hairpin dna covalent bond
On a glass substrate, BSA solution is further added dropwise in sensing bore to be closed, detectable 3 kinds of targets is made after rinsing drying
The DNA microarray of protein；It is detection liquid with the mixed solution of antibody-DNA, is mixed with the sample solution containing target protein
After be added sensing bore, target protein identifies that, based on vucubak effectl alpha effect, this is to antibody-by its corresponding a pair of antibody-DNA simultaneously
DNA on DNA carries out hybridization and forms compound, and then hybridizes with corresponding capture hairpin dna, opens its hairpin structure, from
And cause biotinylation hair fastener H1 and biotinylation hair fastener H2 and cross chain reaction occurs, long-chain is generated on DNA microarray interface
Strand of dna joins object；By biotin-avidin specific reaction, horseradish peroxidase is further fixed to DNA microarray
On, it is catalyzed H2O2Luminol reaction, generates chemiluminescence signal, acquires picture signal by CCD and carries out image analysis.
The principle of this detection system measurement target protein:
The protein chemistry luminescence imaging analysis method based on DNA microarray that the present invention designs, principle is as shown in Figure 1:
Antibody-DNA detection liquid and the sample solution containing target protein are added in each sensing bore on DNA microarray substrate
Mixed solution incubates 1.5 hours at 37 DEG C.In incubation period, target protein is known by its corresponding a pair of antibody-DNA simultaneously
Not and sandwich immunoassay reaction occurs, and then generate vucubak effectl alpha effect, promotes this to the DNA on antibody-DNA apart from close, local is dense
Degree increases, and hybridization occurs and forms compound, which can react with the capture hairpin dna on DNA microarray point, make
A Duan Xulie of hair fastener stem is opened and released to hair fastener.The mixed liquor of biotinylation hair fastener H1 and H2 is added in sensing bore
Afterwards, this section of sequence of capture hairpin dna fixed on DNA microarray release can trigger H1 and H2 generation cross chain reaction, from
And long-chain strand of dna is generated on DNA microarray interface and joins object.Avidin horseradish peroxidase is added dropwise after flushing in sensing bore
Horseradish peroxidase is integrated to microarray sensing site, flushing removes by biotin-avidin specific reaction by enzyme
After removing free enzyme, chemiluminescent substrate is added in sensing bore, chemiluminescence image acquisition is carried out by CCD.In sensing bore
The concentration of the corresponding target protein of the chemiluminescence signal intensity in each micro sensing site is positively correlated, molten using standard
Liquid obtains working curve, by calibration curve method, can measure the concentration of different proteins in sample to be tested simultaneously, realize a variety of
High-sensibility chemical luminescence image analysis while target protein.
Compared with prior art, the present invention having the following characteristics that
Present invention combination DNA microarray, ortho position inductive effect, hybridization chain signal iodine devise a kind of based on DNA
The protein chemistry luminescence imaging analyzing novel methods of microarray.Compared to existing Western Immuno analysis method, have following
Feature:
(1) this method is easy to operate, low in cost using DNA microarray as platform, and since the array has 4 × 12
Sensing bore includes 3 × 3 micro sensing sites in each sensing bore, can analyze 3 kinds of target proteins in 48 samples simultaneously,
With high flux.
(2) this method is reacted by sandwich immunoassay generates vucubak effectl alpha effect, and then causes DNA hybridization and assembling, will be immune anti-
DNA reaction should be converted into be detected, can facilitate in conjunction with various DNA assembling amplifying technique, realize to low abundance proteins
Sensitive Detection.
(3) this method is realized by the sequence design to the capture hairpin dna on the DNA and microarray point on antibody-DNA
DNA sequence dna is changed in the differentiation of different target protein, can facilitate the analysis detection for realizing other more protein, universality
It is good.
(4) this method is not necessarily to additional light source, equipment is simple, and background interference is small using chemiluminescence as read output signal.
Four, Detailed description of the invention
Protein chemistry luminescence imaging analysis method process schematic of Fig. 1 based on DNA microarray
Five, specific embodiment
Embodiment 1: in conjunction with attached drawing 1, illustrate the protein chemistry luminescence imaging analysis method based on DNA microarray to 3 kinds
The detection of target protein
(1) prepared by microarray: sticking prefabricated hydrophobic membrane on aldehyde radical substrate, 4 × 12 sensing bore is separated out, by automatic
Point sample instrument is in each sensing bore midpoint print for different captures hairpin dna (C1, C2 and C3) of 3 kinds of different target protein, shape
At 3 × 3 microarray point, ambient temperature overnight is reacted in wet box.The aldehyde radical on amino and substrate on capture hairpin dna is covalently tied
It closes, rinses drying after reaction, BSA confining liquid is then added dropwise in each sensing bore, 37 DEG C incubate 30 minutes, and flushing is blown
It is dry, it is so repeated 3 times, the DNA microarray that can detect 3 kinds of target proteins simultaneously of system.
(2) Immune discrimination: the standard antigen or test serum of various concentration and 50nM's is directed to 3 kinds of different target protein
Antibody-DNA mixed liquor by 1: 4 mixing, take 6 this mixed liquor of μ L be added dropwise in the sensing bore of DNA microarray, 37 DEG C incubate 90
Minute, antibody and antigen occur sandwich immunoassay and reacts, promote the ortho position the DNA induction on antibody in conjunction with compound is formed, and go forward side by side one
Step hybridizes with the capture hairpin dna at microarray point, rinses drying after reaction.
(3) cross chain reaction: be added dropwise in the different sensing bore of DNA microarray 6 μ L, 0.5 μM of biotinylation hair fastener H1 and
The mixed liquor of H2,37 DEG C incubate 90 minutes, at microarray point by the capture hairpin dna that immune complex is opened can trigger H1,
Cross chain reaction between H2, in micro sensing site, over-assemble forms long string DNA double chain, rinses drying after reaction.
(4) chemiluminescence imaging is analyzed: the diluted Avidin horseradish peroxide of 10 times of 6 μ L being added dropwise in each sensing bore
Compound enzyme, 37 DEG C incubate 45 minutes, and by Avidin-Biotin specific recognition, horseradish peroxidase is integrated to micro sensing
At site, drying is rinsed after reaction, then 6 μ L chemiluminescent substrate H are added in each sensing bore2O2Luminol, horseradish
The Catalyzed Synthesis By Peroxidase system generate chemiluminescence, by CCD acquire chemiluminescence signal, single exposure 3 minutes, according to system
The chemiluminescence intensity in each micro sensing site of meter makes respective standard curve and obtains 3 kinds of target proteins in sample to be tested
Concentration.
Claims (5)
1. a kind of protein chemistry luminescence imaging analysis method based on DNA microarray, it is characterised in that according to different targets
Its corresponding capture hairpin dna of protein design and antibody-DNA；Different capture hairpin dnas is distinguished by auto sample applicator
On point sample to glass substrate, DNA microarray is constructed；It is detection liquid with the mixed solution of antibody-DNA, exists in target protein
When, target protein can sandwich identification form compound simultaneously by its corresponding a pair of antibody-DNA, and then with it is corresponding
The hybridization of capture hairpin dna, open its hairpin structure, cause biotinylation hair fastener H1 and biotinylation hair fastener H2 and hybridization chain occurs
Reaction generates long-chain strand of dna on DNA microarray interface and joins object；By biotin-avidin specific reaction, further will
Horseradish peroxidase is fixed on DNA microarray, is catalyzed H2O2Luminol reaction, generates chemiluminescence signal, is adopted by CCD
Collect picture signal and carries out image analysis.
2. analysis method according to claim 1, it is characterised in that the DNA microarray first passes through hydrophobic pad pasting in aldehyde
4 × 12 sensing bore are constructed on base slide, then are captured in each sensing bore by 3 × 3 mode point systems by auto sample applicator
Hairpin dna and be made.
3. analysis method according to claim 1, it is characterised in that the antibody-DNA is in target corresponding thereto
After the sandwich identification of protein, this can be carried out ortho position to the DNA on antibody-DNA and hybridizes to form compound, and further with it is corresponding
Capture hairpin dna hybridization, open its hairpin structure.
4. analysis method according to claim 1, it is characterised in that the capture hairpin dna discharges after being opened to set out
Block a Duan Xulie of stem, which can trigger biotinylation hair fastener H1 and biotinylation hair fastener H2 and cross chain reaction occurs,
Long-chain strand of dna is generated on the capture hairpin dna being opened joins object.
5. analysis method according to claim 1, it is characterised in that the antibody-DNA mixed solution with containing to be measured
It is added drop-wise in sensing bore, is rinsed after incubating 1.5 hours, then in sensing bore successively after the sample solution mixing of target protein
Biotinylation hair fastener H1 and H2 is added dropwise and incubates 1.5 hours, Avidin horseradish peroxidase incubation 45 minutes, after flushing, In
H is added in sensing bore2O2Luminol solution acquires the chemiluminescence image signal on each DNA microarray point by CCD, most
The concentration of various target proteins in sample is found out by calibration curve method afterwards.
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