CN103389382A - Signal-enhanced test strip biosensor for detecting histone methylation - Google Patents
Signal-enhanced test strip biosensor for detecting histone methylation Download PDFInfo
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Abstract
本发明涉及一种用于检测组蛋白甲基化的试纸条生物传感器。所述试纸条生物传感器包括：具有粘性的底板；硝酸纤维素膜，粘贴在所述底板的中部；所述硝酸纤维素膜上喷有抗组蛋白H3的单克隆抗体和羊抗鼠的IgG抗体，分别形成检测线和质控线；金标垫，粘贴在所述硝酸纤维素膜的一侧的底板上；所述金标垫上喷有c-DNA与抗组蛋白H3的单克隆抗体双标的金纳米颗粒溶液；吸水纸，粘贴在所述硝酸纤维素膜的另一侧的底板上；样品垫，粘贴在所述底板上；检测时，所述样品垫上先滴加组蛋白提取液，然后滴加由DNA与金纳米颗粒偶联形成的增强探针。本发明所述的试纸条生物传感器可简便、快速地检测组蛋白甲基化状况，且通过增强探针使检测信号进一步放大。
The invention relates to a test strip biosensor for detecting histone methylation. The test strip biosensor comprises: a sticky base plate; a nitrocellulose membrane pasted on the middle of the base plate; the nitrocellulose membrane is sprayed with anti-histone H3 monoclonal antibody and goat anti-mouse IgG Antibodies, respectively forming detection lines and quality control lines; gold label pads, pasted on the bottom plate on one side of the nitrocellulose membrane; c-DNA and anti-histone H3 monoclonal antibody doubles are sprayed on the gold label pads The target gold nanoparticle solution; absorbent paper, pasted on the base plate on the other side of the nitrocellulose membrane; a sample pad, pasted on the bottom plate; during detection, the histone extract was first dropped on the sample pad, An enhanced probe formed by coupling DNA to gold nanoparticles was then added dropwise. The test strip biosensor of the invention can detect the histone methylation status simply and quickly, and further amplify the detection signal by enhancing the probe.
Description
Technical field
The present invention relates to a kind ofly for detection of histone methylated test strips biology sensor, particularly relate to the test strips biology sensor that a kind of signal for detection of histone H 3 K9 trimethyl (H3K9me3) in the HeLa cell strengthens.
Background technology
Histone refers to be combined with DNA the general name of the alkaline protein that exists in all Eukaryotic nucleus.Histone has five types, comprises H1, H2A, H2B, H3, H4, can interact with phosphate group electronegative in DNA.In eukaryotic, chromosome is by recurring unit--and nucleosome forms.Each nucleosome comprises that core histones 8 aggressiveness (by each two monomer compositions of H2A, H2B, H3 and H4), length are about DNA (deoxyribonucleic acid) (DNA) and a monomer histone h1 of 200 base-pairs.Nucleosome carries epigenetics information by the modification of DNA or histone, for example DNA methylation, histone methylated, phosphorylation, acetylation, SUMOization and ubiquitin.And the N end of core histones can stretch out from the core of nucleosome, and the covalent modification of histone just often occurs on the lysine (K) or arginine (R) of N end.Lysine side-chain can be by monomethylation (me1), dimethyl (me2) or trimethyl (me3), and the arginine side chain can be by monomethylation or dimethyl.The dissimilar histone methylated different meaning that has in the chromatin of cell.For example H3K4me1 and H3K4me3 are common in euchromatin, and be relevant with transcriptional activation, and H3K9me2 and H3K9me3 are common in heterochromatin, and inhibition is relevant with transcribing.Bibliographical information is arranged, the trimethyl level of H3K9 not only plays an important role building and keep heterochromatin stability, and relevant to important biology event in a lot of cells, such as differentiation direction of cell ageing, tumor regrowth, decider's mesenchymal stem cells MSCs etc.
At present, Western blot is the method commonly used for analysis of cells histone H 3 K9 trimethyl level, and testing result accurately and reliably.But the running time of the method is grown (approximately one day), and complex steps needs the instrument and equipment of multiple costliness and skilled operating personnel, and the reagent toxicity that uses is very large.Therefore, a kind of easy detection method and relevant apparatus that is used for fast histone H 3 K9me3 of research and development is very necessary.
Summary of the invention
The object of the present invention is to provide a kind of test strips biology sensor for detection of histone methylated signal enhancing, can be easy, test set albumen methylation status rapidly, and by strengthening probe, detection signal is further amplified, be applicable to diagnosis and the treatment of diseases related.
The technical solution used in the present invention is:
Test strips biology sensor for detection of histone methylated signal enhancing of the present invention, described test strips biology sensor comprises: the sticking base plate of tool; Nitrocellulose filter, stick on the middle part of described base plate; Be sprayed with the monoclonal antibody of anti-histone H3 and the IgG antibody of sheep anti mouse on described nitrocellulose filter, form respectively detection line and nature controlling line; Gold mark pad, stick on the base plate of a side of described nitrocellulose filter, has between described gold mark pad and described nitrocellulose filter that 2-3mm's is overlapping; Be sprayed with the two target gold nano grain solution of monoclonal antibody of c-DNA and anti-histone H3 on described gold mark pad; Thieving paper, stick on the base plate of opposite side of described nitrocellulose filter, has between described thieving paper and described nitrocellulose filter that 2-3mm's is overlapping; Sample pad, stick on described base plate, has between described sample pad and described gold mark pad that 2-3mm's is overlapping; During detection, first drip the histone extract on described sample pad, then drip the enhancing probe that is formed by DNA and gold nano grain coupling.
The sequence of described DNA is SEQ ID NO.1, and preferably, described DNA is sulfydryl modification.
5 ' the end labeling SH groups of DNA, its sequence is:
5’-SH-atcataagctcatacaatcactaa-3’（SEQ ID NO.1）。
The sequence of described c-DNA is that preferably, described c-DNA is biotin modification with the sequence complementation of described DNA.
5 ' the end mark biotin (biotin) of c-DNA, its sequence is:
5’-biotin-ttagtgattgtatgagcttatgat-3’。
Further feature according to test strips biology sensor of the present invention, described sample pad is following making: glass fibre is soaked in the solution that contains 1% BSA, 2% Triton, 2% PEG4000,20 mM Tris-Ac and 50 mM NaAc, dry the described sample pad of rear formation.
According to the further feature of test strips biology sensor of the present invention, described test strips biology sensor is for detection of histone H 3 K9 trimethyl in the HeLa cell.
Advantage of the present invention is:
The shortcomings such as 1) use the test strips method to detect H3K9me3 in the present invention, easy experimental result with the naked eye just can be observed in 15 minutes fast, thereby had avoided the operation steps of western blot complicated, and the running time is long.
2) introduced an enhancing probe AuNP-DNA who is used for strengthening TZ and CZ colour developing in the present invention, detection signal is further amplified.
3) test strips biology sensor of the present invention has very high sensitivity, just H3K9me3 can be detected in the HeLa of 0.02ug groups of cells protein extract, than the sensitivity of traditional test strips and western blot, has improved respectively 10 times and 15 times.
4) test strips of signal enhancing used in the present invention has very high specificity, and other protein do not produce interference to detection signal.
Description of drawings
Fig. 1 (A) is the detection schematic diagram that traditional test strips is used for H3K9me3.
The test strips that Fig. 1 (B) strengthens for signal is used for the detection schematic diagram of H3K9me3.
Fig. 2 is the result that western blot analyzes histone extract in the HeLa cell.
Fig. 3 is the sensitivity that detects of the inventive method figure as a result.
Fig. 4 is the specificity figure as a result of the inventive method.
Embodiment
Embodiment one: the preparation of the test strips biology sensor for detection of histone methylated signal enhancing of the present invention.
The present invention will be to detect H3K9 in the HeLa cell as example, built this signal of a kind of AuNP-DNA of utilization and strengthened probe the testing result of test strips is carried out the sensor (referring to Fig. 1) that signal amplifies.
The preparation method of the test strips biology sensor for detection of histone methylated signal enhancing of the present invention comprises the steps:
1) oligonucleotide sequence of a pair of complementation of design, be designated as respectively DNA and c-DNA.
5 ' the end labeling SH groups of DNA, its sequence is:
5’-SH-atcataagctcatacaatcactaa-3’（SEQ ID NO.1）。
5 ' the end mark biotin (biotin) of c-DNA, its sequence is:
5’-biotin-ttagtgattgtatgagcttatgat-3’。
2) monoclonal antibody of anti-histone H3K9me3 and NHS-LC-biotin coupling, make antibody by biotinylation.
3) Streptavidin (SA:streptavidin) and AuNPs coupling, form the AuNP-SA conjugate.
4) DNA sequence dna of sulfydryl modification is coupled to gold nano grain (AuNPs) surface by the S-Au key, is formed for strengthening the enhancing probe (AuNP-DNA) of ELISA test strip signal.
5) monoclonal antibody of biotin labeled c-DNA and biotin labeled anti-H3K9me3 and AuNP-SA coupling, form the two target gold nano grains (Dual labelled AuNPs) of c-DNA and antibody.
6) DNA and the two target gold grain solution of antibody are sprayed on glass fibre, form gold mark pad.
7) glass fibre is soaked in the solution that contains 1% BSA, 2% Triton, 2% PEG4000,20 mM Tris-Ac and 50 mM NaAc, dry rear formation sample pad.
8) the IgG antibody of the monoclonal antibody of anti-histone H3 and sheep anti mouse is sprayed on cellulose nitrate (NC) film simultaneously, forms respectively detection line (TZ) and nature controlling line (CZ).
Probe related in experiment is synthetic by Shanghai bioengineering company limited.
Below a specific embodiment:
1, the preparation of gold nano (collaurum) solution:
Add the HAuCl of 100 ml 0.01% in the round-bottomed flask of 500 ml
4Solution, magnetic agitation are heated to boiling; Then add 4 ml 1% sodium citrates in above-mentioned solution, after solution becomes claret, continued to boil 10 minutes, stopped heating continues to stir 15 minutes, obtains gold nano (collaurum) solution.
4 ℃ of colloidal gold solutions keep in Dark Place, and nm of gold is identified by 520 nm maximum absorbance values.
2, preparation AuNP-DNA signal strengthens probe:
With the DNA of 100 μ l deionized water dissolving 1OD, join in concentrated collaurum (15nm) solution of 5 times of volumes, low-speed oscillation, 4 ℃ are spent the night; 1% bovine serum albumin(BSA) sealing after 30 minutes, adds Na
3PO
4SDS with 1%,, respectively to final concentration 0.01M and 0.01%, brought up to 0.15 M with NaCl concentration gradually at 3 days thereafter; 12000 rev/mins centrifugal 20 minutes, abandon supernatant, precipitation is with cleansing solution (0.15 M NaCl, 0.01 M NaH
2PO
4, 0.1% SDS, pH 7.4) wash 4 times after, contain 20 mM Na with 100 μ L
3PO
4, 1% BSA, 0.25% tween and 10% sucrose the solution Eddy diffusion, make suspending liquid.
3, preparation AuNP-SA conjugate:
Add 0.1 M K in the colloidal gold solution of 1 mL
2CO
3(6 μ L) mixes, and adds the SA(Streptavidin of 10 μ g after 5 minutes), room temperature vibration 30 minutes.Add 10 % BSA (100 μ L) sealing 30 minutes, 4 ° of C centrifugal (12 * 10
3Rpm, 10 minutes), obtain AuNP-SA(gold nano grain-Streptavidin) and conjugate.
Abandon supernatant, with the resuspended AuNP-SA conjugate of 100 μ L borate buffers (0.2M, pH 9.0).
4, the monoclonal antibody of the biotinylated anti-histone H3K9me3 of preparation:
Related antibody in experiment, namely the monoclonal antibody of anti-histone H3K9me3 and anti-histone H3, be purchased from Abcam company.
With NHS-LC-Biotin be dissolved in DMSO to final concentration be 10 mM(matching while using).To the monoclonal antibody and the NHS-LC-Biotin solution that add respectively 50 μ g in the PBS of 200 μ L, the relative concentration of NHS-LC-Biotin is excessive in 5 times of antibody.After room temperature oscillating reactions 1 hour, 4 ° of C dialysed overnight in PBS.
5, the two target AuNPs of preparation c-DNA and H3K9me3 monoclonal antibody:
The sequence of the monoclonal antibody of biotinylated anti-H3K9me3 and its sequence of biotinylated c-DNA(and aforesaid DNA is complementary) join in AuNP-SA solution room temperature reaction centrifugal three times (12 * 10 of 4 ° of C after 30 minutes
3Rpm, 10 minutes), abandon supernatant, with 50 μ L borate buffers (0.2M, pH 9.0) resuspended c-DNA and the two target AuNPs of H3K9me3 monoclonal antibody.
6, the structure of test strips biology sensor:
The monoclonal antibody of mouse-anti histone H 3 (0.67 mg/mL, 30 μ L) and the IgG of sheep anti mouse anti-(0.5 mg/mL how, 30 μ L) be sprayed on simultaneously cellulose nitrate (NC, nitrocellulose) on film, form respectively detection line (TZ, test zone) and nature controlling line (CZ, control zone).Glass fibre is soaked and containing 1% BSA, 2% Triton, 2% PEG4000, in the solution of 20 mM Tris-Ac and 50 mM NaAc, dry rear formation sample pad.DNA and the two target gold grain solution of antibody are sprayed on (9 μ L/cm), form gold mark pad on glass fibre.Sample pad, gold mark pad, NC film and thieving paper are attached on the sticking base plate of tool successively, and each several part has overlapping (referring to Fig. 1) of 2-3mm.Then be cut into the wide test strips of 4mm with the plate that cutting cutter will build, dry place preserves.
Embodiment two: the detection method of the test strips biology sensor for detection of histone methylated signal enhancing of the present invention.
The present embodiment adopts the prepared test strips biology sensor of embodiment one to detect histone H 3 K9 trimethyl in the HeLa cell.
To detect H3K9me3 as example, detection method is as follows:
1), with HeLa lysis, use sour extraction process to obtain the histone crude extract.
2) drip the histone extract on sample pad, after 10 minutes, then drip AuNP-DNA, observation experiment result after 5 minutes.
Below a specific embodiment:
1, obtaining of HeLa groups of cells protein crude extract:
A) the HeLa cell is cultivated in containing the DMEM nutrient solution of 10% hyclone.
B) when growing to degree of converging in the double dish of cell at 6cm and be 90%, suck nutrient solution, with PBS, wash 2 times, add cell pyrolysis liquid (10 mM Hepes, pH 7.9,10 mM KCl, 1.5 mM MgCl
2And 0.5 mM beta-mercaptoethanol, with before adding proteinase and inhibitors of phosphatases), add 5 μ L NP-40 on ice after 20 minutes, 4 ° of C centrifugal (12 * 10
3Rpm, 15 minutes), supernatant is cytoplasm protein.
C) abandon supernatant, add nucleus lysate (10 mM Tris-HCl, pH 7.6,420 mM NaCl, 0.5% NP-40, and 1mM DTT, 1mM PMSF, 2mM MgCl2, with before adding proteinase and inhibitors of phosphatases), 4 ° of C centrifugal (12 * 10 after 20 minutes on ice
3Rpm, 15 minutes), supernatant is Nuclear extract.
D) supernatant discarded, add 80 μ L 0.25 M HCl, 4 ° of C shaken overnight, centrifugal (12 * 10
3Rpm, 15 minutes).Draw supernatant, be the histone extract.
2, the H3K9me3 in the ELISA test strip HeLa cell of use signal enhancing:
The histone extract of different amounts is added volume to 60 μ L with PBS, is added drop-wise on sample pad respectively.After 10 minutes, AuNP-DNA solution is added drop-wise on test strips, observation experiment result after 5 minutes.On test strips, the gray-scale value of detection zone red stripes uses image processing software ImageJ 1.37v to draw.
Embodiment three: the sensitivity of H3K9me3 in the ELISA test strip HeLa cell that signal of the present invention strengthens.
H3K9me3 in the test set protein extract:
The histone extract (0,0.15,0.31,0.62,1.3,2.6,5.2 μ g) of different amounts separates with 15% SDS-PAGE separation gel, and 100V is after 40 minutes, and electricity goes on PVDF (polyvinylidene fluoride, Polyvinylidene Fluoride) film.Pvdf membrane sealed 1 hour with 2% skim milk, and 2% skim milk cleans 3 times (10 minutes/time).The monoclonal antibody (1:2000 dilution) that adds anti-H3K9me3, incubated at room 2 hours, 2% skim milk cleans 3 times (10 minutes/time).The IgG(1:4000 dilution of use sheep anti mouse afterwards) incubated at room is 1 hour, and 2% skim milk cleans 3 times (10 minutes/time).Last exposure imaging, photographic fixing.
Testing result as shown in Figure 2.Extract the increase (0.31,0.62,1.3,2.6,5.2 μ g) of liquid measure along with histone, the band color on film is deepened (Fig. 2 A) gradually, and the gray-scale value of band is rising (Fig. 2 B) gradually also.When the amount of histone extract is 0 or 0.15 μ g, can't see any band on film.So use western blot, H3K9me3 can be detected in the histone extract of 0.31 μ g detection HeLa cell.
H3K9me3 in the ELISA test strip histone extract that signal of the present invention strengthens:
Get the histone extract (0,0.02,0.08,0.2,0.5,1.5 μ g) of different amounts, with PBS, add volume to 60 μ L, be added drop-wise on sample pad respectively.After 10 minutes, the AuNP-DNA solution of 2.4 μ L is added drop-wise on test strips, observation experiment result after 5 minutes.On test strips, the gray-scale value of detection zone red stripes uses image processing software ImageJ 1.37v to draw.
Testing result as shown in Figure 3.Extract the increase (0.02,0.08,0.2,0.5,1.5 μ g) of liquid measure along with histone, the redness in T district is deepened (Fig. 3 A(b) gradually), gray-scale value is rising (Fig. 3 B) gradually also.When not containing the histone extract in sample, only have the colour developing of C district, T does not develop the color in district.So the histone extract that uses 0.02 μ g just can detect the H3K9me3 in the HeLa cell.And the gray-scale value in the amount of histone extract and T district has good linear relationship, and the range of linearity of detection is 0.02 μ g-0.5 μ g.Use traditional test strips biology sensor (Fig. 3 A(a)), the histone extract of 0.2 μ g just can detect the H3K9me3 in the HeLa cell.So the H3K9me3 in ELISA test strip HeLa cell of the present invention has improved respectively 10 times and 15 times than the sensitivity of traditional test strips and western blot detection.
Embodiment four: the specificity experiment of the test strips biology sensor for detection of histone methylated signal enhancing of the present invention.
According to the detection method of above-mentioned H3K9me3, use respectively 1.2 μ g Escherichia coli (
E.coli, Escherichia coli) protein extract, cytoplasm protein and Nuclear extract in contrast, are analyzed the present invention and are detected the specificity of H3K9me3.
E.coliThe acquisition methods of extracting solution of protein is as follows: E.coli cultivates at LB(Luria – Beritani) in nutrient culture media, 37 ° of C on shaking table, 220rpm spends the night.Centrifugal (5,000rpm, 10 minutes), collect thalline, adds lysate (50 mM Tris-Cl, pH 8.2 for 1 mM EDTA, 100 mM NaCl) ultrasonic degradation E.coli.Centrifugal (12,000rpm, 15 minutes) collect supernatant afterwards, are the E.coli protein extract.
Testing result as shown in Figure 4.When 1.2 μ g's
E.coliWhen extracting solution of protein, cytoplasm protein or Nuclear extract were added drop-wise on the sample pad of test strips of the present invention, T did not develop the color in district, only had the colour developing of C district.And use the histone extract of 1.2 μ g, with the naked eye in the T district, just can see obvious red stripes.Illustrate that the test strips specificity is good, testing result is not subjected to the interference of other protein.
Sequence table
＜110〉Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health
＜120〉a kind of test strips biology sensor for detection of histone methylated signal enhancing
<130>
<160> 1
<170> PatentIn version 3.4
<210> 1
<211> 24
<212> DNA
＜213〉artificial synthetic
<400> 1
atcataagct catacaatca ctaa 24
Claims (5)
1. test strips biology sensor that strengthens for detection of histone methylated signal, it is characterized in that: described test strips biology sensor comprises:
The sticking base plate of tool;
Nitrocellulose filter, stick on the middle part of described base plate; Be sprayed with the monoclonal antibody of anti-histone H3 and the IgG antibody of sheep anti mouse on described nitrocellulose filter, form respectively detection line and nature controlling line;
Gold mark pad, stick on the base plate of a side of described nitrocellulose filter, has between described gold mark pad and described nitrocellulose filter that 2-3mm's is overlapping; Be sprayed with the two target gold nano grain solution of monoclonal antibody of c-DNA and anti-histone H3 on described gold mark pad;
Thieving paper, stick on the base plate of opposite side of described nitrocellulose filter, has between described thieving paper and described nitrocellulose filter that 2-3mm's is overlapping;
Sample pad, stick on described base plate, has between described sample pad and described gold mark pad that 2-3mm's is overlapping; During detection, first drip the histone extract on described sample pad, then drip the enhancing probe that is formed by DNA and gold nano grain coupling;
Wherein, the sequence of described DNA is SEQ ID NO.1, and the sequence of described c-DNA is with the sequence complementation of described DNA.
2. test strips biology sensor according to claim 1, it is characterized in that: described DNA is sulfydryl modification.
3. test strips biology sensor according to claim 1, it is characterized in that: described c-DNA is biotin modification.
4. test strips biology sensor according to claim 1, it is characterized in that, described sample pad is following making: glass fibre is soaked in the solution that contains 1% BSA, 2% Triton, 2% PEG4000,20 mM Tris-Ac and 50 mM NaAc, dry the described sample pad of rear formation.
5. test strips biology sensor according to claim 1, it is characterized in that: described test strips biology sensor is for detection of histone H 3 K9 trimethyl in the HeLa cell.
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