Method for detecting exosome membrane protein based on telomerase amplification
阅读说明：本技术 一种基于端粒酶扩增的外泌体膜蛋白检测方法 (Method for detecting exosome membrane protein based on telomerase amplification ) 是由 崔大祥 徐艳 徐颖湉 朱君 杨迪诚 于 2021-08-16 设计创作，主要内容包括：本发明属于分子生物检测领域,提供了一种基于端粒酶扩增的外泌体膜蛋白检测方法,以外泌体膜蛋白为靶标,以免疫磁珠捕获外泌体,设计带有识别序列的发夹结构探针,并利用端粒酶扩增的方式进一步放大信号,实现外泌体膜蛋白的可视化检测。本发明还包括该发夹结构探针及其应用。本发明的分离方法便捷,所用材料部分已商品化,生物安全性高。本发明的检测过程适合临床检测需求,不需借助专业仪器,反应仅需水浴锅进行,适应基层医疗检测需求,成本低廉、检测灵敏、特异性高且可视化,可以很好的对外泌体膜蛋白进行分析。(The invention belongs to the field of molecular biological detection, and provides a method for detecting exosome membrane protein based on telomerase amplification. The invention also comprises the hairpin structure probe and application thereof. The separation method is convenient and fast, the used materials are partially commercialized, and the biological safety is high. The detection process of the invention is suitable for clinical detection requirements, does not need to use professional instruments, only needs a water bath for reaction, meets the requirements of basic medical detection, has low cost, sensitive detection, high specificity and visualization, and can well analyze exosome membrane protein.)
1. A method for detecting an exosome membrane protein based on telomerase amplification is characterized by comprising the following steps:
(1) magnetic bead capture exosomes:
the method comprises the steps of coating an exosome membrane protein antibody with magnetic beads and capturing an exosome, wherein carboxylated magnetic beads are activated and then coupled with the exosome membrane protein antibody, and then the activated magnetic beads and the exosome are incubated to form a mixture I;
(2) hairpin structure probe recognition exosomes:
taking the single-chain recognition sequence to incubate with the block sequence to enable the probe sequence to form a hairpin structure, then mixing with the mixture I, washing and resuspending to form a mixture II,
in the hairpin structure probe, a single-stranded recognition sequence comprises an exosome membrane protein aptamer, a spacer sequence and a telomerase recognition primer sequence from 5 'end to 3' end, and a block sequence comprises a telomerase primer sequence complementary sequence and a sequence partially complementary with the exosome membrane protein aptamer from 5 'end to 3' end;
(3) amplification of telomerase:
adding a template containing telomerase and dNTPs into the mixture II, amplifying the telomerase, washing and resuspending to obtain a mixture IV;
(4) and (3) detection:
and adding a developing reagent into the mixture IV, and recording the change of absorbance at 405 nm by using an ultraviolet spectrophotometer.
2. The method for detecting an exosome membrane protein based on telomerase amplification according to claim 1,
the exosome membrane protein antibody coated by the magnetic beads is any one of CD63, CD81, CD9, ALIX, ANNEXIN, EpCAM and RAB 5.
3. The method for detecting an exosome membrane protein based on telomerase amplification according to claim 1,
the single-chain recognition sequence is selected from any one of SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9 and SEQ ID NO 11;
the block sequence is selected from any one of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10 and SEQ ID NO 12;
and mixing the single-chain recognition sequence and the block sequence in a molar ratio of 1:1, incubating, and hybridizing to form the hairpin structure recognition probe.
4. The method for detecting an exosome membrane protein based on telomerase amplification according to claim 1,
the tail end of the telomerase identification primer sequence is TGTT or AGTT.
5. The method for detecting an exosome membrane protein based on telomerase amplification according to claim 1,
the step of coating the exosome membrane protein antibody by the magnetic beads is to wash 10 mg of carboxylated magnetic beads by MES buffer solution with pH of 6.0, dissolve the washed carboxylated magnetic beads in 0.5 mL of MES buffer solution with pH of 6.0, add 10 mg/mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 12 mg/mL of N-hydroxysuccinimide, mix the mixture evenly, and activate the mixture for 30min at room temperature; magnetic separation and washing of the activated magnetic beads, dissolving in MES buffer solution, pH6.0, adding 2mg/mL antibody, mixing well, reacting overnight at 4 ℃, washing well with PBS containing 0.1% Tween20 and pH7.4, and suspending in PBS containing 0.1% Tween20 and 0.1% BSA, pH7.4 for later use; or
The step of capturing the exosomes is to separate and extract the exosomes by an ultracentrifugation method, fully mix 50 muL 0.5 mg/mL magnetic beads with 50 muL exosomes, incubate for 1hr at room temperature, magnetically separate, fully clean, resuspend the precipitate in 50 muL Tb buffer solution containing 22 mM Tris-HCl, 120 mM Na Cl, 50 mM K Cl, 0.85 mM Ca Cl2，6 mM Mg Cl26.8% glycerol, pH 7.8, as mixture I; or
The step of identifying exosomes by using the hairpin structure probe is to incubate a single-chain identification sequence and a block sequence for 5 min at a molar ratio of 1: 190 ℃, slowly reduce the temperature to room temperature, enable the probe sequence to form a hairpin structure, identify the probe by using the hairpin structure, fully mix 10 muL of the hairpin structure identification probe with a mixture I, react for 1hr at room temperature, fully clean by magnetic separation, and wash for three times by using telomerase extension buffer solution, wherein the telomerase extension buffer solution contains 20 mM Tris-H Cl, 1.5 mM Mg Cl2, 63 mM K Cl, 0.005%, v/v, Tween-20 and 1 mM EGTA, and the precipitate is resuspended as a mixture II by using 100 muL of the telomerase extension buffer solution; or
The telomerase amplification step is that 100 mu L of the mixture II obtained in the step (2) is added with 20 mu L of telomerase extract and 1 mM of dNTPs, the mixture is mixed evenly and reacted for 1hr at 30 ℃, after the reaction is finished, the supernatant is removed by magnetic separation, the mixture is washed for three times by 10 mM of PBS buffer II, and the PBS buffer II contains 10 mM of Na2 HPO4，2 mM K H2PO4137 mM Na Cl and 50 mM K Cl, and resuspending the precipitate with 100. mu.L of PBS buffer II to obtain a mixture IV; or
And (3) adding Hemin, hydrogen peroxide and ABTS into the mixture IV obtained in the step (3), uniformly mixing, observing the color change of the solution according to the difference of target concentration, and recording the change of absorbance at 405 nm by using an ultraviolet spectrophotometer.
6. The method for detecting an exosome membrane protein based on telomerase amplification according to any of claims 1-5,
the aptamer recognition exosome membrane protein is any one of PSMA, HER-2, EpCAM, CD63, CD81 and MUC 1.
7. A hairpin structure probe with an identification sequence is characterized in that the hairpin structure probe is composed of two single-stranded DNAs, one single-stranded probe comprises an aptamer for identifying membrane proteins and a telomerase identification sequence, and the other single-stranded probe is a block sequence and is partially complementary with the aptamer and the telomerase identification sequence;
wherein the recognition single-stranded sequence is from 5，End to 3，The end comprises an exosome membrane protein aptamer, a spacer sequence and a telomerase recognition primer sequence;
block sequence from 5，End to 3，The end comprises a telomerase primer sequence complementary sequence and an exosome membrane protein aptamer partial complementary sequence, and the telomerase primer sequence complementary sequence and the exosome membrane protein aptamer partial complementary sequence are hybridized to form a hairpin structure recognition probe.
8. The hairpin probe having a recognition sequence according to claim 7,
the combination of the single-chain recognition sequence and the block sequence is selected from the following combinations:
SEQ ID NO 1 and SEQ ID NO 2;
SEQ ID NO 3 and SEQ ID NO 4;
SEQ ID NO 5 and SEQ ID NO 6;
SEQ ID NO 7 and SEQ ID NO 8;
SEQ ID NO 9 and SEQ ID NO 10;
SEQ ID NO 11 and SEQ ID NO 12.
9. The use of the hairpin probe of claim 7 or 8 wherein the hairpin probe of claim 7 or 8 is used in the preparation of reagents for the visual detection of exosome membrane proteins.
10. Use of the telomerase amplification based exosome membrane protein detection method according to any of claims 1-5, characterized in that it is used for exosome membrane protein visualization detection.
11. The application of claim 10, wherein the exosome is captured by immunomagnetic beads by using an exosome membrane protein as a target, a hairpin structure probe with a recognition sequence is designed, signals are further amplified by using a telomerase amplification mode, and the color change of a solution is observed according to the difference of target concentration to obtain a visual detection result of the exosome membrane protein.
Technical Field
The invention belongs to the field of molecular biological detection, relates to a method for detecting the content of an exosome membrane protein, and particularly relates to a method for detecting the exosome membrane protein based on telomerase amplification. The invention relates to a method for visually detecting a repeated product rich in guanine (G) by taking prostate cancer markers miRNAs as targets, designing a hairpin structure recognition probe complementary with the miRNAs, exposing a terminal telomerase recognition primer after recognizing the targets, utilizing the telomerase recognition primer to carry out nucleic acid amplification, and combining the repeated product rich in guanine (G) with blood crystal.
Background
The liquid biopsy technology can detect biomarkers in body fluid (such as blood, plasma, serum, urine and gastric juice), and is a novel noninvasive diagnosis technology. Since a series of cancer components are released into blood during the occurrence and development of cancer, the liquid biopsy can realize the clinical applications of early screening, molecular typing, prognosis, medication guidance, relapse monitoring and the like of cancer through the analysis of the cancer components. In the current clinical research, the liquid biopsy technique is mainly applied to the detection of free Circulating Tumor Cells (CTCs), circulating tumor dna (ctdna), exosome and circulating rna (circulating rna), and the like.
Exosomes are membrane vesicle structures secreted by living cells with diameters of about 30-150 nm. Exosomes are mainly derived from late endosomes and naturally occur in body fluids such as blood, saliva, urine, and breast milk. Exosomes are involved in regulating cancer progression, cancer cell proliferation and migration, neovascularization, escape of cell death, invasion and metastasis. The exosome surface is rich in a large number of tumor-specific proteins, and the exosome surface is rich in nucleic acid content, so that abundant information is provided for judging the development and metastasis of cancers. Thus, cancer-source-related exosomes have become potential biomarkers to detect cancer progression.
Disclosure of Invention
Aiming at the existing detection requirements, the invention aims to provide a method for detecting exosome membrane protein based on telomerase amplification.
Yet another object of the present invention is to: a hairpin probe having a recognition sequence is provided.
Yet another object of the present invention is to: provides the application of the hairpin structure probe with the recognition sequence.
The purpose of the invention is realized by the following scheme: an exosome membrane protein detection method based on telomerase amplification is characterized in that exosome membrane protein is used as a target, an immunomagnetic bead is used for capturing exosome, a hairpin structure probe with a recognition sequence is designed, signals are further amplified by using the telomerase amplification mode, and the exosome membrane protein is visually detected.
The invention provides an exosome membrane protein detection method based on telomerase amplification, which comprises the following steps:
(1) magnetic bead capture exosomes:
the method comprises the steps of coating an exosome membrane protein antibody with magnetic beads and capturing an exosome, wherein carboxylated magnetic beads are activated and then coupled with the exosome membrane protein antibody, and then the activated magnetic beads and the exosome are incubated to form a mixture I;
(2) hairpin structure probe recognition exosomes:
taking the single-chain recognition sequence to incubate with a block (closed) sequence to enable the probe sequence to form a hairpin structure, then mixing with the mixture I, washing and re-suspending to form a mixture II;
(3) amplification of telomerase:
adding a template containing telomerase and dNTPs into the mixture II, amplifying the telomerase, washing and resuspending to obtain a mixture IV;
(4) and (3) detection:
a chromogenic reagent was added to the mixture IV and the change in absorbance at 405 nm was recorded using an ultraviolet spectrophotometer.
ABTS is a mediator substance, also known as 2, 2-diaza-bis (3-ethyl-benzothiazole-6-sulfonic acid), 2,2' -Azinobis- (3-ethylbenzthiazoline-6-sulfosalt), which can be used to determine laccase enzyme activity, and the rate of ABTS oxidation by laccase can be used to determine the laccase enzyme activity. The application comprises the following steps: free chlorine spectral reagent, chromogenic substrate for enzyme-linked immunosorbent assay, substrate for peroxidase, which produces a soluble green chelate, and the like, for ELISA assay.
Preferably, the magnetic bead coated exosome membrane protein antibody is any one of CD63, CD81, CD9, ALIX, ANNEXIN, EpCAM and RAB 5.
Preferably, the hairpin probe is composed of two single-stranded DNAs, wherein the recognition single-stranded sequence is from 5，End to 3，The end mainly comprises an exosome membrane protein aptamer, a spacer sequence and a telomerase recognition primer sequence; block sequence from 5，End to 3，The terminal mainly comprises a telomerase primer sequence complementary sequence and an exosome membrane protein aptamer partial complementary sequence, and the telomerase primer sequence complementary sequence and the exosome membrane protein aptamer partial complementary sequence are hybridized to form a hairpin structure recognition probe.
Preferably, in the hairpin structure probe, the recognition single-stranded sequence mainly comprises an exosome membrane protein aptamer, a spacer sequence and a telomerase recognition primer sequence from the 5 'end to the 3' end,
the single-chain recognition sequence is selected from any one of SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9 and SEQ ID NO 11;
the block sequence comprises a telomerase primer sequence complementary sequence and an exosome membrane protein aptamer part complementary sequence from the 5 'end to the 3' end,
the block sequence is selected from any one of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10 and SEQ ID NO 12;
and mixing the single-chain recognition sequence and the block sequence in a molar ratio of 1:1, incubating, and hybridizing to form the hairpin structure recognition probe.
Preferably, the telomerase recognition sequence ends in a TGTT or AGTT.
Specifically, the method of the present invention comprises the steps of:
(1) magnetic bead capture exosomes:
a) magnetic bead coating of exosome membrane protein antibody: after 10 mg of the carboxylated magnetic beads are washed by MES buffer (pH6.0), dissolved in 0.5 mL of MES buffer (pH6.0), added with 10 mg/mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 12 mg/mL of N-hydroxysuccinimide (NHS), mixed uniformly and activated for 30min at room temperature; magnetic separation and washing of the activated magnetic beads, dissolving in MES buffer (pH6.0), adding 2mg/mL antibody, mixing well, reacting overnight at 4 ℃, washing well with PBS (pH7.4) containing 0.1% Tween20, and suspending in PBS (pH7.4) containing 0.1% Tween20 and 0.1% BSA for later use;
b) capturing exosomes: separating and extracting exosomes by an ultracentrifugation method, fully mixing 50 muL 0.5 mg/mL magnetic beads and 50 muL exosomes, incubating at room temperature for 1hr, performing magnetic separation, fully cleaning, and resuspending precipitates in 50 muL Tb buffer solution (22 mM Tris-HCl, 120 mM Na Cl, 50 mM K Cl, 0.85 mM Ca Cl)2，6 mM Mg Cl26.8% glycerol, pH 7.8), noted as mixture 1;
(2) hairpin structure probe recognition exosomes: incubating the single-chain recognition sequence and the block sequence at a molar ratio of 1: 190 ℃ for 5 min, slowly cooling to room temperature to enable the probe sequence to form a hairpin structure, recognizing the probe of the hairpin structure, fully mixing 10 muL of the hairpin structure recognition probe with the mixture I, reacting at room temperature for 1hr, fully cleaning after magnetic separation, and adopting telomerase extension buffer (Te buffer: 20 mM Tris-HCl, 1.5 mM Mg Cl)2Washing with 63 mM K Cl, 0.005% (v/v) Tween-20, 1 mM EGTA) for three times, and re-rotating the precipitate with 100 μ L of Te buffer solution, and recording as a mixture II;
(3) amplification of telomerase: adding 20 μ L telomerase extract and 1 mM dNTPs into 100 μ L mixture II obtained in step (2), mixing, reacting at 30 deg.C for 1hr, removing supernatant by magnetic separation, and adding 10 mM PBS buffer II (10 mM Na)2 HPO4，2 mM K H2PO4137 mM Na Cl, 50 mM K Cl) and the precipitate is re-spun with 100. mu.L PBS buffer II to obtain a mixture IV;
(4) and (3) detection: adding Hemin, hydrogen peroxide and (H) into the mixture IV obtained in the step (3)2O2) And ABTS, mixing uniformly, observing the color change of the solution according to the difference of target concentration, and recording the change of absorbance at 405 nm by using an ultraviolet spectrophotometer.
Preferably, the aptmer recognizes the exosome membrane protein as any one of PSMA, HER-2, EpCAM, CD63, CD81, MUC 1.
The invention takes the exosome membrane protein as a target, captures exosomes by immunomagnetic beads, designs a hairpin structure probe with a recognition sequence, and further amplifies signals by using a telomerase amplification mode to realize the visual detection of the exosome membrane protein. After the hairpin structure probe is combined with the target, the telomerase recognition sequence at the tail end of the probe is exposed. After telomerase (telomerase) is combined with a recognition primer, a template region of self RNA is taken as a template, a 6-base repetitive sequence (AGGGTT) is added at the tail end of the primer under the condition of the existence of dNTP, the sequence can be combined with hemin to form a compound with peroxidase-like activity, and the content of miRNAs is quantitatively analyzed in a chemiluminescence way with high selectivity, high sensitivity, simplicity and rapidness.
The invention also provides a hairpin structure probe with an identification sequence, wherein the hairpin structure probe consists of two single-stranded DNAs, one single-stranded probe comprises an aptamer for identifying membrane protein and a telomerase identification (primer) sequence, and the other single-stranded probe is a block sequence which is partially complementary with the aptamer and the telomerase identification sequence;
wherein the recognition single-stranded sequence is from 5 to 3, and the end comprises an exosome membrane protein aptamer, a spacer sequence and a telomerase recognition primer sequence;
and the block sequence is from 5 to 3, the end of the block sequence comprises a telomerase primer sequence complementary sequence and an exosome membrane protein aptamer partial complementary sequence, and the telomerase primer sequence complementary sequence and the exosome membrane protein aptamer partial complementary sequence are hybridized to form the hairpin structure recognition probe.
Preferably, the combination of the single-stranded recognition sequence and the block sequence is selected from, but not limited to, the following combinations:
SEQ ID NO 1(DNA1) and SEQ ID NO 2(DNA 2);
SEQ ID NO 3(DNA3) and SEQ ID NO 4(DNA 4);
SEQ ID NO 5(DNA5) and SEQ ID NO 6(DNA 6);
EQ ID NO 7(DNA7) and SEQ ID NO 8(DNA 8);
SEQ ID NO 9(DNA9) and SEQ ID NO 10(DNA 10);
SEQ ID NO 11(DNA11) and SEQ ID NO 12(DNA 12).
The invention also provides application of the hairpin structure probe of the sequence in preparation of an exosome membrane protein visual detection reagent.
To achieve the purpose, in the technical scheme of the invention, the exosome membrane protein antibody is coupled after the activation of the carboxylated magnetic beads so as to rapidly capture the exosome. Designing a hairpin structure probe, wherein the probe consists of two single-stranded DNAs, one single-stranded probe comprises an aptamer (aptamer) for identifying membrane proteins and a telomerase identification sequence, and the other single-stranded probe is a block sequence which is partially complementary with the aptamer and the telomerase identification sequence. After the hairpin structure probe is fully combined with the exosome membrane protein, the stem-loop structure of the probe is opened, and 3 is leaked，Telomerase recognition primers. After adding telomerase, telomerase binds to the exposed primer sequence, which can form a repeated guanine-rich sequence at the end of the primer in the presence of dntps. Further, hemin (hemin) is added to form a compound with peroxidase activity, and the compound catalyzes 2,2' -biazonitrogen-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) to develop color, so that the content of the exosome membrane protein can be visually determined.
The invention also provides application of the telomerase amplification based exosome membrane protein detection method, and the telomerase amplification based exosome membrane protein detection method is used for exosome membrane protein visual detection.
Preferably, the exosome membrane protein is taken as a target, an immunomagnetic bead is used for capturing exosomes, a hairpin structure probe with a recognition sequence is designed, signals are further amplified by a telomerase amplification mode, and the color change of a solution is observed according to the difference of target concentration, so that the visual detection result of the exosome membrane protein is obtained. The preliminary result can be judged according to the color of the solution, and a qualitative result can be obtained by naked eyes.
As a preferred embodiment of the invention, the prostate cancer marker PSMA is taken as a target, a hairpin structure recognition probe which is complementary to the PSMA is designed, and a terminal telomerase recognition primer is exposed after the hairpin structure recognition probe is recognized with the target. Identifying primer by telomerase and amplifying nucleic acid to obtain repeated product rich in guanine (G), and performing visual detection after combining with hemin.
The invention has the advantages that:
(1) the invention designs a hairpin structure probe to identify an exposed priming sequence behind a target membrane protein, induce telomerase amplification to further amplify signals, and carry out exosome membrane protein visual detection through magnetic separation. The separation method is convenient, the used materials are partially commercialized, and the biological safety is high.
(2) The detection process of the invention does not need to use professional instruments, and the reaction only needs to be carried out by a water bath, thereby meeting the requirement of primary medical detection.
(3) The invention has the advantages of low cost, sensitive detection, high specificity and visualization. Can well analyze the exosome membrane protein and is suitable for clinical detection requirements.
Drawings
FIG. 1 shows the UV absorbance of the PSMA content of the exosome membrane protein measured in example 1.
Detailed Description
The technical solution of the present invention is further described below with reference to the accompanying drawings. The following specific examples are further illustrative of the present invention and do not limit the scope of the invention.
Example 1
1. Magnetic bead capture exosomes
(1) Magnetic bead coating of exosome membrane protein antibody: after 10 mg of the carboxylated magnetic beads were washed with MES buffer (pH 6.0), they were dissolved in 0.5 mL of MES buffer (pH 6.0), and then 10 mg/mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 12 mg/mL of N-hydroxysuccinimide (NHS) were added thereto, and they were mixed well and activated at room temperature for 30 min. The activated magnetic beads were washed by magnetic separation, dissolved in MES buffer (pH 6.0), mixed well with 2mg/mL of antibody, reacted overnight at 4 ℃, washed well with PBS containing 0.1% Tween20 (pH 7.4), and resuspended in PBS containing 0.1% Tween20 and 0.1% BSA (pH 7.4) for use.
(2) Capturing exosomes: separating and extracting exosome by an ultracentrifugation method, fully mixing 50 muL 0.5 mg/mL magnetic beads and 50 muL exosomes, incubating at room temperature for 1hr, performing magnetic separation, fully cleaning,the pellet was resuspended in 50 μ L Tb buffer (22 mM Tris-HCl, 120 mM Na Cl, 50 mM K Cl, 0.85 mM Ca Cl2，6 mM Mg Cl26.8% glycerol, pH 7.8), noted as mixture 1.
2. Hairpin structure probe recognition exosomes:
and (3) taking 10 mu L of single-chain recognition sequence DNA1 (100 mu M) and 10 mu L of block sequence DNA2 (100 mu M), incubating for 5 min at 90 ℃, and slowly cooling to room temperature to enable the probe sequence to form a hairpin structure and recognize the probe by the hairpin structure. Taking 10 mu L of hairpin structure recognition probe, fully mixing with the mixture I, reacting at room temperature for 1hr, fully cleaning by magnetic separation, and adopting telomerase extension buffer (Te buffer: 20 mM Tris-H Cl, 1.5 mM Mg Cl)263 mM K Cl, 0.005% (v/v) Tween-20, 1 mM EGTA) and the pellet was re-spun with 100 μ L of Te buffer, denoted as mixture II.
3. Amplification of telomerase:
mu.L of the mixture obtained in step 2 was added with 20. mu.L of telomerase extract and 1 mM of dNTPs, mixed well, and reacted at 30 ℃ for 1 hr. The supernatant was removed by magnetic separation using 10 mM PBS buffer II (10 mM Na)2 HPO4，2 mM K H2PO4137 mM Na Cl, 50 mM K Cl) and the pellet was re-spun with 100. mu.L of PBS buffer II.
4. And (3) detection:
mu.L of 20. mu.M hemin solution was added to 100. mu.L of the mixture obtained in step 3, mixed well and incubated at room temperature for 10 min.
To the mixture was added 1. mu.L of 40 mM hydrogen peroxide (H)2O2) And 50 mu L of 2 mM ABTS, wherein after the mixture is uniformly mixed, the color of the solution is changed from colorless to dark blue-green, an ultraviolet spectrophotometer is adopted to record the change of absorbance at 405 nm, the attached figure 1 shows that the ultraviolet absorption value of the content of the exosome specific membrane protein PSMA is measured in the embodiment 1, and the method can be used for measuring the content of the exosome specific membrane protein as shown in the figure 1.
Therefore, the method successfully identifies a large amount of the exosome membrane proteins, has obvious difference with a control, and can obtain a qualitative result by observing the color of the solution.
Example 2
1. Magnetic bead capture exosomes
(1) Magnetic bead coating of exosome membrane protein antibody: after 10 mg of the carboxylated magnetic beads were washed with MES buffer (pH 6.0), they were dissolved in 0.5 mL of MES buffer (pH 6.0), and then 10 mg/mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 12 mg/mL of N-hydroxysuccinimide (NHS) were added thereto, and they were mixed well and activated at room temperature for 30 min. The activated magnetic beads were washed by magnetic separation, dissolved in MES buffer (pH 6.0), mixed well with 2mg/mL of antibody, reacted overnight at 4 ℃, washed well with PBS containing 0.1% Tween20 (pH 7.4), and resuspended in PBS containing 0.1% Tween20 and 0.1% BSA (pH 7.4) for use.
(2) Capturing exosomes: separating and extracting exosomes by an ultracentrifugation method, fully mixing 50 muL 0.5 mg/mL magnetic beads and 50 muL exosomes, incubating at room temperature for 1hr, performing magnetic separation, fully cleaning, and resuspending precipitates in 50 muL Tb buffer solution (22 mM Tris-HCl, 120 mM Na Cl, 50 mM K Cl, 0.85 mM Ca Cl)2，6 mM Mg Cl26.8% glycerol, pH 7.8), noted as mixture 1.
2. Hairpin structure probe recognition exosomes:
and (3) taking 10 mu L of single-chain recognition sequence DNA3 (100 mu M) and 10 mu L of block sequence DNA4 (100 mu M), incubating for 5 min at 90 ℃, and slowly cooling to room temperature to enable the probe sequence to form a hairpin structure and recognize the probe by the hairpin structure. Taking 10 mu L of hairpin structure recognition probe, fully mixing with the mixture I, reacting at room temperature for 1hr, fully cleaning by magnetic separation, and adopting telomerase extension buffer (Te buffer: 20 mM Tris-H Cl, 1.5 mM Mg Cl)263 mM K Cl, 0.005% (v/v) Tween-20, 1 mM EGTA) and the pellet was re-spun with 100 μ L of Te buffer, denoted as mixture II.
3. Amplification of telomerase:
mu.L of the mixture obtained in step 2 was added with 20. mu.L of telomerase extract and 1 mM of dNTPs, mixed well, and reacted at 30 ℃ for 1 hr. The supernatant was removed by magnetic separation using 10 mM PBS buffer II (10 mM Na)2 HPO4，2 mM K H2PO4137 mM Na Cl, 50 mM K Cl) and the pellet was re-spun with 100. mu.L of PBS buffer II.
4. And (3) detection:
mu.L of 20. mu.M hemin solution was added to 100. mu.L of the mixture obtained in step 3, mixed well and incubated at room temperature for 10 min. mu.L of 40 mM hydrogen peroxide (H) was added to the above mixture2O2) And 50. mu.L of 2 mM ABTS, and after mixing well, recording the change in absorbance at 405 nm using an ultraviolet spectrophotometer.
Example 3
1. Magnetic bead capture exosomes
(1) Magnetic bead coating of exosome membrane protein antibody: after 10 mg of the carboxylated magnetic beads were washed with MES buffer (pH 6.0), they were dissolved in 0.5 mL of MES buffer (pH 6.0), and then 10 mg/mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 12 mg/mL of N-hydroxysuccinimide (NHS) were added thereto, and they were mixed well and activated at room temperature for 30 min. The activated magnetic beads were washed by magnetic separation, dissolved in MES buffer (pH 6.0), mixed well with 2mg/mL of antibody, reacted overnight at 4 ℃, washed well with PBS containing 0.1% Tween20 (pH 7.4), and resuspended in PBS containing 0.1% Tween20 and 0.1% BSA (pH 7.4) for use.
(2) Capturing exosomes: separating and extracting exosomes by an ultracentrifugation method, fully mixing 50 muL 0.5 mg/mL magnetic beads and 50 muL exosomes, incubating at room temperature for 1hr, performing magnetic separation, fully cleaning, and resuspending precipitates in 50 muL Tb buffer solution (22 mM Tris-HCl, 120 mM Na Cl, 50 mM K Cl, 0.85 mM Ca Cl)2，6 mM Mg Cl26.8% glycerol, pH 7.8), noted as mixture 1.
2. Hairpin structure probe recognition exosomes:
and (3) taking 10 mu L of single-chain recognition sequence DNA5 (100 mu M) and 10 mu L of block sequence DNA6 (100 mu M), incubating for 5 min at 90 ℃, and slowly cooling to room temperature to enable the probe sequence to form a hairpin structure and recognize the probe by the hairpin structure. Taking 10 mu L of hairpin structure recognition probe, fully mixing with the mixture I, reacting at room temperature for 1hr, fully cleaning by magnetic separation, and adopting telomerase extension buffer (Te buffer: 20 mM Tris-H Cl, 1.5 mM Mg Cl)2，63 mM K Cl，0.005%(v/v) Tween-20, 1 mM EGTA) three times, and the pellet was re-spun with 100 μ L of Te buffer, as mixture II.
3. Amplification of telomerase:
mu.L of the mixture obtained in step 2 was added with 20. mu.L of telomerase extract and 1 mM of dNTPs, mixed well, and reacted at 30 ℃ for 1 hr. The supernatant was removed by magnetic separation using 10 mM PBS buffer II (10 mM Na)2 HPO4，2 mM K H2PO4137 mM Na Cl, 50 mM K Cl) and the pellet was re-spun with 100. mu.L of PBS buffer II.
4. And (3) detection:
mu.L of 20. mu.M hemin solution was added to 100. mu.L of the mixture obtained in step 3, mixed well and incubated at room temperature for 10 min. mu.L of 40 mM hydrogen peroxide (H) was added to the above mixture2O2) And 50. mu.L of 2 mM ABTS, and after mixing well, recording the change in absorbance at 405 nm using an ultraviolet spectrophotometer.
Example 4
1. Magnetic bead capture exosomes
(1) Magnetic bead coating of exosome membrane protein antibody: after 10 mg of the carboxylated magnetic beads were washed with MES buffer (pH 6.0), they were dissolved in 0.5 mL of MES buffer (pH 6.0), and then 10 mg/mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 12 mg/mL of N-hydroxysuccinimide (NHS) were added thereto, and they were mixed well and activated at room temperature for 30 min. The activated magnetic beads were washed by magnetic separation, dissolved in MES buffer (pH 6.0), mixed well with 2mg/mL of antibody, reacted overnight at 4 ℃, washed well with PBS containing 0.1% Tween20 (pH 7.4), and resuspended in PBS containing 0.1% Tween20 and 0.1% BSA (pH 7.4) for use.
(2) Capturing exosomes: separating and extracting exosomes by an ultracentrifugation method, fully mixing 50 muL 0.5 mg/mL magnetic beads and 50 muL exosomes, incubating at room temperature for 1hr, performing magnetic separation, fully cleaning, and resuspending precipitates in 50 muL Tb buffer solution (22 mM Tris-HCl, 120 mM Na Cl, 50 mM K Cl, 0.85 mM Ca Cl)2，6 mM Mg Cl26.8% glycerol, pH 7.8), noted as mixture 1.
2. Hairpin structure probe recognition exosomes:
and (3) taking 10 mu L of single-chain recognition sequence DNA7 (100 mu M) and 10 mu L of block sequence DNA8 (100 mu M), incubating for 5 min at 90 ℃, and slowly cooling to room temperature to enable the probe sequence to form a hairpin structure and recognize the probe by the hairpin structure. Taking 10 mu L of hairpin structure recognition probe, fully mixing with the mixture I, reacting at room temperature for 1hr, fully cleaning by magnetic separation, and adopting telomerase extension buffer (Te buffer: 20 mM Tris-H Cl, 1.5 mM Mg Cl)263 mM K Cl, 0.005% (v/v) Tween-20, 1 mM EGTA) and the pellet was re-spun with 100 μ L of Te buffer, denoted as mixture II.
3. Amplification of telomerase:
mu.L of the mixture obtained in step 2 was added with 20. mu.L of telomerase extract and 1 mM of dNTPs, mixed well, and reacted at 30 ℃ for 1 hr. The supernatant was removed by magnetic separation using 10 mM PBS buffer II (10 mM Na)2 HPO4，2 mM K H2PO4137 mM Na Cl, 50 mM K Cl) and the pellet was re-spun with 100. mu.L of PBS buffer II.
4. And (3) detection:
mu.L of 20. mu.M hemin solution was added to 100. mu.L of the mixture obtained in step 3, mixed well and incubated at room temperature for 10 min. To the mixture was added 1. mu.L of 40 mM hydrogen peroxide (H)2O2) And 50. mu.L of 2 mM ABTS, and after mixing well, recording the change in absorbance at 405 nm using an ultraviolet spectrophotometer.
Example 5
1. Magnetic bead capture exosomes
(1) Magnetic bead coating of exosome membrane protein antibody: after 10 mg of the carboxylated magnetic beads were washed with MES buffer (pH 6.0), they were dissolved in 0.5 mL of MES buffer (pH 6.0), and then 10 mg/mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 12 mg/mL of N-hydroxysuccinimide (NHS) were added thereto, and they were mixed well and activated at room temperature for 30 min. The activated magnetic beads were washed by magnetic separation, dissolved in MES buffer (pH 6.0), mixed well with 2mg/mL of antibody, reacted overnight at 4 ℃, washed well with PBS containing 0.1% Tween20 (pH 7.4), and resuspended in PBS containing 0.1% Tween20 and 0.1% BSA (pH 7.4) for use.
(2) Capturing exosomes: separating and extracting exosomes by an ultracentrifugation method, fully mixing 50 muL 0.5 mg/mL magnetic beads and 50 muL exosomes, incubating at room temperature for 1hr, performing magnetic separation, fully cleaning, and resuspending precipitates in 50 muL Tb buffer solution (22 mM Tris-HCl, 120 mM Na Cl, 50 mM K Cl, 0.85 mM Ca Cl)2，6 mM Mg Cl26.8% glycerol, pH 7.8), noted as mixture 1.
2. Hairpin structure probe recognition exosomes:
and (3) taking 10 mu L of single-chain recognition sequence DNA9 (100 mu M) and 10 mu L of block sequence DNA10 (100 mu M), incubating for 5 min at 90 ℃, and slowly cooling to room temperature to enable the probe sequence to form a hairpin structure and recognize the probe by the hairpin structure. Taking 10 mu L of hairpin structure recognition probe, fully mixing with the mixture I, reacting at room temperature for 1hr, fully cleaning by magnetic separation, and adopting telomerase extension buffer (Te buffer: 20 mM Tris-H Cl, 1.5 mM Mg Cl)263 mM K Cl, 0.005% (v/v) Tween-20, 1 mM EGTA) and the pellet was re-spun with 100 μ L of Te buffer, denoted as mixture II.
3. Amplification of telomerase:
mu.L of the mixture obtained in step 2 was added with 20. mu.L of telomerase extract and 1 mM of dNTPs, mixed well, and reacted at 30 ℃ for 1 hr. The supernatant was removed by magnetic separation using 10 mM PBS buffer II (10 mM Na)2 HPO4，2 mM K H2PO4137 mM Na Cl, 50 mM K Cl) and the pellet was re-spun with 100. mu.L of PBS buffer II.
4. And (3) detection:
mu.L of 20. mu.M hemin solution was added to 100. mu.L of the mixture obtained in step 3, mixed well and incubated at room temperature for 10 min. mu.L of 40 mM hydrogen peroxide (H) was added to the above mixture2O2) And 50. mu.L of 2 mM ABTS, and after mixing well, recording the change in absorbance at 405 nm using an ultraviolet spectrophotometer.
Example 6
1. Magnetic bead capture exosomes
(1) Magnetic bead coating of exosome membrane protein antibody: after 10 mg of the carboxylated magnetic beads were washed with MES buffer (pH 6.0), they were dissolved in 0.5 mL of MES buffer (pH 6.0), and then 10 mg/mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 12 mg/mL of N-hydroxysuccinimide (NHS) were added thereto, and they were mixed well and activated at room temperature for 30 min. The activated magnetic beads were washed by magnetic separation, dissolved in MES buffer (pH 6.0), mixed well with 2mg/mL of antibody, reacted overnight at 4 ℃, washed well with PBS containing 0.1% Tween20 (pH 7.4), and resuspended in PBS containing 0.1% Tween20 and 0.1% BSA (pH 7.4) for use.
(2) Capturing exosomes: separating and extracting exosomes by an ultracentrifugation method, fully mixing 50 muL 0.5 mg/mL magnetic beads and 50 muL exosomes, incubating at room temperature for 1hr, performing magnetic separation, fully cleaning, and resuspending precipitates in 50 muL Tb buffer solution (22 mM Tris-HCl, 120 mM Na Cl, 50 mM K Cl, 0.85 mM Ca Cl)2，6 mM Mg Cl26.8% glycerol, pH 7.8), noted as mixture 1.
2. Hairpin structure probe recognition exosomes:
and (3) taking 10 mu L of single-chain recognition sequence DNA11 (100 mu M) and 10 mu L of block sequence DNA12 (100 mu M), incubating for 5 min at 90 ℃, and slowly cooling to room temperature to enable the probe sequence to form a hairpin structure and recognize the probe by the hairpin structure. Taking 10 mu L of hairpin structure recognition probe, fully mixing with the mixture I, reacting at room temperature for 1hr, fully cleaning by magnetic separation, and adopting telomerase extension buffer (Te buffer: 20 mM Tris-H Cl, 1.5 mM Mg Cl)263 mM K Cl, 0.005% (v/v) Tween-20, 1 mM EGTA) and the pellet was re-spun with 100 μ L of Te buffer, denoted as mixture II.
3. Amplification of telomerase:
mu.L of the mixture obtained in step 2 was added with 20. mu.L of telomerase extract and 1 mM of dNTPs, mixed well, and reacted at 30 ℃ for 1 hr. The supernatant was removed by magnetic separation using 10 mM PBS buffer II (10 mM Na)2 HPO4，2 mM K H2PO4137 mM Na Cl, 50 mM K Cl) and the pellet was re-spun with 100. mu.L of PBS buffer II.
4. And (3) detection:
1 μ L20 μ M heminAdding 100 μ L of the mixed solution obtained in step 3 into the solution, mixing uniformly, and incubating at room temperature for 10 min. mu.L of 40 mM hydrogen peroxide (H) was added to the above mixture2O2) And 50. mu.L of 2 mM ABTS, and after mixing well, recording the change in absorbance at 405 nm using an ultraviolet spectrophotometer.
It will be appreciated by those skilled in the art that the invention can be embodied in many other specific forms without departing from the spirit or scope thereof. Having described embodiments, it should be understood that the preferred embodiments of the present invention can be modified and changed by those skilled in the art within the spirit and scope of the present invention as defined by the appended claims, which should not be limited to the embodiments described above.
<110> Shanghai nanotechnology and applied national center for engineering research Ltd
<120> method for detecting exosome membrane protein based on telomerase amplification
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