Trace MUC1 fluorescence detection method based on chain type hybridization reaction and fluorescent carbon quantum dots
阅读说明：本技术 基于链式杂交反应及荧光碳量子点的痕量muc1荧光检测方法 (Trace MUC1 fluorescence detection method based on chain type hybridization reaction and fluorescent carbon quantum dots ) 是由 杨大威 缪鹏 陈锡峰 孟凡渝 于 2019-09-19 设计创作，主要内容包括：本发明公开了一种基于链式杂交反应及荧光碳量子点的痕量MUC1荧光检测方法,具体为：先合成碳量子点,然后将颈环DNA H1、H2修饰至碳量子点上,再与氧化石墨烯混合；MUC1不存在时,修饰有碳量子点的H1、H2吸附至氧化石墨烯表面,由于荧光共振能量转移效应,碳量子点荧光猝灭；当MUC1存在时,MUC1与适配体结合,并促发H1、H2发生链式杂交反应形成链式杂交产物,脱离氧化石墨烯的表面,最终碳量子点荧光得到恢复；从而通过荧光检测实现MUC1含量的测定。本方法通过链式杂交反应实现的信号的放大,大大提高了MUC1检测的灵敏度；同时本方法合成的荧光碳量子点荧光性质稳定,极大提高了本方法的稳定性和重复性。(The invention discloses a trace MUC1 fluorescence detection method based on chain type hybridization reaction and fluorescent carbon quantum dots, which comprises the steps of firstly synthesizing the carbon quantum dots, then modifying neck ring DNAs H1 and H2 on the carbon quantum dots, and then mixing the carbon quantum dots with graphene oxide, adsorbing H1 and H2 modified with the carbon quantum dots to the surface of the graphene oxide when MUC1 does not exist, quenching the fluorescence of the carbon quantum dots due to fluorescence resonance energy transfer effect, combining MUC1 with an aptamer and promoting H1 and H2 to perform chain type hybridization reaction to form a chain type hybridization product when MUC1 exists, separating from the surface of the graphene oxide, and finally recovering the fluorescence of the carbon quantum dots, so that the determination of the MUC1 content is realized through the fluorescence detection.)
The method is characterized by comprising the steps of firstly synthesizing carbon quantum dots, then modifying neck ring DNA H1 and H2 on the carbon quantum dots, mixing the carbon quantum dots with graphene oxide, adsorbing H1 and H2 modified with the carbon quantum dots to the surface of the graphene oxide when MUC1 does not exist, quenching the fluorescence of the carbon quantum dots due to the fluorescence resonance energy transfer effect, combining MUC1 and an aptamer when MUC1 exists, promoting H1 and H2 to perform chain type hybridization reaction to form a chain type hybridization product, separating from the surface of the graphene oxide, and finally recovering the fluorescence, so that the determination of the content of MUC1 is realized through fluorescence detection.
2. The method for detecting trace MUC1 fluorescence based on chain hybridization and fluorescent carbon quantum dots according to claim 1, wherein the MUC1 is bound to the aptamer to cause a conformational change in the aptamer, the aptamer exposes the recognition sequence, and the recognition sequence triggers H1 and H2 to perform chain hybridization to form a chain hybridization product.
3. The method for detecting trace MUC1 fluorescence based on chain hybridization reaction and fluorescent carbon quantum dots according to claim 1, wherein the method comprises the following steps:
1) synthesizing carbon quantum dots;
2) pretreating neck ring DNA H1 and H2;
3) modifying the pretreated neck ring DNA H1 and H2 to a carbon quantum dot;
4) h1 and H2 modified with carbon quantum dots are mixed with graphene oxide;
5) adding MUC1 aptamer into the MUC1 sample to be detected, then adding the mixture obtained in the step 4) into the MUC1 sample to be detected and the MUC1 aptamer mixed solution, and detecting the fluorescence intensity.
4. The method for detecting trace MUC1 fluorescence based on chain hybridization reaction and fluorescent carbon quantum dots according to claim 3, wherein the step 1) is specifically as follows: firstly, grinding and mixing citric acid and cysteine according to a molar ratio of 2:1, and reacting in a microwave oven for 4min to obtain the carbon quantum dots.
5. The method for detecting trace MUC1 fluorescence based on chain hybridization reaction and fluorescent carbon quantum dots according to claim 3, wherein the step 2) is specifically as follows: dissolving the neck ring DNA H1, H2 and MUC1 aptamer in a Tris buffer solution, heating the solution to 95 ℃ in a metal bath, keeping the temperature for 5 minutes, and naturally cooling the solution to room temperature; the DNA is then diluted to the desired concentration and stored for future use.
6. The method for detecting trace MUC1 fluorescence based on chain hybridization reaction and fluorescent carbon quantum dots according to claim 3, wherein the step 3) is specifically as follows: firstly, activating the synthesized carbon quantum dots by EDC and NHS, and shaking for 30 minutes at 25 ℃; then, the end-aminated H1 and H2 were added to the activated carbon quantum dot solution, shaken at room temperature for 2 hours, and then left overnight at 4 ℃ to hydrolyze the unreacted EDC, and finally, carbon quantum dots modified with H1 and H2 were obtained.
7. The method for detecting trace MUC1 fluorescence based on chain hybridization reaction and fluorescent carbon quantum dots according to claim 3, wherein the step 4) is specifically as follows: and mixing the carbon quantum dots modified by H1 and H2 with graphene oxide, and incubating for 15min at 25 ℃ to quench fluorescence.
8. The method for detecting trace MUC1 fluorescence based on chain hybridization reaction and fluorescent carbon quantum dots according to claim 3, wherein the step 5) is specifically as follows: adding a proper amount of MUC1 aptamer into a MUC1 sample to be detected, and mixing and incubating for 30 minutes at 37 ℃; then, the mixture obtained in step 4) was added thereto, incubated for 15 minutes, and the fluorescence intensity thereof was measured.
9. The method for detecting trace MUC1 fluorescence based on chain hybridization reaction and fluorescent carbon quantum dots according to claim 8, wherein the fluorescence intensity detection is performed by an FLS-1000 fluorescence spectrometer, the excitation wavelength is 355nm, the emission wavelength is 420nm, and the slit width is 3nm and 4nm respectively.
Technical Field
The invention relates to the field of trace MUC1 detection, in particular to trace MUC1 fluorescence detection methods based on chain hybridization reaction and fluorescent carbon quantum dots.
Background
Mucin (MUC1) is a high molecular weight, high glycosylation protein, exists in epithelial cells, forms a complete transmembrane domain through a gel matrix, mucin (MUC1) is a cell surface glycoprotein, a polypeptide skeleton of the mucin is composed of 3 parts of a transmembrane segment, a transmembrane segment and an intracellular segment, the transmembrane segment and the intracellular segment contain 31 and 69 amino acids respectively, the extracellular segment contains a plurality of continuous repetitive sequences, each repetitive sequence contains 20 amino acids, MUC1 plays an important role in a signal transmission process, MUC1 can enable E-cadherin to be expressed down, the latter is calcium ion-dependent cell adhesion molecules, mediates intercellular binding and plays an inhibiting role in tumor metastasis, the down-regulated expression of the E-cadherin is a step of enhancing invasiveness of tumor cells, is MUC1 is highly expressed in human epithelial cell adenocarcinoma, such as breast cancer, gastric cancer, lung cancer, prostate cancer, ovarian cancer and pancreatic cancer, when the down-regulated expression of the E-cadherin reaches , the MUC1 is a great degree, the early diagnosis sensitivity can be detected by an extracellular colorimetric method, and the detection method can be used as a traditional early diagnosis marker, a plurality of early detection of early cancer, the early detection of the traditional colorimetric detection of early cancer, the early.
Disclosure of Invention
The invention aims to solve the technical problem of providing trace MUC1 fluorescence detection methods based on chain hybridization reaction and fluorescent carbon quantum dots aiming at the defects in the prior art.
In order to solve the technical problems, the invention adopts the technical scheme that trace MUC1 fluorescence detection methods based on chain hybridization reaction and fluorescent carbon quantum dots are adopted, the method specifically comprises the steps of firstly synthesizing the carbon quantum dots, then modifying the carbon quantum dots with the neck ring DNAH1 and H2, mixing the modified carbon quantum dots with graphene oxide, adsorbing H1 and H2 modified with the carbon quantum dots to the surface of the graphene oxide when MUC1 does not exist, quenching the fluorescence of the carbon quantum dots due to the fluorescence resonance energy transfer effect, combining MUC1 with an aptamer when MUC1 exists, promoting the chain hybridization of H1 and H2 to form a chain hybridization product, separating from the surface of the graphene oxide, and finally recovering the fluorescence, so that the determination of the content of MUC1 is realized through the fluorescence detection.
Preferably, the MUC1 causes a conformational change in the aptamer upon binding to the aptamer, the aptamer exposes the recognition sequence, and the recognition sequence facilitates chain hybridization of H1 and H2 to form a chain hybridization product.
Preferably, the method comprises the steps of:
1) synthesizing carbon quantum dots;
2) pretreating neck ring DNA H1 and H2;
3) modifying the pretreated neck ring DNA H1 and H2 to a carbon quantum dot;
4) h1 and H2 modified with carbon quantum dots are mixed with graphene oxide;
5) adding MUC1 aptamer into the MUC1 sample to be detected, then adding the mixture obtained in the step 4) into the MUC1 sample to be detected and the MUC1 aptamer mixed solution, and detecting the fluorescence intensity.
Preferably, the step 1) is specifically: firstly, grinding and mixing citric acid and cysteine according to a molar ratio of 2:1, and reacting in a microwave oven for 4min to obtain the carbon quantum dots.
Preferably, the step 2) is specifically: dissolving the neck ring DNA H1, H2 and MUC1 aptamer in a Tris buffer solution, heating the solution to 95 ℃ in a metal bath, keeping the temperature for 5 minutes, and naturally cooling the solution to room temperature; the DNA is then diluted to the desired concentration and stored for future use.
Preferably, the step 3) is specifically: firstly, activating the synthesized carbon quantum dots by EDC and NHS, and shaking for 30 minutes at 25 ℃; then, the end-aminated H1 and H2 were added to the activated carbon quantum dot solution, shaken at room temperature for 2 hours, and then left overnight at 4 ℃ to hydrolyze the unreacted EDC, thereby obtaining modified carbon quantum dots H1 and H2.
Preferably, the step 4) is specifically: and mixing the carbon quantum dots modified by H1 and H2 with graphene oxide, and incubating for 15min at 25 ℃ to quench fluorescence.
Preferably, the step 5) is specifically: adding a proper amount of MUC1 aptamer into a MUC1 sample to be detected, and mixing and incubating for 30 minutes at 37 ℃; then, the mixture obtained in step 4) was added thereto, incubated for 15 minutes, and the fluorescence intensity thereof was measured.
Preferably, the fluorescence intensity detection is performed by using an FLS-1000 fluorescence spectrometer, wherein the excitation wavelength is 355nm, the emission wavelength is 420nm, and the slit widths are 3nm and 4nm respectively.
The invention has the beneficial effects that: the method realizes signal amplification through chain type hybridization reaction, and greatly improves the sensitivity of MUC1 detection; meanwhile, the fluorescent carbon quantum dots synthesized by the method have stable fluorescence property, the stability and the repeatability of the method are greatly improved, and the cost is greatly reduced; in addition, the method is also suitable for detecting MUC1 in complex biological samples such as serum and the like.
Drawings
FIG. 1 is a schematic representation of the MUC1 fluorescence detection principle in examples of the present invention;
FIG. 2 is a graph of the validation results of the feasibility of the test system in examples of the present invention;
FIG. 3 is a graph showing the results of fluorescence intensity measurements of different concentrations of MUC1 in examples of the present invention;
fig. 4 is a graph of interference validation results in embodiments of the present invention.
Detailed Description
The present invention is further described in conjunction with the following examples to enable those skilled in the art to practice the invention in light of the above teachings.
It should be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of or more other elements or combinations thereof.
The trace MUC1 fluorescence detection method based on chain hybridization reaction and fluorescent carbon quantum dots specifically comprises the steps of synthesizing carbon quantum dots with high fluorescence intensity and good stability, modifying neck ring DNA H1 and H2 on the carbon quantum dots, mixing the carbon quantum dots with graphene oxide, adsorbing H1 and H2 modified with the carbon quantum dots to the surface of the graphene oxide when MUC1 does not exist, quenching the fluorescence of the carbon quantum dots due to fluorescence resonance energy transfer effect, combining MUC1 with an aptamer to cause the change of the aptamer conformation when MUC1 exists, exposing an identification sequence from the aptamer, promoting the chain hybridization reaction of H1 and H2 by the identification sequence to form a chain hybridization product, further separating from the surface of the graphene oxide, and finally recovering the fluorescence of the carbon quantum dots, so that the determination of the MUC1 content is realized through fluorescence detection.
The detection limit of the detection system is as low as 10fg/mL through the signal amplification effect mediated by the chain hybridization reaction. The method realizes signal amplification through chain hybridization reaction, and greatly improves the sensitivity of MUC1 detection; meanwhile, the fluorescent carbon quantum dots synthesized by the method have stable fluorescence property, and the stability and the repeatability of the method are greatly improved.
In embodiments, the trace MUC1 fluorescence detection method based on chain hybridization reaction and fluorescent carbon quantum dots specifically comprises the following steps:
1) synthesizing carbon quantum dots with strong fluorescence intensity and stable fluorescence: firstly, fully grinding and mixing citric acid and cysteine according to the molar ratio of 2:1, and reacting in a microwave oven for 4min to obtain the carbon quantum dots with strong fluorescence intensity and stable fluorescence.
2) Pretreatment of neck ring DNA H1 and H2: dissolving the neck ring DNA H1, H2 and MUC1 Aptamer (Aptamer) in a Tris buffer solution, heating the solution to 95 ℃ in a metal bath, keeping the temperature for 5 minutes, and naturally and slowly cooling the solution to room temperature; the DNA is then diluted to the desired concentration and stored for future use.
3) Modifying the pretreated neck ring DNA H1 and H2 to a carbon quantum dot: firstly, activating the synthesized carbon quantum dots by EDC and NHS, and slowly shaking for 30 minutes at 25 ℃; then adding the end aminated H1 and H2 into the activated carbon quantum dot solution, slowly shaking for 2 hours at room temperature, and then standing overnight at 4 ℃ to hydrolyze unreacted EDC, thereby finally obtaining the carbon quantum dots of modified H1 and H2.
4) H1 and H2 modified with carbon quantum dots are mixed with graphene oxide: and mixing the carbon quantum dots modified by H1 and H2 with graphene oxide, and incubating for 15min at 25 ℃ to quench fluorescence.
5) Adding MUC1 aptamer into a MUC1 sample to be detected, then adding the mixture obtained in the step 4) into a MUC1 sample to be detected and a MUC1 aptamer mixed solution, and performing fluorescence intensity detection, wherein in the embodiment, the MUC1 sample to be detected is prepared MUC1 solutions with different concentrations: adding appropriate amount of MUC1 aptamer into MUC1 with different concentrations respectively, and mixing and incubating for 30 minutes at 37 ℃; then, the mixtures obtained in step 4) were added thereto, respectively, and incubated for 15 minutes to measure the fluorescence intensity thereof. The detection results are shown in FIG. 3.
In a preferred embodiment, the fluorescence intensity detection is performed by FLS-1000 fluorescence spectrometer, wherein the excitation wavelength is 355nm, the emission wavelength is 420nm, and the slit width is 3nm and 4nm respectively.
Referring to fig. 1, the principle of fluorescence detection of MUC1 in this embodiment is schematically illustrated, wherein, part of fig. 1a is the principle of carbon dot preparation and carbon dot modification H1, H2, wherein — COOH on the carbon dot is derived from unreacted-COOH on citric acid; FIG. 1b is a schematic representation of MUC1 in part by modification of the carbon sites of H1, H2.
Referring to fig. 2, which is a graph of the verification result of the feasibility of the detection system in this embodiment, the feasibility of the detection system in this embodiment is verified, wherein fig. 2a is a fluorescence spectrum measured by adding 1ng/mL MUC1 into the system; FIG. 2b is a graph of the fluorescence spectrum measured in the absence of MUC1 in the system (with the addition of the same amount of buffer as in FIG. 2 a). The feasibility of the assay system for MUC1 detection can be demonstrated from the figure.
Referring to fig. 3, the fluorescence intensity detection result of step 5 of this embodiment is shown. FIG. 3a is a graph of the fluorescence spectra obtained with different concentrations of
FIG. 4 is a graph showing the results of interference verification of the system of the present invention, wherein FIG. 4a is a comparison graph of fluorescence intensities obtained from MUC1 in a detected amount (10pg/mL) of a target protein relative to an excess (100pg/mL) of other interfering proteins, bovine Serum albumin, human Serum albumin, PDGF-BB, FIG. 4b is a comparison graph of fluorescence intensities of MUC1 in different concentrations in Buffer and serum.error bars indicate relative standard deviations of three independent measurements.FIG. 4a verifies the specificity of the method by using of the excess interfering proteins.A significant difference in fluorescence signals exists between MUC1 assay and control experiment.thus, the experiment results show that the method has high specificity, and the high selectivity of the proposed method is verified by steps.FIG. 4b verifies that the detection method has similar detection effects in samples as in Buffer by detecting the fluorescence intensities of MUC1 in different concentrations in Buffer (Buffer) and Serum (Serum).
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable to various fields of endeavor for which the invention pertains, and further modifications may readily be made thereto by those skilled in the art, and the invention is therefore not limited to the details shown and described without departing from the -generic concept defined by the claims and their equivalents.