CN110734960B - Trace MUC1 fluorescence detection method based on chain type hybridization reaction and fluorescent carbon quantum dots - Google Patents
Trace MUC1 fluorescence detection method based on chain type hybridization reaction and fluorescent carbon quantum dots Download PDFInfo
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N2333/4701—Details
- G01N2333/4725—Mucins, e.g. human intestinal mucin
Abstract
The invention discloses a trace MUC1 fluorescence detection method based on chain type hybridization reaction and fluorescent carbon quantum dots, which specifically comprises the following steps: firstly, synthesizing carbon quantum dots, then modifying the neck ring DNAs H1 and H2 to the carbon quantum dots, and then mixing the carbon quantum dots with graphene oxide; when MUC1 does not exist, H1 and H2 modified with carbon quantum dots are adsorbed to the surface of graphene oxide, and the fluorescence of the carbon quantum dots is quenched due to the fluorescence resonance energy transfer effect; when MUC1 exists, the MUC1 is combined with the aptamer, H1 and H2 are promoted to perform chain type hybridization reaction to form a chain type hybridization product, the chain type hybridization product is separated from the surface of the graphene oxide, and finally the fluorescence of the carbon quantum dots is recovered; thereby realizing the determination of the MUC1 content through fluorescence detection. 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 properties, and the stability and the repeatability of the method are greatly improved.
Description
Technical Field
The invention relates to the field of trace MUC1 detection, in particular to a trace MUC1 fluorescence detection method based on chain type hybridization reaction and fluorescent carbon quantum dots.
Background
Mucins (MUC 1) are a class of high molecular weight, highly glycosylated proteins that are present in epithelial cells and form an integral transmembrane domain through the gel matrix. Mucin (MUC 1) is one of cell surface glycoproteins, and the polypeptide skeleton thereof consists of 3 parts of an extracellular segment, a transmembrane segment and an intracellular segment, wherein the transmembrane segment and the intracellular segment respectively contain 31 and 69 amino acids; the extracellular domain contains multiple consecutive repeats, each of which contains 20 amino acids. MUC1 plays an important role in the signal transduction process, and in addition, MUC1 can down-regulate the expression of E-cadherin, which is a calcium ion-dependent cell adhesion molecule, mediates the binding between cells and plays an inhibiting role in tumor metastasis, and the down-regulation of the expression of the E-cadherin is one of the steps of enhancing the invasiveness of tumor cells. MUC1 is highly expressed in human epithelial cell adenocarcinomas such as breast cancer, gastric cancer, lung cancer, prostate cancer, ovarian cancer and pancreatic cancer, and its presence in blood can also be detected to some extent. Therefore, MUC1 can be used as an effective tumor early diagnosis marker, and the realization of quick and sensitive detection of the marker is of great significance for early diagnosis of tumors. At present, the traditional MUC1 detection method mainly comprises an enzyme-linked immunosorbent assay, an electrochemical method, a mass spectrometry method, a colorimetric method and a fluorescence method. However, the conventional method has problems of low sensitivity, complicated operation, and the like.
Disclosure of Invention
The invention aims to solve the technical problem of providing a trace MUC1 fluorescence detection method 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: a trace MUC1 fluorescence detection method based on chain type hybridization reaction and fluorescent carbon quantum dots comprises the following steps: firstly, synthesizing carbon quantum dots, then modifying neck ring DNAs H1 and H2 to the carbon quantum dots, and mixing the carbon quantum dots with graphene oxide; when MUC1 does not exist, H1 and H2 modified with carbon quantum dots are adsorbed to the surface of graphene oxide, and the fluorescence of the carbon quantum dots is quenched due to the fluorescence resonance energy transfer effect; when MUC1 exists, the MUC1 is combined with the aptamer, and H1 and H2 are promoted to generate chain type hybridization to form a chain type hybridization product, the chain type hybridization product is separated from the surface of the graphene oxide, and finally the fluorescence is recovered; thereby realizing the determination of the MUC1 content through fluorescence detection.
Preferably, 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 promotes the H1 and H2 to perform a chain hybridization reaction 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, citric acid and cysteine are ground and mixed according to the molar ratio of 2:1, and react for 4min in a microwave oven 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 to 95 ℃ in a metal bath, keeping for 5 minutes, and naturally cooling 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 ℃; and then adding the H1 and H2 with the aminated ends into the activated carbon quantum dot solution, shaking for 2 hours at room temperature, and then standing overnight at 4 ℃ to hydrolyze unreacted EDC, thereby finally obtaining the carbon quantum dots for modifying 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 beneficial effects of the invention are: the method of the invention realizes the amplification of signals through chain type hybridization reaction, thereby greatly improving 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 diagram of the MUC1 fluorescence detection principle in an embodiment of the present invention;
FIG. 2 is a graph illustrating the validation of the feasibility of the assay system in an embodiment of the present invention;
FIG. 3 is a graph showing the results of fluorescence intensity measurements for different concentrations of MUC1 in an embodiment of the present invention;
fig. 4 is a diagram of interference verification results in an embodiment of the invention.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
The method for detecting trace MUC1 fluorescence based on chain hybridization reaction and fluorescent carbon quantum dots comprises the following steps: firstly, synthesizing carbon quantum dots with high fluorescence intensity and good stability, then modifying neck ring DNAs H1 and H2 onto the carbon quantum dots, and mixing the carbon quantum dots with graphene oxide; when MUC1 does not exist, H1 and H2 modified with carbon quantum dots are adsorbed to the surface of graphene oxide, and the fluorescence of the carbon quantum dots is quenched due to the fluorescence resonance energy transfer effect; when MUC1 exists, the MUC1 is combined with the aptamer to cause the change of the aptamer conformation, the aptamer exposes the recognition sequence, the recognition sequence promotes H1 and H2 to generate chain type hybridization reaction to form a chain type hybridization product, and then the chain type hybridization product is separated from the surface of the graphene oxide, and finally the fluorescence of the carbon quantum dots is recovered; thereby realizing the determination of the MUC1 content 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 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, and the stability and the repeatability of the method are greatly improved.
In one embodiment, 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 a 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 carbon quantum dots: firstly, activating the synthesized carbon quantum dots by EDC and NHS, and slowly shaking for 30 minutes at 25 ℃; and then adding the H1 and H2 with the aminated ends 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 for modifying 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 in advance with MUC1 solutions with different concentrations: respectively adding appropriate amount of MUC1 aptamer into MUC1 with different concentrations, 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, which is a schematic diagram of the fluorescence detection principle of MUC1 in this embodiment, wherein, a part in fig. 1a is a schematic diagram of the carbon dot preparation and carbon dot modification of H1 and H2, wherein — COOH on a carbon dot is derived from unreacted-COOH on citric acid; FIG. 1b is a schematic diagram showing the principle of MUC1 by modifying the carbon sites of H1 and H2.
Referring to fig. 2, a result diagram of verifying the feasibility of the detection system in this embodiment is shown, and this embodiment verifies the feasibility of the detection system, where a curve a in fig. 2 is a fluorescence spectrum measured by adding 1ng/mL MUC1 to the system; curve b in FIG. 2 is the fluorescence spectrum measured in the absence of MUC1 in the system (with the addition of the same amount of buffer as in curve a in FIG. 2). 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 fluorescence spectra obtained with different concentrations of MUC 1. The concentrations of MUC1 solutions with different concentrations to be detected are respectively 10fg/mL,100fg/mL,1pg/mL,10pg/mL,100pg/mL,1ng/mL and 10ng/mL; the curves are that the wave crests in the graph correspond from bottom to top in sequence. FIG. 3b is a linear-scale statistical plot of fluorescence intensity for MUC1 at different concentrations. The target protein MUC1 is combined with the aptamer to cause the change of the aptamer conformation, thereby exposing the recognition sequence, promoting the chain type hybridization reaction and recovering the fluorescence. The content of MUC1 can be estimated by the amount of change in fluorescence intensity measured by a fluorescence spectrometer. FIG. 3a shows the fluorescence spectra obtained for different concentrations of MUC 1. The fluorescence signal intensity gradually increases with increasing MUC1 concentration within a certain range. As shown in FIG. 3b, the fluorescence intensity is linear with the logarithm of MUC1 concentration in the range of 10fg/mL to 100 pg/mL. The regression equation is y =3527x +14721 (R) 2 =0.997, n = 3), wherein y is the fluorescence signal intensity value and x is the logarithm of the MUC1 concentration.
Fig. 4 is a diagram showing the interference verification result of the system of the present invention. FIG. 4a shows the measured amount of the target protein MUC1 (10 pg/mL) relative to the other interfering proteins in excess (100 pg/mL): fluorescence intensity contrast images obtained from bovine serum albumin, human serum albumin, PDGF-BB; FIG. 4b is a graph showing the comparison of fluorescence intensity of MUC1 at different concentrations in buffer and serum. Error bars represent the relative standard deviation of three independent measurements. FIG. 4a verifies the specificity of the method by using some excess of interfering proteins. There was a significant difference in fluorescence signal between the MUC1 assay and the control experiment. Therefore, experimental results show that the method has high specificity, and further confirms the high selectivity of the proposed method. FIG. 4b demonstrates that the detection method has similar detection effect in Serum samples and Buffer solution by detecting the fluorescence intensity of MUC1 in Buffer solution (Buffer) and Serum (Serum) at different concentrations.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Claims (3)
1. The application of the trace MUC1 fluorescence detection method based on chain type hybridization reaction and fluorescent carbon quantum dots in the preparation of the sensor is characterized in that the method specifically comprises the following steps: firstly, synthesizing carbon quantum dots, then modifying neck ring DNAs H1 and H2 to the carbon quantum dots, and mixing the carbon quantum dots with graphene oxide; when MUC1 does not exist, H1 and H2 modified with carbon quantum dots are adsorbed to the surface of graphene oxide, and the fluorescence of the carbon quantum dots is quenched due to the fluorescence resonance energy transfer effect; when MUC1 exists, the MUC1 is combined with the aptamer, H1 and H2 are promoted to generate chain type hybridization reaction to form a chain type hybridization product, the chain type hybridization product is separated from the surface of the graphene oxide, and finally the fluorescence is recovered; thereby realizing the determination of the MUC1 content through fluorescence detection;
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;
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 carbon quantum dots;
the step 2) is specifically as follows: dissolving the neck ring DNA H1, H2 and MUC1 aptamer in a Tris buffer solution, heating to 95 ℃ in a metal bath, keeping for 5 minutes, and naturally cooling to room temperature; then diluting the DNA to the required concentration, and storing for later use;
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 ℃; adding the H1 and H2 with aminated ends into the activated carbon quantum dot solution, shaking for 2 hours at room temperature, standing overnight at 4 ℃ to hydrolyze unreacted EDC, and finally obtaining carbon quantum dots for modifying H1 and H2;
the step 4) is specifically as follows: mixing the carbon quantum dots modified by H1 and H2 with graphene oxide, and incubating for 15min at 25 ℃ to quench fluorescence;
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 was measured.
2. The use of the method for detecting trace MUC1 fluorescence based on chain hybridization and fluorescent carbon quantum dots according to claim 1 in the preparation of sensors, wherein the combination of MUC1 and aptamers causes the conformational change of aptamers, the aptamers expose the recognition sequences, and the recognition sequences promote H1 and H2 to perform chain hybridization to form chain hybridization products.
3. The use of the chain hybridization reaction and fluorescent carbon quantum dot-based trace MUC1 fluorescence detection method in the preparation of sensors according to claim 2, wherein the fluorescence intensity detection adopts an FLS-1000 fluorescence spectrometer, wherein the excitation wavelength is 355nm, the emission wavelength is 420nm, and the slit width is 3nm and 4nm respectively.
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| Fluorescent detection of Mucin 1 protein based on aptamer functionalized biocompatible carbon dots and graphene oxide;Yanjun Ding等;《Anal. Methods》;20150630;第7792-7798页 * |
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