CN111778315A - Gold nanoparticle sensor based on hairpin locking deoxyribozyme probe and application of gold nanoparticle sensor in MUC1 detection - Google Patents
Gold nanoparticle sensor based on hairpin locking deoxyribozyme probe and application of gold nanoparticle sensor in MUC1 detection Download PDFInfo
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Abstract
The invention discloses a gold nanoparticle sensor based on a hairpin locking deoxyribozyme probe and application of the gold nanoparticle sensor in detecting MUC1, wherein the gold nanoparticle sensor comprises a MUC1 aptamer probe, a hairpin locking deoxyribozyme probe and gold nanoparticles, and the MUC1 aptamer probe can identify MUC1 and release an aptamer DNA sequence; the hairpin locking deoxyribozyme probe sequentially comprises a first connecting sequence, a first binding sequence, a substrate sequence, a second connecting sequence, a deoxyribozyme sequence and a second binding sequence, wherein the first connecting sequence and the second connecting sequence are complementary to enable the hairpin locking deoxyribozyme probe to form a hairpin structure, the substrate sequence comprises a cutting site, the first binding sequence and the second binding sequence are complementary to an aptamer DNA sequence to enable the deoxyribozyme sequence to form an active secondary structure in a catalytic core, and the 5' end of the hairpin locking deoxyribozyme probe is connected with a fluorescent group capable of being quenched by gold nanoparticles; the 3' end of the hairpin locking deoxyribozyme probe is connected with the gold nanoparticle.
Description
Technical Field
The invention belongs to the technical field of biological analysis, and relates to a gold nanoparticle (AuNPs) sensor based on a hairpin locking deoxyribozyme (DNAzyme) probe and application thereof in detecting MUC 1.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The tumor associated antigen mucin 1(mucin 1, MUC1) is a transmembrane glycoprotein comprising a 31 amino acid hydrophobic transmembrane domain, a 69 amino acid cytoplasmic domain, and an extracellular repeat region consisting of nearly identical repeats (20 amino acids each). MUC1 achieves the mediation of anti-tumor immune responses by activating cytotoxic T-lymphocytes (CTL), killer Human Leukocyte Antigens (HLA) and different types of tumor cells by both Major Histocompatibility Complex (MHC) restricted and non-MHC restricted modes. It is overexpressed in many malignant tumors, such as gastric cancer, breast cancer, pancreatic cancer. In addition, MUC1 was also found in the blood of adenocarcinoma patients, which makes it possible to apply serum analysis of MUC1 for the prevention and early diagnosis of cancer.
In the past, the methods for detecting MUC1 have been dominated by enzyme-linked immunoassays. However, the dependence on antibodies is difficult to overcome, such as instability, complexity and high cost. Ferreira et al screened an anti-MUC 1 DNA aptamer that might recognize a 20 amino acid tandem repeat of a highly immunodominant and novel peptide region. Thus, a number of detection strategies for the combined detection of MUC1 have been established based on aptamer (aptamer) based fluorescence, electrochemical, colorimetric and piezoelectric techniques. However, through the research of the inventor of the invention, the further application of the methods still has the problem of undesirable sensitivity.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a gold nanoparticle sensor based on a hairpin locking deoxyribozyme probe and application thereof in detecting MUC1, wherein the gold nanoparticle sensor has higher sensitivity and good reproducibility for the detection of MUC 1.
In order to achieve the purpose, the technical scheme of the invention is as follows:
in one aspect, a gold nanoparticle sensor based on a hairpin-locked deoxyribozyme probe comprises a MUC1 aptamer probe, a hairpin-locked deoxyribozyme probe and a gold nanoparticle;
the MUC1 aptamer probe comprises an aptamer DNA sequence and a hairpin DNA sequence which are sequentially connected, wherein the aptamer DNA sequence and the hairpin DNA sequence form a hairpin structure, and the MUC1 aptamer probe can recognize MUC1 and release the aptamer DNA sequence;
the hairpin locking deoxyribozyme probe is a single-stranded DNA sequence and sequentially comprises a first connecting sequence, a first binding sequence, a substrate sequence, a second connecting sequence, a deoxyribozyme sequence and a second binding sequence from 5' end to 3', the first connecting sequence and the second connecting sequence are complementary to enable the hairpin locking deoxyribozyme probe to form a hairpin structure, the substrate sequence comprises a cutting site, the first binding sequence and the second binding sequence can be complementary to an aptamer DNA sequence, the first binding sequence and the second binding sequence can enable the deoxyribozyme sequence to form an active secondary structure in a catalytic core after being complementary to the aptamer DNA sequence, and the 5' end of the hairpin locking deoxyribozyme probe is connected with a fluorescent group capable of being quenched by gold nanoparticles; the 3' end of the hairpin locking deoxyribozyme probe is connected with the gold nanoparticle.
In the absence of MUC1, the hairpin-locked deoxyribozyme probe can form a hairpin structure through intramolecular hybridization, so that the catalytic activity of a DNAzyme chain is inhibited, and FAM fluorescence can be effectively quenched by AuNPs. In the presence of MUC1, firstly opening a MUC1 aptamer probe, releasing a probe complex containing an aptamer DNA sequence, wherein the probe complex can open a hairpin structure of a hairpin locking deoxyribozyme probe, form an active secondary structure in a catalytic core, and perform self-cleavage under the action of magnesium ions as a coenzyme, so that the gold nanoparticles release a fluorescent group, and then recover fluorescence.
In another aspect, the gold nanoparticle sensor based on the hairpin locking deoxyribozyme probe is applied to detection of MUC 1.
In a third aspect, a method for detecting MUC1 is provided, the gold nanoparticle sensor based on the hairpin locking deoxyribozyme probe is provided, MUC1 in a sample to be detected is combined with the MUC1 aptamer probe, a probe complex containing an aptamer DNA sequence is released, the probe complex opens a hairpin structure of the hairpin locking deoxyribozyme probe, an active secondary structure is formed in a catalytic core, a substrate sequence is subjected to self-cleavage under the action of magnesium ions as a coenzyme, a fluorescent group is separated from the gold nanoparticle, and fluorescence detection is performed on the separated fluorescent group.
In a fourth aspect, the detection kit comprises the gold nanoparticle sensor based on the hairpin locking deoxyribozyme probe, a magnesium salt and a buffer solution.
The invention has the beneficial effects that:
1. the invention designs a gold nanoparticle sensor based on a hairpin locking deoxyribozyme probe for detecting tumor-associated antigen mucin 1, AuNPs serve as a platform to support hairpin locking DNAzyme to form a three-dimensional orbit, and the high-density combination of the DNAzyme on the surface of the AuNPs enhances the movement of an aptamer molecular beacon in the three-dimensional orbit. The fluorescent group is released from the AuNPs surface, and the fluorescence is enhanced. The method can enable the aptamer molecular beacon to move along the AuNPs autonomously, and the signal is amplified continuously.
2. The gold nanoparticle sensor based on the hairpin locking deoxyribozyme probe provided by the invention has high sensitivity and good specificity; MUC1 induces fluorescence signal amplification, which greatly improves the sensitivity of the method and simplifies the procedure. Only MUC1 can be recognized and combined by MUC1 aptamer probe to trigger DNAzyme self-cleavage reaction, so that the high specificity of the method is ensured.
2. The gold nanoparticle sensor based on the hairpin locking deoxyribozyme probe provided by the invention can be used for sensitively detecting MUC1 in an actual sample, the detection limit of the method can reach 0.36fg/mL, and the gold nanoparticle sensor has good reproducibility in the serum of the actual sample.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a schematic diagram of embodiment 1 of the present invention;
FIG. 2 is a representation of AuNPs and hairpin-locked DNAzyme functionalized AuNPs of example 1 of the present invention, wherein A is a Transmission Electron Microscopy (TEM) image of AuNPs, and B is an ultraviolet absorption spectrum of hairpin-locked DNAzyme functionalized AuNPs;
FIG. 3 is a schematic diagram showing the results of the principle verification of example 1 of the present invention, wherein A is the analysis of the reaction products under different experimental conditions by native polyacrylamide gel electrophoresis, lanes 1 and 2, 3 represent the cleavage reaction products in the presence and absence of MUC1, respectively, and B is a fluorescence spectrum;
FIG. 4 is a graph showing the results of the sensitivity detection in example 1 of the present invention, wherein A is a fluorescence spectrum (the concentration of the curve from bottom to top is 0, 1fg/mL, 10fg/mL, 100fg/mL, 1pg/mL, 10pg/mL, 100pg/mL, 1ng/mL), and B is a standard curve of fluorescence intensity versus concentration;
FIG. 5 is a diagram showing the result of specific detection in example 1 of the present invention, wherein A is a fluorescence spectrum and B is a histogram of fluorescence intensity.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
In view of the problem of poor sensitivity of the existing MUC1 detection, the invention provides a gold nanoparticle sensor based on a hairpin locking deoxyribozyme probe and application of the gold nanoparticle sensor in MUC1 detection.
The invention provides a gold nanoparticle sensor based on a hairpin-locked deoxyribozyme probe, which comprises a MUC1 aptamer probe, a hairpin-locked deoxyribozyme probe and gold nanoparticles;
the MUC1 aptamer probe comprises an aptamer DNA sequence and a hairpin DNA sequence which are sequentially connected, wherein the aptamer DNA sequence and the hairpin DNA sequence form a hairpin structure, and the MUC1 aptamer probe can recognize MUC1 and release the aptamer DNA sequence;
the hairpin locking deoxyribozyme probe is a single-stranded DNA sequence and sequentially comprises a first connecting sequence, a first binding sequence, a substrate sequence, a second connecting sequence, a deoxyribozyme sequence and a second binding sequence from 5' end to 3', the first connecting sequence and the second connecting sequence are complementary to enable the hairpin locking deoxyribozyme probe to form a hairpin structure, the substrate sequence comprises a cutting site, the first binding sequence and the second binding sequence can be complementary to an aptamer DNA sequence, the first binding sequence and the second binding sequence can enable the deoxyribozyme sequence to form an active secondary structure in a catalytic core after being complementary to the aptamer DNA sequence, and the 5' end of the hairpin locking deoxyribozyme probe is connected with a fluorescent group capable of being quenched by gold nanoparticles; the 3' end of the hairpin locking deoxyribozyme probe is connected with the gold nanoparticle.
In the absence of MUC1, the hairpin-locked deoxyribozyme probe can form a hairpin structure through intramolecular hybridization, so that the catalytic activity of a DNAzyme chain is inhibited, and FAM fluorescence can be effectively quenched by AuNPs. In the presence of MUC1, firstly opening a MUC1 aptamer probe, releasing a probe complex containing an aptamer DNA sequence, wherein the probe complex can open a hairpin structure of a hairpin locking deoxyribozyme probe, form an active secondary structure in a catalytic core, and perform self-cleavage under the action of magnesium ions as a coenzyme, so that the gold nanoparticles release a fluorescent group, and then recover fluorescence.
In some embodiments of this embodiment, the 3' end of the hairpin-locked deoxyribozyme probe is linked to the gold nanoparticle by an S-Au bond.
In some embodiments of this embodiment, the second joining sequence is poly-T.
In some embodiments of this embodiment, the fluorophore is FAM.
In some embodiments of this embodiment, the MUC1 aptamer probe has the sequence: GGT CCC ATA GGTTTC CTA GTT GAC GAC CTA TGG GAC CGGTGT GCT
The sequence of the hairpin-locked deoxyribozyme probe is as follows:
CAAAAAAGC ACA CCG GCT rAGT CTT TTT TTT TGA TCCGAG CCG GAC GAA GCC CCATAG GT。
in another embodiment of the invention, the application of the gold nanoparticle sensor based on the hairpin locking deoxyribozyme probe in the detection of MUC1 is provided.
The application of the invention in detecting MUC1 aims at diagnosis and treatment of non-diseases.
In a third embodiment of the present invention, a method for detecting MUC1 is provided, where MUC1 in a sample to be detected is combined with a MUC1 aptamer probe to release a probe complex containing an aptamer DNA sequence, the probe complex opens a hairpin structure of the hairpin locking deoxyribozyme probe and forms an active secondary structure in a catalytic core, and a substrate sequence is self-cleaved under the action of magnesium ions as a coenzyme, such that a fluorophore is separated from the gold nanoparticle, and the separated fluorophore is subjected to fluorescence detection.
The method of detecting MUC1 of the present invention is aimed at diagnosis and treatment of non-diseases.
In some embodiments of this embodiment, the process of forming the hairpin structure is: contacting the probe with a solution containing Tris-HCl and MgCl2The hybridization buffer solution is uniformly mixed, subjected to denaturation reaction in a water bath kettle at 90-100 ℃, and then cooled to room temperature. The probe is a MUC1 aptamer probe or a hairpin locking deoxyribozyme probe.
In some embodiments of this embodiment, the steps are: adding the MUC1 aptamer probe into a sample to be detected, incubating at 36-38 ℃, then adding the gold nanoparticles and the magnesium salt connected with the MUC1 aptamer probe, continuing incubating at 36-38 ℃, and then performing fluorescence detection.
In one or more embodiments, the incubation is continued followed by dilution and then fluorescence detection.
In one or more embodiments, the MUC1 aptamer probe is reacted in the sample to be tested for 10-20 min.
In one or more embodiments, the incubation is continued for 80-100 min after adding the gold nanoparticles linked to the MUC1 aptamer probe and the magnesium salt.
The magnesium salt in the present invention refers to a compound having a divalent magnesium ion as a cation, and examples thereof include magnesium nitrate, magnesium chloride, and magnesium sulfate. The effect of adopting magnesium chloride is better.
According to a fourth embodiment of the invention, a detection kit is provided, which comprises the gold nanoparticle sensor based on the hairpin locking deoxyribozyme probe, a magnesium salt and a buffer solution.
In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the technical solution of the present invention will be described in detail below with reference to specific examples and comparative examples.
Example 1
Preparation of hairpin-locked DNAzyme modified AuNPs 1mL of AuNPs solution (5.7 × 10)12particles/mL) was added 6nmol of hairpin-locked DNAzyme probe (FAM-CAAAAAAGC ACA CCG GCT rAGT CTT TTT TTT TGATCCGAG CCG GAC GAA GCC CCA TAG GT-SH, sequence shown in SEQ ID NO. 1), incubated at room temperature for 16h, and then incubated in the presence of 10mM phosphate (NaH)2PO4/Na2HPO4) And 0.1M NaCl in PBS buffer (pH 7.0) and left standing at room temperature for another 36 hours. The hairpin-locked DNAzyme probes not assembled on the surface of the AuNPs were then removed by three high-speed centrifugation (13000rpm, 30min) of the mixture of AuNPs and DNAzyme, and the resulting pellet was resuspended in 250. mu.L of PBS buffer and finally stored at 4 ℃ until use. In the hairpin-locked DNAzyme probe-AuNPs solution, the concentration of the hairpin-locked DNAzyme probe was about 18. mu.M.
Mg2+Amplification of self-cleavage cycle signal by the dependent hairpin-locked DNAzyme probe: the synthesized DNA oligonucleotide sequences were dissolved in Tris-EDTA buffer. Simultaneous aptamer probe (GGT CCC ATA GGT TTC CTA GTT GAC GAC CTATGG GAC CGGTGT GCT)The sequence is shown as SEQ ID NO. 2) and the hairpin locked DNAzyme probe was diluted to 1. mu.M with ultrapure water. With 10mM Tris-HCl (pH 8.0) and 1.5mM MgCl2The hybridization buffer solution is mixed uniformly, and denaturation reaction is carried out for 5min in a water bath kettle at the temperature of 95 ℃, and then the mixture is slowly cooled to room temperature to form a hairpin structure. mu.L of aptamer probe was added to a recognition binding reaction (25. mu.L) of MUC1 polypeptide and aptamer probe, containing different concentrations of MUC1 polypeptide. Incubation with 37 ℃ for 15min, 1.5. mu.L hairpin-locked DNAzyme modified AuNPs and MgCl2Adding into a reaction system, reacting for 90min at 37 ℃ to perform Mg2+The self-cleavage of the dependent hairpin-locked DNAzyme probe induces recovery of quenching fluorescence by AuNPs. The reaction product (30. mu.L) was diluted with ultrapure water to 60. mu.L, and the fluorescence spectrum was measured at room temperature using a fluorescence spectrophotometer.
Gel electrophoresis analysis: the reaction products were analyzed by electrophoresis on a 12% native polyacrylamide gel in the presence and absence of MUC1, respectively.
And (3) specific analysis: 50nM cytochrome C, 50nM AFP, 50nM CEA and 1ng/mL MUC1 were used to explore the specificity of this approach.
Analysis of the actual samples: mu.L of the reaction system (containing different concentrations of MUC1 polypeptide, 5% human serum, 2. mu.M aptamer probe, 1.5. mu.L hairpin-locked DNAzyme modified AuNPs) was reacted at 37 ℃ for 90min, and then fluorescence detection was performed in accordance with the above method.
The principle is shown in figure 1: in the embodiment, a hairpin-locked DNAzyme probe is designed, and a Sulfhydryl (SH) group modified at the 3' end of the hairpin-locked DNAzyme probe can be covalently bonded on the surface of AuNPs through S-Au to form a nanostructure based on the hairpin-locked DNAzyme probe. The hairpin locked DNAzyme probe is modified with FAM fluorescent group at the 5' end and consists of 4 basic structural parts including a binding sequence with aptamer probe and Mg2+Dependent 10-23DNAzyme sequences, substrate sequences comprising cleavage sites and linker sequences. The length of the sequence that binds to the aptamer probe should be considered when designing hairpin-locked DNAzyme probes, as it affects the activation of dnazymes and the recovery of aptamer probes. 9+9 binding arms were selected. Linker sequence consisting of poly-T for the ligation of 10-23DNAzyme and its substrateLinked together and hybridized with poly-A to form a hairpin structure. In the absence of MUC1, hairpin-locked DNAzyme probes can form hairpin structures by intramolecular hybridization, thereby inhibiting the catalytic activity of DNAzyme strands, and FAM fluorescence can be efficiently quenched by AuNPs. In the presence of MUC1, the aptamer probe hairpin structure can be opened to form a MUC1-aptamer probe complex with exposed DNA fragments as hybridized to the hairpin-locked DNAzyme probe, and the MUC1-aptamer probe complex can open the hairpin-locked DNAzyme and form an active secondary structure in the catalytic core to produce an "active" DNAzyme, which is then treated with Mg2+Self-cleavage of the ribonucleotide moiety with the aid of (a). Due to the lower binding force, the two shorter fragments formed by cleavage were separated from the MUC1-aptamer probe complex and the fluorophore on the AuNPs was released to restore their fluorescence. Meanwhile, the MUC1-aptamer probe complex is released and combined with the adjacent hairpin-locked DNAzyme to participate in more activation, cleavage and release cycles, so that the cyclic utilization of the MUC1-aptamer probe complex is realized, the fluorescence signal is obviously enhanced, and the detection sensitivity is very high.
Experimental verification of principle
As shown in FIG. 2A, AuNPs have an average diameter of about 10nm and thus have good dispersibility in aqueous solutions. In this example, ultraviolet absorption spectra of AuNPs and hairpin-locked DNAzyme probe-modified AuNPs were measured by an ultraviolet absorption spectrophotometer, respectively. As shown in FIG. 2B, the ultraviolet absorption spectrum of AuNPs at water temperature is red-shifted from 519nm to 524nm, and the result shows that the hairpin-locked DNAzyme probe is successfully connected with AuNPs to realize the functionalization of the AuNPs.
This example first validates the design using a 12% native polyacrylamide gel. The results show that upon addition of aptamer probe-MUC1, self-cleavage products of hairpin-locked DNAzyme can be observed (fig. 3A), whereas no cleavage is observed in the absence of the target MUC 1. Indicating that after hybridization, the target acts as an allosteric effector, with Mg2+The homogeneous DNAzyme chain conformation is triggered to switch to the active DNAzyme structure responsible for cleavage in a dependent manner and specific cleavage capacity is demonstrated. By adding different concentrations of miR141 in vitro target binding studies of the probes were performed. The results in fig. 3B show that in the presence of MUC1, FAM fluorescence signals with a characteristic emission peak of 520nm were observed (fig. 3B), in contrast to the absence of MUC1, where no significant FAM fluorescence signals were observed (fig. 3B). These results clearly show that the nanosensor designed in this example can be used to detect MUC 1. In conclusion, the technical scheme is completely feasible.
Sensitivity detection
The sensitivity of the sensor was investigated by measuring the fluorescence signal of FAM at different concentrations of MUC1, as shown in fig. 4. As shown in FIG. 4A, the fluorescence intensity of FAM increased as the concentration of MUC1 increased from 1fg/mL to 1 ng/mL. Meanwhile, in order to evaluate the detection sensitivity of the sensor, in the embodiment, after the logarithm of the concentration of MUC1 is taken, a good linear relationship between the logarithm of the concentration and the fluorescence intensity can be observed in the concentration range of 1fg/mL to 1 ng/mL. As shown in fig. 4B, the linear correlation equation is F5671.92 +932.227log 10C (R)20.9847) where F and C represent the fluorescence intensity of FAM and the concentration of MUC1 (ng/mL), respectively. After calculation (defined as 3 σ/K, where σ is the standard deviation of the blank sample and K is the slope of the linear regression equation), the lower limit of detection for this detection method is 0.36 fg/mL. Compared with the existing electrochemical luminescence detection method (2.80fg/mL), the sensitivity of the method is improved by at least 1 order of magnitude; compared with a fluorescence analysis method of 75 mu g/mL, the method has the advantage that the sensitivity is improved by 8 orders of magnitude.
Specificity detection
To evaluate the selectivity of the method, this example used Cytochrome C (Cytochrome C, concentration 50nM), alpha-fetoprotein (AFP, concentration 50nM) and carcinoembryonic antigen (CEA, concentration 50nM) as non-specific proteins. These three proteins were all proteins not relevant to this experiment. Theoretically, none of these proteins could be recognized by aptamer probes to bind to initiate a DNAzyme self-cleavage reaction. As shown in FIG. 5, no significant FAM fluorescence signal was detected under the same reaction conditions corresponding to cytochrome C, AFP, CEA and the control group. Conversely, an enhanced FAM fluorescence signal was detected corresponding to MUC1 (concentration 1 ng/mL). These results demonstrate that the method is very specific for the detection of MUC 1.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> university of Shandong Master
<120> gold nanoparticle sensor based on hairpin locking deoxyribozyme probe and application thereof in MUC1 detection
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Claims (10)
1. A gold nanoparticle sensor based on a hairpin locking deoxyribozyme probe is characterized by comprising a MUC1 aptamer probe, a hairpin locking deoxyribozyme probe and gold nanoparticles;
the MUC1 aptamer probe comprises an aptamer DNA sequence and a hairpin DNA sequence which are sequentially connected, wherein the aptamer DNA sequence and the hairpin DNA sequence form a hairpin structure, and the MUC1 aptamer probe can recognize MUC1 and release the aptamer DNA sequence;
the hairpin locking deoxyribozyme probe is a single-stranded DNA sequence and sequentially comprises a first connecting sequence, a first binding sequence, a substrate sequence, a second connecting sequence, a deoxyribozyme sequence and a second binding sequence from 5' end to 3', the first connecting sequence and the second connecting sequence are complementary to enable the hairpin locking deoxyribozyme probe to form a hairpin structure, the substrate sequence comprises a cutting site, the first binding sequence and the second binding sequence can be complementary to an aptamer DNA sequence, the first binding sequence and the second binding sequence can enable the deoxyribozyme sequence to form an active secondary structure in a catalytic core after being complementary to the aptamer DNA sequence, and the 5' end of the hairpin locking deoxyribozyme probe is connected with a fluorescent group capable of being quenched by gold nanoparticles; the 3' end of the hairpin locking deoxyribozyme probe is connected with the gold nanoparticle.
2. The gold nanoparticle sensor according to claim 1, wherein the 3' end of the hairpin-locked deoxyribozyme probe is connected with the gold nanoparticle through an S-Au bond.
3. The hairpin-locked deoxyribozyme probe-based gold nanoparticle sensor according to claim 1, wherein the second linker sequence is poly-T.
4. The hairpin-locked deoxyribozyme probe-based gold nanoparticle sensor according to claim 1, wherein the fluorophore is FAM.
5. The hairpin locked deoxyribozyme probe-based gold nanoparticle sensor according to claim 1, wherein the sequence of the MUC1 aptamer probe is: GGT CCC ATA GGT TTC CTA GTT GAC GAC CTA TGG GACCGGTGT GCT, respectively;
the sequence of the hairpin-locked deoxyribozyme probe is as follows: CAAAAAAGC ACA CCG GCT rAGT CTT TTT TTT TGATCCGAG CCG GAC GAA GCC CCA TAG GT.
6. Use of the gold nanoparticle sensor based on the hairpin-locked deoxyribozyme probe according to any one of claims 1 to 5 for detecting MUC 1.
7. A method for detecting MUC1, which is characterized in that the gold nanoparticle sensor based on the hairpin locking deoxyribozyme probe as claimed in any one of claims 1 to 5 is provided, MUC1 in a sample to be detected is combined with a MUC1 aptamer probe, a probe complex containing an aptamer DNA sequence is released, the probe complex opens a hairpin structure of the hairpin locking deoxyribozyme probe and forms an active secondary structure in a catalytic core, a substrate sequence is subjected to self-cleavage under the action of magnesium ions as a coenzyme, a fluorescent group is separated from the gold nanoparticle, and the separated fluorescent group is subjected to fluorescence detection.
8. The method of claim 7 for detecting MUC1, wherein the hairpin formation is performed by: contacting the probe with a solution containing Tris-HCl and MgCl2The hybridization buffer solution is uniformly mixed, subjected to denaturation reaction in a water bath kettle at 90-100 ℃, and then cooled to room temperature.
9. The method of detecting MUC1 of claim 7, further comprising the steps of: adding the MUC1 aptamer probe into a sample to be detected, incubating at 36-38 ℃, then adding the gold nanoparticles and the magnesium salt connected with the MUC1 aptamer probe, continuing incubating at 36-38 ℃, and then performing fluorescence detection.
10. A detection kit, which is characterized by comprising the gold nanoparticle sensor based on the hairpin locking deoxyribozyme probe as claimed in any one of claims 1 to 5, a magnesium salt and a buffer solution.
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