CN111778315B - 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-locked deoxyribozyme probe and application thereof in detecting MUC1, wherein the gold nanoparticle sensor comprises a MUC1 aptamer probe, the hairpin-locked 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 MUC1 detection.
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, muc 1) 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 almost identical repeats (20 amino acids each for re-reading). MUC1 activates cytotoxic T-lymphocytes (CTL) and Human Leukocyte Antigen (HLA) different tumor cells by a Major Histocompatibility Complex (MHC) restriction mode and a non-MHC restriction mode, so as to mediate anti-tumor immune response. It is overexpressed in many malignant tumors, such as gastric cancer, breast cancer, pancreatic cancer. Furthermore, MUC1 is also found in the blood of adenocarcinoma patients, which makes it possible to apply the serum analysis of MUC1 to the prevention and early diagnosis of cancer.
In the past, the methods for detecting MUC1 have been dominated by several 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 could recognize 20 amino acid tandem repeats of a highly immunodominant and novel peptide region. Therefore, a large number of detection strategies for detecting MUC1 by combining fluorescence, electrochemistry, colorimetry and piezoelectric technologies based on aptamers (aptamers) are established at present. 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-locked deoxyribozyme probe and an application thereof in detecting MUC1, wherein the gold nanoparticle sensor has higher sensitivity and good repeatability for the detection of MUC1.
In order to achieve the purpose, the technical scheme of the invention is as follows:
on one hand, the gold nanoparticle sensor based on the hairpin locking deoxyribozyme probe comprises a MUC1 aptamer probe, the 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.
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 compound containing an aptamer DNA sequence, opening a hairpin structure of a hairpin locking deoxyribozyme probe in the probe compound, forming an active secondary structure in a catalytic core, and performing self-cleavage under the action of magnesium ions as a coenzyme, so that the gold nanoparticles release a fluorescent group, and then recovering fluorescence.
In another aspect, the gold nanoparticle sensor based on the hairpin locking deoxyribozyme probe is applied to the detection of MUC1.
In a third aspect, a method for detecting MUC1 is provided, wherein 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 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.
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 beneficial effects of the invention are as follows:
1. the invention designs a gold nanoparticle sensor based on a hairpin locking deoxyribozyme probe, which is used for detecting tumor-associated antigen mucin 1, and 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 operation process. Only MUC1 can be identified and combined by the MUC1 aptamer probe to further 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 included to illustrate an exemplary embodiment of the invention and not to limit the invention.
FIG. 1 is a schematic view 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 Microscope (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 result of the principle verification of example 1 of the present invention, wherein A is the analysis of the reaction product under different experimental conditions by native polyacrylamide gel electrophoresis, lanes 1 and 2, 3 represent the cleavage reaction product 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, 1 ng/mL), and B is a standard curve of fluorescence intensity versus concentration;
FIG. 5 is a graph 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 thereof 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-locked 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 the 5' end to the 3', the hairpin-locked deoxyribozyme probe can form a hairpin structure after the first connecting sequence is complementary to the second connecting sequence, the substrate sequence comprises a cleavage site, the first binding sequence and the second binding sequence can be complementary to an aptamer DNA sequence, the deoxyribozyme sequence can form an active secondary structure in a catalytic core after the first binding sequence and the second binding sequence are complementary to the aptamer DNA sequence, and the 5' end of the hairpin-locked 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 the FAM fluorescence can be effectively quenched by AuNPs. In the presence of MUC1, firstly opening a MUC1 aptamer probe, releasing a probe compound containing an aptamer DNA sequence, opening a hairpin structure of a hairpin locking deoxyribozyme probe in the probe compound, forming an active secondary structure in a catalytic core, and performing self-cleavage under the action of magnesium ions as a coenzyme, so that the gold nanoparticles release a fluorescent group, and then recovering fluorescence.
In some embodiments of this embodiment, the 3' end of the hairpin-locked deoxyribozyme probe is linked to a gold nanoparticle via 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 GGT TTC 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 CCA TAG 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 the 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 under the action of magnesium ions as a coenzyme, a substrate sequence is self-cleaved to separate a fluorophore from the gold nanoparticle, and the separated fluorophore is subjected to fluorescence detection.
The method of detecting MUC1 of the present invention is directed to 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 MgCl 2 The hybridization buffer solution is mixed evenly, and then the mixture is subjected to denaturation reaction in a water bath kettle at the temperature of 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 to 20min.
In one or more embodiments, the incubation is continued for 80-100 min after addition of the gold nanoparticles and magnesium salt linked to the MUC1 aptamer probe.
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 invention has better effect by adopting magnesium chloride.
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 (5.7X 10) 12 particles/mL) was added 6nmol of hairpin-locked DNAzyme probe (FAM-CAAAAAAGC ACA CCG GCT rAGT CTT TTT TGA TCCGAG CCG GAC GAA GCC CCA TACG GT-SH, sequence shown in SEQ ID NO. 1), incubated at room temperature for 16h, then incubated with 10mM phosphate (NaH) in 2 PO 4 /Na 2 HPO 4 ) And 0.1M NaCl in PBS buffer (pH 7.0) and left standing at room temperature for another 36 hours. Then, hairpin-locked DNAzyme probes not assembled on the surface of the AuNPs are removed by three times of high speed centrifugation (13000rpm, 30min) of the mixture of AuNPs and DNAzyme, and the resulting pellet is resuspended in 250. Mu.L of PBS buffer and finally stored at 4 ℃ for further use. In the hairpin-locked DNAzyme probe-AuNPs solution, the concentration of the hairpin-locked DNAzyme probe was about 18. Mu.M.
Mg 2+ Dependent amplification of hairpin-locked DNAzyme probes from cleavage cycle signal: the synthesized DNA oligonucleotide sequences were dissolved in Tris-EDTA buffer. At the same time, aptamer probe (GGT CCC ATA GGT TTC CTA GTT GAC GAC CTA TGG GAC CGGTGT GCT, sequence shown in SEQ ID NO. 2) and hairpin-locked DNAzyme probe were diluted with ultrapure water to1 μ M. With a solution containing 10mM Tris-HCl (pH 8.0) and 1.5mM MgCl 2 The 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 MUC1 polypeptide and aptamer probe recognition binding reaction (25. Mu.L), containing different concentrations of MUC1 polypeptide. Incubation with 37 ℃ for 15min, 1.5. Mu.L hairpin-locked DNAzyme modified AuNPs and MgCl 2 Adding into a reaction system, reacting for 90min at 37 ℃, and carrying out Mg 2+ The self-cleavage induced recovery of quenching fluorescence by AuNPs was induced by the dependent hairpin locked DNAzyme probe. 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 12% native polyacrylamide gel electrophoresis 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 (including 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 consistent with the above method.
The principle is shown in figure 1: in the embodiment, a hairpin locked DNAzyme probe is designed, and a thiol (SH) group modified at the 3' end of the 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 Mg 2+ 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 DNAzyme and the recovery of aptamer probe. And 9+9 binding arms are selected. A linker sequence consisting of poly-T, for linking together the 10-23DNAzyme and its substrate, which hybridizes with poly-A to form a hairpin structure. In the absence of MUC1The hairpin-locked DNAzyme probe can form a hairpin structure by intramolecular hybridization, thereby inhibiting the catalytic activity of the DNAzyme chain, 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 having an exposed DNA fragment as a probe that hybridizes 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 Mg 2+ Self-cleavage of the ribonucleotide moiety with the aid of (2). 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 its fluorescence. Meanwhile, the MUC1-aptamer probe complex is released and combined with the adjacent hairpin-locked DNAzyme to participate in more cycles of activation, cleavage and release, 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
According to TEM, as shown in FIG. 2A, auNPs have an average diameter of about 10nm, and thus have good dispersibility in aqueous solution. 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 target MUC1. Indicating that after hybridization, the target acts as an allosteric effector with Mg 2+ The homogeneous DNAzyme chain conformation is triggered in a dependent manner to switch to the active DNAzyme structure responsible for cleavage and to exhibit specific cleavage capacity. In vitro target binding studies of the probes were performed by adding different concentrations of miR-141. The results in FIG. 3B indicate that MU is presentIn the case of C1, a FAM fluorescence signal having a characteristic emission peak of 520nm was observed (FIG. 3B), in contrast to which, in the absence of MUC1, no significant FAM fluorescence signal was observed (FIG. 3B). These results clearly show that the nanosensor designed in this example can be used to detect MUC1. In conclusion, the technical scheme is completely feasible.
Sensitivity detection
The fluorescence signal of FAM was measured at different MUC1 concentrations and the sensitivity of the sensor was investigated, as shown in fig. 4. As shown in FIG. 4A, the fluorescence intensity of FAM increased with increasing MUC1 concentration 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 relation 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 F =5671.92+ 932.227log10C (R =5671.92 +) 2 = 0.9847), wherein 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 0.36fg/mL. Compared with the existing electrochemical luminescence detection method (2.80 fg/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 50 nM), alpha-fetoprotein (AFP, concentration 50 nM) and carcinoembryonic antigen (CEA, concentration 50 nM) 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 can be detected corresponding to MUC1 (concentration 1 ng/mL). These results demonstrate that the method has good specificity for the detection of MUC1.
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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<170> PatentIn version 3.3
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caaaaaagca caccggctra gtcttttttt ttgatccgag ccggacgaag ccccataggt 60
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ggtcccatag gtttcctagt tgacgaccta tgggaccggt gtgct 45
Claims (5)
1. A gold nanoparticle sensor based on a hairpin-locked deoxyribozyme probe is characterized by comprising 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 gold nanoparticles;
the sequence of the MUC1 aptamer probe is as follows: GGT CCCATAGGT TTC CTAGTT GAC GAC CTA TGG GAC CGG TGT GCT;
the sequence of the hairpin-locked deoxyribozyme probe is as follows: CAAAAAAGC ACACCG GCT rAGT CTT TTT TGATCC GAG CCG GAC GAAGCC CCATAG GT.
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. A detection kit, characterized by comprising the gold nanoparticle sensor based on the hairpin-locked deoxyribozyme probe according to any one of claims 1 to 4, a magnesium salt, and a buffer solution.
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| CN113687084A (en) * | 2021-08-26 | 2021-11-23 | 四川大学华西医院 | Method for detecting protein based on target recognition induction deoxyribozyme release strategy, application and kit |
| CN114397343B (en) * | 2022-03-25 | 2022-06-14 | 南京邮电大学 | Tumor marker activity detection kit, detection method and application thereof |
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