CN113624823A - Signal probe based on tetrahedral nano-structure DNA, preparation method and application thereof - Google Patents
Signal probe based on tetrahedral nano-structure DNA, preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a signal probe based on tetrahedral nano-structure DNA and a preparation method and application thereof, wherein the signal probe comprises the following components: an aligned tetrahedral nanostructure DNA and a complex comprising an inverted tetrahedral nanostructure DNA and a biotin-modified aptamer, the inverted tetrahedral nanostructure DNA having three of its vertices extended with the biotin-modified aptamer; the other vertex of the inverted tetrahedral nanostructure DNA can hybridize to one of the vertices of the regular tetrahedral nanostructure DNA; the aptamer can be identified and combined with circulating tumor cells to be detected. The probe is used for realizing the identification, capture and detection of the circulating tumor cells, so that the cost is reduced, the detection is efficient and stable, and the diagnosis method based on the technology has high application value.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a tetrahedral nano-structure DNA-based signal probe and a preparation method and application thereof.
Background
Malignant tumor is a disease with high death rate at home and abroad, and has more and more obvious adverse effects on the development of social economy. At present, the traditional tumor diagnosis and monitoring means mainly comprise imaging examination, puncture pathology biopsy, traditional blood tumor marker detection and the like. However, the imaging examination has high requirements for operators, and generally has good distinguishing capability only for tumors with the length of more than 1 cm; cytological or histological needle biopsies are traumatic, resulting in low patient acceptance; traditional hematological tumor marker detection results in poor accuracy due to lack of reliable markers. Circulating Tumor Cells (CTCs) are tumor cells from a primary tumor focus and are separated from a basement membrane to enter peripheral blood or bone marrow due to spontaneous or diagnosis and treatment operations, and are closely related to early screening, auxiliary staging, personalized medicine, prognosis evaluation, relapse metastasis early warning and the like of tumors, but the extremely low content of the CTCs in blood brings great difficulty in capture and detection.
With the development of science and technology, the inspection medicine has a new development trend, on one hand, highly integrated and large-scale automatic inspection instruments come out in succession, thereby greatly widening the range of clinical inspection, improving the working efficiency and improving the immunochemical inspection to a new level; on the other hand, simple and integrated biosensor detection techniques are rapidly developing. Because there are many complicated steps before and after analysis by an automated instrument, a lot of time is consumed, and a detection result cannot be obtained quickly, so that a miniaturized, simple-to-operate and quick instant test technology attracts people's attention. The method has the characteristics that the complex operation is simplified, the complex equipment is small and portable, and the detection result can be quickly obtained.
A biosensor refers to a type of sensor that is constructed of a biologically active material and a corresponding transducer, and is capable of measuring a particular chemical or biological substance. An electrochemical biosensor is a type of biosensor, which is generally formed by using basic electrodes such as an ion selective electrode, a gas sensitive electrode or a solid electrode as conversion elements, and a transducer converts chemical or biological information generated by molecular recognition into electric signals, such as current, voltage and resistance, and the signals are amplified and output. The method has the unique advantages of high analysis speed, easy miniaturization, high sensitivity, simple operation, capability of realizing real-time online monitoring and the like, and is widely applied to a plurality of fields of clinical examination, food quality control, industrial analysis, environmental monitoring, medicine analysis and the like. In recent years, the research of electrochemical sensors has become one of the research hotspots in the research of modern electroanalytical chemistry, which is crosswise integrated with the discipline fields of biology, informatics, medicine, materials science and the like.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a signaling probe based on tetrahedral nanostructure DNA, and a preparation method and use thereof, which are used to solve the problems in the prior art, to capture and detect circulating tumor cells, to perform low-cost and high-efficiency detection, and to obtain a stable diagnostic standard with application value.
To achieve the above objects and other related objects, the present invention includes the following technical solutions.
The invention provides in a first aspect a signaling probe based on tetrahedral nanostructure DNA, the signaling probe comprising: an aligned tetrahedral nanostructure DNA and a complex comprising an inverted tetrahedral nanostructure DNA and a biotin-modified aptamer, the inverted tetrahedral nanostructure DNA having one of the biotin-modified aptamers extending from each of three vertices thereof; the other vertex of the inverted tetrahedral nanostructure DNA can hybridize to one of the vertices of the regular tetrahedral nanostructure DNA; the aptamer can be identified and combined with circulating tumor cells to be detected.
Preferably, the side length of a tetrahedron in the regular tetrahedral nanostructure DNA can be changed by adjusting the number of DNA base pairs. More preferably, the edge length of the tetrahedron in the regular tetrahedral nanostructure DNA is formed by complementary pairing of 17 pairs of bases.
Preferably, the regular tetrahedral nanostructure DNA comprises a1、B1、C1And D1Four chains.
Preferably, B in the regular tetrahedral nano-structured DNA1、C1And D1The three chains are modified with sulfhydryl at 5'.
Preferably, A in the regular tetrahedral nano-structured DNA1The single strand has a first DNA recognition sequence extending 3' from the vertex that is complementary to a DNA recognition sequence extending from one of the vertices in the inverted tetrahedral nanostructure DNA.
Preferably, the regular tetrahedral nanostructure DNA is formed by self-assembly under conditions of denaturation at 95 ℃ followed by annealing at 4 ℃.
More preferably, the regular tetrahedral nanostructure DNA comprises a1、B1、C1And D1Four chains, the nucleotide sequences of which are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.
Preferably, A1：
5'-ACATTCCTAAGTCTGAAACATTACAGCTTGCTACACGAGAAGAGCCGCCATAGTATTTTTTTTTTTCAACATCAGTCTGATAAGC-3'。(SEQ ID NO.1)
Preferably, B1：
5'-TATCACCAGGCAGTTGACAGTGTAGCAAGCTGTAATAGATGCGAGGGTCCAATAC-3'。(SEQ ID NO.2)
Preferably, C1:
5'-TCAACTGCCTGGTGATAAAACGACACTACGTGGGAATCTACTATGGCGGCTCTTC-3'。(SEQ ID NO.3)
Preferably, D1：
5'-TTCAGACTTAGGAATGTGCTTCCCACGTAGTGTCGTTTGTATTGGACCCTCGCAT-3'。(SEQ ID NO.4)
Preferably, the inverted tetrahedral nanostructure DNA comprises a2、B2、C2And D2Four chains.
Preferably, the side length of a tetrahedron in the inverted tetrahedral nanostructure DNA can be changed by adjusting the number of DNA base pairs. More preferably, the edge length of the tetrahedron in the inverted tetrahedral nanostructure DNA is formed by complementary pairing of 17 pairs of bases. Preferably, in the inverted tetrahedral nano-structured DNAB2、C2And D2The 3 'ends of the three strands each extend beyond the apex with a second DNA recognition sequence complementary to the DNA recognition sequence 3' to the aptamer.
Preferably, A in the inverted tetrahedral nano-structured DNA2The single strand has a third DNA recognition sequence extending 3' from the apex, said third DNA recognition sequence being base complementary to said first DNA recognition sequence.
Preferably, the inverted tetrahedral nanostructure DNA is formed by denaturation at 95 ℃ followed by self-assembly under annealing conditions at 4 ℃.
Preferably, the inverted tetrahedral nanostructure DNA comprises a2、B2、C2And D2Four chains, the nucleotide sequences of which are respectively shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8.
Preferably, A2：
5'-ACATTCCTAAGTCTGAAACATTACAGCTTGCTACACGAGAAGAGCCGCCATAGTATTTTTTTTTTGCTTATCAGACTGATGTTGA-3'。(SEQ ID NO.5)
Preferably, B2:
5'-TATCACCAGGCAGTTGACAGTGTAGCAAGCTGTAATAGATGCGAGGGTCCAATACCTGACCACGAGCTCCATTAC-3'。(SEQ ID NO.6)
Preferably, C2：
5'-TCAACTGCCTGGTGATAAAACGACACTACGTGGGAATCTACTATGGCGGCTCTTCCTGACCACGAGCTCCATTAC-3'。(SEQ ID NO.7)
Preferably, D2：
5'-TTCAGACTTAGGAATGTGCTTCCCACGTAGTGTCGTTTGTATTGGACCCTCGCATCTGACCACGAGCTCCATTAC-3'。(SEQ ID NO.8)
Preferably, the nucleotide sequence of the biotin-modified aptamer is shown as SEQ ID NO. 9.
Preferably, the nucleotide sequence of the accounting aptamer is:
5'-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTGTTTTTGTAATGGAGCTCGTGGTCAG-3'。(SEQ ID NO.9)
preferably, the biotin-modified aptamer means that both 3 'and 5' of the aptamer have biotin attached.
The second aspect of the invention also discloses an electrochemical detection method, which comprises the following steps:
1) incubating the circulating tumor cells to be detected and the compound together to obtain the circulating tumor cells to be detected combined with the compound;
modifying the surface of the working electrode with the DNA of the regular tetrahedron nano structure;
2) hybridizing the orthotetrahedral nanostructure DNA and the inverted tetrahedral nanostructure DNA in the complex to capture circulating tumor cells on the working electrode;
3) adding oxidoreductase and corresponding substrate for electrochemical detection.
Preferably, the complex comprises inverted tetrahedral nanostructure DNA and a biotin-modified aptamer capable of recognizing and binding to the circulating tumor cells to be detected.
Preferably, three vertices of the inverted tetrahedral nanostructure DNA are each extended with one of the biotin-modified aptamers, and another vertex of the inverted tetrahedral nanostructure DNA is capable of being cross-linked with one vertex of the regular tetrahedral nanostructure DNA.
Preferably, the regular tetrahedral nanostructure DNA comprises a1、B1、C1And D1Four chains, the nucleotide sequences of which are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.
Preferably, the inverted tetrahedral nanostructure DNA comprises a2、B2、C2And D2Four chains, the nucleotide sequences of which are respectively shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8.
Preferably, the nucleotide sequence of the biotin-modified aptamer is shown as SEQ ID NO. 9.
Preferably, the regular tetrahedral nano-structure DNA is modified on the surface of the substrate electrode through Au-S bonds.
Preferably, the chloroauric acid is directly reduced into the nanogold by adopting a constant potential deposition method and is modified on the substrate electrode, and then the nanogold is bonded with the DNA of the regular tetrahedron nanostructure.
Preferably, the working electrode is a carbon electrode.
Preferably, the working electrode is a screen-printed electrode. More preferably, the screen printed electrodes are multi-channel screen printed electrodes.
The third aspect of the invention also discloses the application of the signaling probe based on the tetrahedral nano-structure DNA in circulating tumor cell detection.
The fourth aspect of the invention also discloses the application of the orthotetrahedral nano-structure DNA and the compound in the preparation of a signaling probe.
The fifth aspect of the invention also discloses an electrochemical biosensor, which comprises a working electrode, a regular tetrahedral nano-structure DNA and a compound, wherein the regular tetrahedral nano-structure DNA is modified on the working electrode; the complex comprises inverted tetrahedral nanostructure DNA and a biotin-modified aptamer; wherein three vertices of said inverted tetrahedral nanostructure DNA are each extended with one of said biotin-modified aptamers, and another vertex of said inverted tetrahedral nanostructure DNA is capable of hybridizing with one vertex of said regular tetrahedral nanostructure DNA.
Preferably, said orthotetrahedral nanostructure DNA comprises a1、B1、C1And D1Four chains, the nucleotide sequences of which are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.
Preferably, the inverted tetrahedral nanostructure DNA comprises a2、B2、C2And D2Four chains, the nucleotide sequences of which are respectively shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8.
Preferably, the nucleotide sequence of the biotin-modified aptamer is shown as SEQ ID NO. 9.
Preferably, the electrochemical biosensor further comprises a reference electrode and a counter electrode.
The invention also discloses the application of the electrochemical biosensor for detecting circulating tumor cells.
The above-described detection in the present application is a detection method for non-disease diagnosis purposes.
DNA is not only a carrier of genetic information, but also natural nano materials and structural elements, and has super-strong coding and self-assembly capabilities. The tetrahedral nanostructure DNA scaffold has a rigid structure, is beneficial to maintaining better vertical orientation of the top probe, and the existence of the tetrahedron enables the longitudinal and transverse distances to be easily regulated and controlled through the size of the tetrahedron. The top of the tetrahedral nano-structure DNA can be combined with a plurality of ligand molecules or signal molecules to realize multivalent capture and signal amplification, then the chemical signal and the electric signal of the reaction product are converted, and the relationship of the concentration of the target object to be detected is converted according to the detected electric signal to realize detection.
The applicant in the application constructs a signal probe, a working electrode and an electrochemical biosensor with a DNA double-tetrahedron nanostructure based on the concept, and the signal probe, the working electrode and the electrochemical biosensor are used for realizing the identification, capture and detection of circulating tumor cells, so that the cost is reduced, the detection is efficient and stable, and the diagnosis method based on the technology has high application value.
Drawings
FIG. 1 shows an assay using agarose gel electrophoresis to demonstrate double tetrahedral nanostructure DNA synthesis.
FIG. 2 is a graph showing the effect of affinity of the complex for target circulating tumor cells using fluorescence microscopy.
FIG. 3 shows the current time curves obtained for the detection of circulating tumor cells using screen-printed electrodes modified with different probes.
FIG. 4 shows the current signal variation curve corresponding to the detection of different MCF-7 cell numbers by the double-tetrahedron nanostructure DNA-modified silk-screen printing electrode in the technical scheme of the application.
FIG. 5 shows the current signal values of the screen-printed electrodes modified by the double tetrahedral nano-structure DNA in the technical scheme of the application when different cells are detected.
FIG. 6 shows the current signal curve corresponding to the double tetrahedral nano-structure DNA modified silk-screen printing electrode in the technical scheme of the application when MCF-7 cells with different concentrations of Hela cells are detected.
FIG. 7 shows the corresponding current signal values when detecting MCF-7 target cells in a whole blood sample for the screen-printed electrode modified by the double-tetrahedral nano-structured DNA in the technical scheme of the application.
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. 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. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
DNA is not only a carrier of genetic information, but also natural nano materials and structural elements, and has super-strong coding and self-assembly capabilities. The tetrahedral nanostructure DNA scaffold has a rigid structure, is beneficial to maintaining better vertical orientation of the top probe, and the existence of the tetrahedron enables the longitudinal and transverse distances to be easily regulated and controlled through the size of the tetrahedron. The top of the tetrahedral nano-structure DNA can be combined with a plurality of ligand molecules or signal molecules to realize multivalent capture and signal amplification, then the chemical signal and the electric signal of the reaction product are converted, and the relationship of the concentration of the target object to be detected is converted according to the detected electric signal to realize detection. The applicant in the application constructs a signal probe and an electrochemical biosensor which can form a double-DNA double-tetrahedron nano structure based on the concept, and the signal probe and the electrochemical biosensor are used for capturing and detecting circulating tumor cells, so that the cost is reduced, the detection is efficient and stable, and a diagnosis method based on the technology has high application value.
In the examples of the present application, the applicant specifically provides a signaling probe based on tetrahedral nanostructure DNA, comprising: an aligned tetrahedral nanostructure DNA and a complex comprising an inverted tetrahedral nanostructure DNA and a biotin-modified aptamer, the inverted tetrahedral nanostructure DNA having one of the biotin-modified aptamers extending from each of three vertices thereof; the other vertex of the inverted tetrahedral nanostructure DNA can hybridize to one of the vertices of the regular tetrahedral nanostructure DNA; the aptamer can be identified and combined with circulating tumor cells to be detected.
In a preferred embodiment, the side length of a tetrahedron in the regular tetrahedral nanostructure DNA can be varied in its length by adjusting the number of DNA base pairs. In a more specific embodiment, the edge length of the tetrahedron in the regular tetrahedral nanostructure DNA is formed from 17 pairs of base complementary pairings.
The regular tetrahedral nano-structure DNA comprises A1、B1、C1And D1Four chains.
In a preferred embodiment, B in the regular tetrahedral nanostructure DNA1、C1And D1The three chains are modified with sulfhydryl at 5'.
In a preferred embodiment, the tetrahedron is placed right side upA in nanostructured DNA1The single strand has a first DNA recognition sequence extending 3' from the vertex that is complementary to a DNA recognition sequence extending from one of the vertices in the inverted tetrahedral nanostructure DNA.
In a preferred embodiment, the regular tetrahedral nanostructure DNA is formed by self-assembly under conditions of denaturation at 95 ℃ followed by annealing at 4 ℃.
In a preferred embodiment, the orthotetrahedral nanostructure DNA comprises A1、B1、C1And D1Four chains, the nucleotide sequences of which are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.
Specifically, A1：
5'-ACATTCCTAAGTCTGAAACATTACAGCTTGCTACACGAGAAGAGCCGCCATAGTATTTTTTTTTTTCAACATCAGTCTGATAAGC-3'。(SEQ ID NO.1)
Specifically, B1：
5'-TATCACCAGGCAGTTGACAGTGTAGCAAGCTGTAATAGATGCGAGGGTCCAATAC-3'。(SEQ ID NO.2)
Specifically, C1:
5'-TCAACTGCCTGGTGATAAAACGACACTACGTGGGAATCTACTATGGCGGCTCTTC-3'。(SEQ ID NO.3)
In particular, D1：
5'-TTCAGACTTAGGAATGTGCTTCCCACGTAGTGTCGTTTGTATTGGACCCTCGCAT-3'。(SEQ ID NO.4)
In a preferred embodiment, the inverted tetrahedral nanostructure DNA comprises A2、B2、C2And D2Four chains.
In a preferred embodiment, the tetrahedral edge length in the inverted tetrahedral nanostructure DNA can be varied by adjusting the number of DNA base pairs to change its length.
In a more preferred embodiment, the edge length of the tetrahedron in the inverted tetrahedral nanostructure DNA is formed by complementary pairing of 17 pairs of bases.
In a preferred embodiment, said inverted tetrahedronB in nanostructured DNA2、C2And D2The 3 'ends of the three strands each extend beyond the apex with a second DNA recognition sequence complementary to the DNA recognition sequence 3' to the aptamer.
In a preferred embodiment, A is in the inverted tetrahedral nanostructure DNA2The single strand has a third DNA recognition sequence extending 3' from the apex, said third DNA recognition sequence being base complementary to said first DNA recognition sequence.
In a preferred embodiment, the inverted tetrahedral nanostructure DNA is formed by denaturation at 95 ℃ followed by self-assembly under annealing conditions at 4 ℃.
In a preferred embodiment, the inverted tetrahedral nanostructure DNA comprises A2、B2、C2And D2Four chains, the nucleotide sequences of which are respectively shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8.
Specifically, A2：
5'-ACATTCCTAAGTCTGAAACATTACAGCTTGCTACACGAGAAGAGCCGCCATAGTATTTTTTTTTTGCTTATCAGACTGATGTTGA-3'。(SEQ ID NO.5)
Specifically, B2:
5'-TATCACCAGGCAGTTGACAGTGTAGCAAGCTGTAATAGATGCGAGGGTCCAATACCTGACCACGAGCTCCATTAC-3'。(SEQ ID NO.6)
Specifically, C2：
5'-TCAACTGCCTGGTGATAAAACGACACTACGTGGGAATCTACTATGGCGGCTCTTCCTGACCACGAGCTCCATTAC-3'。(SEQ ID NO.7)
In particular, D2：
5'-TTCAGACTTAGGAATGTGCTTCCCACGTAGTGTCGTTTGTATTGGACCCTCGCATCTGACCACGAGCTCCATTAC-3'。(SEQ ID NO.8)
In a preferred embodiment, the nucleotide sequence of the biotin-modified aptamer is as shown in SEQ ID NO. 9.
Specifically, the nucleotide sequence of the accounting aptamer is:
5'-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTGTTTTTGTAATGGAGCTCGTGGTCAG-3'。(SEQ ID NO.9)
in a preferred embodiment, the biotin-modified aptamer is one in which both 3 'and 5' of the aptamer are linked to biotin.
In the embodiments of the present application, the applicant further specifically provides an electrochemical biological detection method, including the following steps:
1) incubating the circulating tumor cells to be detected and the compound together to obtain the circulating tumor cells to be detected combined with the compound;
modifying the surface of the working electrode with the DNA of the regular tetrahedron nano structure;
2) hybridizing the orthotetrahedral nanostructure DNA and the inverted tetrahedral nanostructure DNA in the complex to capture circulating tumor cells on the working electrode;
3) adding oxidoreductase and corresponding substrate for electrochemical detection.
For the above electrochemical biological detection method, in a preferred embodiment, the complex comprises inverted tetrahedral nanostructure DNA and biotin-modified aptamer capable of recognizing and binding to the circulating tumor cells to be detected.
For the above electrochemical biological detection method, in a preferred embodiment, three vertices of the inverted tetrahedral nanostructure DNA are extended with the biotin-modified aptamer, and another vertex of the inverted tetrahedral nanostructure DNA can be hybridized and connected with one vertex of the aligned tetrahedral nanostructure DNA.
For the above electrochemical bioassay method, in a preferred embodiment, the regular tetrahedral nanostructure DNA comprises A1、B1、C1And D1Four chains, the nucleotide sequences of which are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.
For the above electrochemical bioassay method, in a preferred embodiment, the inverted tetrahedral nanostructure DNA comprises A2、B2、C2And D2Four chains, the nucleotide sequences of which are respectively shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8.
For the electrochemical biological detection method, in a preferred embodiment, the tetrahedron-shaped nano-structured DNA is modified on the surface of the working electrode through Au-S bonds.
For the above electrochemical biological detection method, in a preferred embodiment, the working electrode is a carbon electrode.
For the above electrochemical biological detection method, in a preferred embodiment, the working electrode is a screen-printed electrode. In a more preferred embodiment, the screen printed electrodes are multi-channel screen printed electrodes.
For the electrochemical biological detection method, in a preferred embodiment, the chloroauric acid is directly reduced to the nanogold by a constant potential deposition method and is decorated on the working electrode, and then the nanogold is bonded with the regular tetrahedral nanostructure DNA.
For the above electrochemical biological detection method, in a preferred embodiment, the nucleotide sequence of the biotin-modified aptamer is shown in SEQ ID NO.9
In an embodiment of the present application, applicants further provide an electrochemical biosensor comprising a working electrode, an orthotetrahedral nanostructure DNA, and a complex; the positive tetrahedral nanostructure DNA is modified on the working electrode; the complex comprises inverted tetrahedral nanostructure DNA and a biotin-modified aptamer; wherein three vertices of said inverted tetrahedral nanostructure DNA are each extended with one of said biotin-modified aptamers, and another vertex of said inverted tetrahedral nanostructure DNA is capable of hybridizing with one vertex of said regular tetrahedral nanostructure DNA.
With respect to the electrochemical biosensor described above, in a preferred embodiment, the electrochemical biosensor further comprises a reference electrode and a counter electrode.
For the electrochemical biosensor described above, in a preferred embodiment, the regular tetrahedral nanostructure DNA is modified on the surface of the working electrode by Au — S bonds.
In a preferred embodiment of the above electrochemical biosensor, the working electrode is a carbon electrode.
In a preferred embodiment of the above electrochemical biosensor, the working electrode is a screen-printed electrode. A 16-channel screen printed electrode is particularly employed as in the present embodiment.
In a preferred embodiment, the electrochemical biosensor directly reduces chloroauric acid into nanogold by a constant potential deposition method, modifies the nanogold on the working electrode, and then bonds with the regular tetrahedral nanostructure DNA.
In a preferred embodiment of the electrochemical biosensor, the regular tetrahedral nano-structured DNA comprises A1、B1、C1And D1Four chains.
In a preferred embodiment, the electrochemical biosensor is a tetrahedron nanostructure DNA with B being regularly arranged1、C1And D1The three chains are modified with sulfhydryl at 5'.
In a preferred embodiment, the electrochemical biosensor is a tetrahedron nanostructure DNA1The single strand has a first DNA recognition sequence extending 3' from the vertex that is complementary to a DNA recognition sequence extending from one of the vertices in the inverted tetrahedral nanostructure DNA.
In a preferred embodiment, the regular tetrahedral nano-structured DNA is formed by denaturation at 95 ℃ and then self-assembly under annealing at 4 ℃.
In a more preferred embodiment, the regular tetrahedral nano-structured DNA comprises A1、B1、C1And D1Four chains, the nucleotide sequences of which are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.
In a preferred embodiment of the electrochemical biosensor, the inverted tetrahedral nano-structured DNA comprises A2、B2、C2And D2Four chains.
In a preferred embodiment, the length of the side of the tetrahedron in the inverted tetrahedral nanostructure DNA can be changed by adjusting the base pairing number of the DNA.
In a more preferred embodiment, the edge length of the tetrahedron in the inverted tetrahedral nanostructure DNA is formed by complementary pairing of 17 pairs of bases.
The electrochemical biosensor is characterized in that, in a preferred embodiment, B in the inverted tetrahedral nanostructure DNA2、C2And D2The 3 'ends of the three strands each extend beyond the apex with a second DNA recognition sequence complementary to the DNA recognition sequence 3' to the aptamer.
The electrochemical biosensor, in a preferred embodiment, comprises a in the inverted tetrahedral nanostructure DNA2The single strand has a third DNA recognition sequence extending 3' from the apex, said third DNA recognition sequence being base complementary to said first DNA recognition sequence.
In a preferred embodiment, the inverted tetrahedral nanostructure DNA is formed by denaturation at 95 ℃ and then self-assembly under annealing at 4 ℃.
In a more preferred embodiment, the inverted tetrahedral nano-structured DNA comprises A2、B2、C2And D2Four chains, the nucleotide sequences of which are respectively shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8.
In a preferred embodiment of the electrochemical biosensor, the nucleotide sequence of the biotin-modified aptamer is shown in SEQ ID NO. 9.
In a preferred embodiment of the electrochemical biosensor, the biotin-modified aptamer is a nucleic acid aptamer having biotin attached to both 3 'and 5' ends thereof.
Also disclosed in the present application is the use of a signaling probe as described above or an electrochemical biosensor as described above for detecting circulating tumor cells.
The above-described detection in the present application is a detection method for non-disease diagnosis purposes.
In the specific embodiment of the present application, the preparation method of the orthotetrahedral nanostructure DNA comprises: four single strands forming the tetrahedrally aligned nanostructured DNA are self-assembled. In a preferred embodiment, the regular tetrahedral nanostructure DNA is formed by self-assembly under conditions of denaturation at 95 ℃ followed by annealing at 4 ℃.
In the specific embodiment of the present application, the preparation method of the inverted tetrahedral nano-structure DNA comprises: four single strands forming the inverted tetrahedral nanostructure DNA self-assemble. In a preferred embodiment, the inverted tetrahedral nanostructure DNA is formed by denaturation at 95 ℃ followed by self-assembly under annealing conditions at 4 ℃.
In the specific embodiment of the application, the preparation method of the compound comprises the following steps: self-assembling the four single strands of each inverted tetrahedral nanostructure DNA forming the complex with a biotin-modified aptamer. In a preferred embodiment, the complex is formed under conditions of denaturation at 95 ℃ followed by annealing at 4 ℃.
The electrochemical sensor is constructed and the circulating tumor cells are detected based on the following principles:
in the working electrode modified with the DNA with the double-tetrahedron nano structure, the compound has a specific recognition effect on circulating tumor cells, and the compound is captured by the working electrode modified with the DNA with the regular tetrahedron nano structure; when the signal molecules modified with the avidin are placed in hydrogen peroxide for electrochemical test, the signal molecules are integrated through the action of the biotin and the avidin, the hydrogen peroxide is catalytically decomposed by the signal molecules, biochemical signals are converted into electric signals, and the content of corresponding circulating cells can be detected and obtained according to current signals.
The technical solutions mentioned above in the present application are further explained and illustrated by specific examples and implementation effect data.
The materials and equipment used in the present application in the following specific embodiments are as follows:
chloroauric acid (Hydrogen tetrachloroaurate (III) hydrate (HAuCl)4) Purchased from carbofuran corporation; reagents such as potassium ferrocyanide, potassium chloride, 30% hydrogen peroxide and the like are purchased from chemical reagents of national medicine group, ltd; reagents such as buffer solution PBS, 0.5% tyrosine solution (0.1M PBS) and the like are purchased from Biotechnology engineering (Shanghai) GmbH; all solutions were made up in Milli-Q water (18 M.OMEGA.cm resistance). Electrophoresis apparatus was purchased from Bio-Rad, the PCR apparatus was a Peltier thermal cycler PTC-200(MJ. research Inc., SA), and the UV gel apparatus was a Box (gene Company Limited); an HSBS16x series multichannel electrochemical workstation, a CHI 760E electrochemical workstation, an electronic balance, a pH meter, a magnetic heating stirrer, a normal temperature centrifuge and a vortex mixer; DNA strands were purchased from Shanghai organisms.
In this application, the substrate used comprises 3, 3', 5, 5' -Tetramethylbenzidine (TMB) and hydrogen peroxide.
In the present application, the counter electrode is a carbon electrode and the reference electrode is a silver electrode. During electrochemical detection, a cyclic voltammetry graph and a time current curve are scanned, wherein the initial voltage of the cyclic voltammetry is-0.3V, the highest voltage is 0.7V, the lowest voltage is-0.3V, and the scanning speed is 0.1V/s. The initial potential of the time-current method is 0.1V, the sampling interval is 0.5s, the running time is 100s, and the rest time is 3 s.
Synthesis of orthotetrahedral nanostructured DNA (abbreviated UTDF): mixing four single chains forming the tetrahedral nano-structure DNA in the TM buffer in equal proportion to prepare the tetrahedral nano-structure, putting the prepared sample into a PCR instrument, keeping the temperature at 95 ℃ for 5min, then quickly cooling to 4 ℃, and keeping the temperature at 4 ℃ for more than 5 min. Specifically, the final concentration was 1. mu.M. In the actual and synthetic process, each single-stranded DNA is dissolved, quantified under an ultraviolet spectrophotometer, and the molar extinction coefficient is obtained from an IDTDNA website and diluted to 100 mu M.
Synthesis of inverted tetrahedral nanostructure DNA (abbreviated ITDF): the same procedure as for UTDF synthesis is used, except that the DNA single strands used are different.
Synthesis of complexes of ITDF with biotin-modified nucleic acid aptamers: the same procedure as for ITDF synthesis, but with the addition of each chain between syntheses: the tetrahedral nanostructure DNA was identical in concentration of four single strands and in a molar ratio of 1:3 to the concentration of biotin-modified aptamers (abbreviated aptamers). Specifically, the four single strands of tetrahedral nanostructure DNA were at a concentration of 1. mu.M, and the aptamers were at a concentration of 3. mu.M, respectively.
Synthesis of modified double tetrahedral nanostructure DNA: UTDF was hybridized with the complex at 1:1 molar. Specifically, 20. mu.L of each was taken and hybridized at 37 ℃ for 2 hours.
And (3) characterizing each synthetic structure by a gel electrophoresis method. During characterization, 8 wt% polyacrylamide is adopted, electrophoresis is carried out for 120min at a constant voltage of 100V, and then Gel red staining, ultraviolet irradiation and photographing are carried out. The photograph of the effect is shown in fig. 1. As can be seen from fig. 1: the technical scheme in the application really synthesizes the DNA with the double-tetrahedron nano structure.
And verifying the specific binding property of the complex to the circulating tumor cells MCF-7 by adopting a fluorescence microscope. A complex of the formed ITDF with an aptamer and 105The MCF-7 cells were incubated on ice for 30min and washed 3 times by centrifugation in PBS to remove unbound probe. The culture medium was resuspended and centrifuged cells, and observed under a fluorescence microscope, and the results are shown in FIG. 2. FIG. 2(a) shows MCF-7 target cells; FIG. 2(b) is an ITDF and aptamer complex formed by hybridization of a Cy-3 fluorophore modified aptamer and inverted tetrahedral nanostructure DNA, wherein both 3 'and 5' of the Cy-3 fluorophore modified aptamer are connected with Cy 3; panel (c) is an integrated image of panels (a) and (b), with the probes coincident with the cell sites, demonstrating that complexes of ITDF and aptamer bind to the cell surface with good affinity for MCF-7 cells.
Detecting the circulating tumor cells MCF-7 by adopting working electrodes modified by different probe structures:
a) the specific method for forming the electrode in the present application is as follows: 16-channel screen-printed carbon electrode was placed in 0.06mg/mL HAuC14In the solution, electrodeposition is carried out under the condition of-200 mV, and the running time is 150 s; after electrodeposition, the electrode strips were removed and the electrode surface was rinsed thoroughly with Milli-Q water, N2And drying the residual water drops to obtain the nano-gold modified 16-channel screen printing electrode.
b) Different probes capture MCF-7 cells:
b1 capture of MCF-7 cells based on the probes obtained according to the protocol of the present application:
dripping 10 mu L of synthesized right tetrahedron nano-structure DNA (UTDF) on the surface of a nano-gold modified 16-channel screen printing electrode, incubating overnight in a wet box, thoroughly washing the surface of the electrode by using deionized water, washing out unbound tetrahedron nano-structure DNA nano-structure, and drying residual water drops by using nitrogen to finish the assembly of the UTDF on the working electrode interface. Dropping 10 μ LITFD/biotin-modified aptamer/MCF-7 onto UTDF-modified 16-channel silk-screen-printed electrode, incubating in a wet chamber for 30min to allow cell-bound ITDF/aptamers to hybridize and capture with the UTDF probe on the electrode, washing the electrode with PBS to remove the non-captured MCF-7, and washing with N2Blow-drying, incubating with 3 mu L of SA-poly HRP signal molecule solution (1 mu g/mL) modified with avidin at room temperature for 15min, binding the signal molecules to MCF-7 under the action of biotin-avidin, fully washing the reacted electrode with PBS, washing away the unbound SA-poly HRP signal molecules, and finally, determining a time-current curve to represent the detection capability of the double-tetrahedron DNA probe on the CTC.
b2 capture of MCF-7 cells based on a probe constructed from UTDF:
dripping 10 mu L of synthesized right tetrahedron nano-structure DNA (UTDF) on the surface of a nano-gold modified 16-channel screen printing electrode, incubating overnight in a wet box, thoroughly washing the surface of the electrode by using deionized water, washing out unbound tetrahedron nano-structure DNA nano-structure, and drying residual water drops by using nitrogen to finish the assembly of the UTDF on the working electrode interface.
Providing a comparison aptamer having a nucleotide sequence:
5'-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTGTTTTTGCTTATCAGACTGATGTTGA-3', as shown in SEQ ID NO. 11.
Comparing aptamers to 105The individual MCF-7 cells were incubated on ice for 30min and washed 3 times by centrifugation in PBS to remove unbound probe and form a control aptamer/MCF-7 mixture. Dripping 10 μ L of the aptamer/MCF-7 onto a gold-coated electrode modified with UTDF, incubating in a wet chamber for 30min to allow the aptamer bound to the cell to hybridize with the capture probe on the electrode for capture, washing the electrode with PBS to wash away the uncaptured MCF-7, and washing with N2Blow-drying, incubating with 3 mu L of SA-poly HRP signal molecule solution (1 mu g/mL) modified with avidin at room temperature for 15min, binding the signal molecules to MCF-7 under the action of biotin-avidin, fully washing the reacted electrode with PBS, washing away the unbound SA-poly HRP signal molecules, and finally, determining a time-current curve to represent the detection capability of the double-tetrahedron DNA probe on the CTC.
b3 capture of MCF-7 cells based on aptamer probes constructed on comparative single stranded DNA:
providing a comparison single-stranded DNA, wherein the comparison single-stranded DNA is connected with a sulfydryl at the 5' end. Specifically, the nucleotide sequences of the aligned single-stranded DNAs are:
5'-ACATTCCTAAGTCTGAAACATTTTTTTTTTTCAACATCAGTCTGATAAGC-3', as shown in SEQ ID NO. 10.
The control single stranded DNA may be directly modified on the electrode substrate. The control single-stranded DNA is capable of hybridizing to a control aptamer having a biotin modification.
Dripping 10 μ L of contrast single-stranded DNA onto the surface of gold-printed electrode, incubating overnight in a wet box, washing thoroughly the electrode surface with deionized water, washing away unbound DNA tetrahedral nanostructures, and incubating with N2And drying the residual water drops to finish the assembly of the contrast single-stranded DNA on the interface of the printed electrode.
Comparing aptamers to 105The MCF-7 cells were incubated on ice for 30min and then in PBSThe unbound probe was removed by 3 washes by centrifugation to form a comparative aptamer/MCF-7 mixture. Dripping 10 μ L of the aptamer/MCF-7 onto a gold-printed electrode modified with single-stranded DNA, incubating in a wet chamber for 30min to allow hybridization between the aptamer bound to the cell and the capture probe on the electrode, washing the electrode with PBS to remove the uncaptured MCF-7, and washing with N2Blow-drying, incubating with 3 mu L of SA-poly HRP solution (1 mu g/mL) modified with avidin for 15min at room temperature, binding signal molecules to MCF-7 under the action of biotin-avidin, fully washing the reacted electrode with PBS, washing away the SA-poly HRP signal molecules which are not bound, and finally characterizing the detection capability of the probe on CTC by measuring a time-current curve.
The specific result is shown in fig. 3, and it can be seen from fig. 3 that the current signal value corresponding to the detection of the circulating cells by using the probe and the electrode according to the technical scheme of the present application is significantly higher than that of the other two groups.
The detection range and detection limit of the electrode in the present application are considered. Maintaining the number of MCF-7 cells at 1, 102，103，104，105Within each range, a corresponding current signal value is detected. As can be seen from fig. 4, as the number of cells increases, the current intensity increases and the equation of the correlation curve log (y) 0.26952log (x) -0.12895, R is plotted20.98655. Wherein Y is the current value and X is the MCF-7 cell number.
Verification of specific detection of the electrode on circulating tumor cells in the present application:
a. separately detect 105Hela cell, 103MCF-7 cells, 103MCF-7 and 105The HeLa cell mixture is specifically shown in FIG. 5. As can be seen from FIG. 5, the current value corresponding to Hela cells is substantially the same as the background signal value, and the current signal value corresponding to the mixed solution is similar to the current signal value when MCF-7 cells exist alone, thus proving that the electrochemical biosensor and the detection system have good specificity.
b. Will 105Hela cells were compared with 0,102，103，104Mixing MCF-7 cells, and detecting current signals under different conditionsNumber values, results are shown in FIG. 6. The curves in FIG. 6 are from bottom to top; a is 0, b is 10 MCF-7 cells, c is 102MCF-7 cells with a d of 103MCF-7 cells, e is 104And MCF-7 cells. As can be seen from FIG. 6, the current intensity gradually increased as the number of target cells increased.
c. 10, 100, 500, 5000 MCF-7 target cells were added to the whole blood sample and their corresponding current signals were measured, the results are shown in FIG. 7. As can be seen from fig. 7, the results demonstrate that the results of each set of experiments are consistent with the results of the cells in the PBS solution.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Sequence listing
<120> tetrahedral nanostructure DNA-based signaling probe, preparation method and application thereof
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 85
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
acattcctaa gtctgaaaca ttacagcttg ctacacgaga agagccgcca tagtattttt 60
ttttttcaac atcagtctga taagc 85
<210> 2
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tatcaccagg cagttgacag tgtagcaagc tgtaatagat gcgagggtcc aatac 55
<210> 3
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tcaactgcct ggtgataaaa cgacactacg tgggaatcta ctatggcggc tcttc 55
<210> 4
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ttcagactta ggaatgtgct tcccacgtag tgtcgtttgt attggaccct cgcat 55
<210> 5
<211> 85
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
acattcctaa gtatgaaaca ttacagcttg ctacacgaga agagccgcca tagtattttt 60
tttttgctta tcagactgat gttga 85
<210> 6
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
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<210> 7
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
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cacgagctcc attac 75
<210> 8
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
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cacgagctcc attac 75
<210> 9
<211> 73
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<213> Artificial Sequence (Artificial Sequence)
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agctcgtggt cag 73
<210> 10
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
acattcctaa gtctgaaaca tttttttttt tcaacatcag tctgataagc 50
<210> 11
<211> 72
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
cactacagag gttgcgtctg tcccacgttg tcatgggggg ttggcctgtt tttgcttatc 60
agactgatgt ta 72
Claims (10)
1. A signaling probe based on tetrahedral nanostructure DNA, the signaling probe comprising: an aligned tetrahedral nanostructure DNA and a complex comprising an inverted tetrahedral nanostructure DNA and a biotin-modified aptamer, the inverted tetrahedral nanostructure DNA having one of the biotin-modified aptamers extending from each of three vertices thereof; the other vertex of the inverted tetrahedral nanostructure DNA can hybridize to one of the vertices of the regular tetrahedral nanostructure DNA; the aptamer can be identified and combined with circulating tumor cells to be detected.
2. The signaling probe of claim 1, wherein the orthotetrahedral nanostructure DNA comprises a1、B1、C1And D1Four chains, the nucleotide sequences of which are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4; and/or the inverted tetrahedral nano-structured DNA comprises A2、B2、C2And D2Four chains, the nucleotide sequences of which are respectively shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8; and/or the nucleotide sequence of the biotin-modified aptamer is shown as SEQ ID NO. 9.
3. An electrochemical biological detection method, comprising the steps of:
1) incubating the circulating tumor cells to be detected and the compound together to obtain the circulating tumor cells to be detected combined with the compound;
modifying the surface of the working electrode with the DNA of the regular tetrahedron nano structure;
2) hybridizing the orthotetrahedral nanostructure DNA and the inverted tetrahedral nanostructure DNA in the complex to capture circulating tumor cells on the working electrode;
3) adding oxidoreductase and corresponding substrate for electrochemical detection.
4. The electrochemical biological detection method of claim 3, wherein the complex comprises inverted tetrahedral nanostructure DNA and biotin-modified aptamer capable of recognizing and binding to the circulating tumor cells to be detected; and/or, three vertices of the inverted tetrahedral nanostructure DNA are each extended with the biotin-modified aptamer, and another vertex of the inverted tetrahedral nanostructure DNA is capable of hybridizing with one vertex of the regular tetrahedral nanostructure DNA.
5. The electrochemical bioassay method of claim 3 wherein the regular tetrahedral nanostructure DNA comprises A1、B1、C1And D1Four chains, the nucleotide sequences of which are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4; and/or the inverted tetrahedral nano-structured DNA comprises A2、B2、C2And D2Four chains, the nucleotide sequences of which are respectively shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8; and/or the DNA of the regular tetrahedron nanostructure is modified on the surface of the working electrode through Au-S bonds; and/or, the working electrode is a carbon electrode; and/or the working electrode is a screen printing electrode; and/or, directly reducing chloroauric acid into nanogold by adopting a constant potential deposition method, modifying the nanogold on the working electrode, and then bonding the nanogold and the working electrode with the DNA of the regular tetrahedron nanostructure.
6. The electrochemical bioassay method as set forth in claim 5, wherein the nucleotide sequence of the biotin-modified nucleic acid aptamer is represented by SEQ ID NO. 9.
7. An electrochemical biosensor comprising a working electrode, an orthotetrahedral nanostructure DNA, and a complex, the orthotetrahedral nanostructure DNA being modified on the working electrode; the complex comprises inverted tetrahedral nanostructure DNA and a biotin-modified aptamer; wherein three vertices of said inverted tetrahedral nanostructure DNA are each extended with one of said biotin-modified aptamers, and another vertex of said inverted tetrahedral nanostructure DNA is capable of hybridizing with one vertex of said regular tetrahedral nanostructure DNA.
8. The electrochemical biosensor of claim 7, further comprising a reference electrode and a counter electrode; and/or the DNA of the regular tetrahedron nanostructure is modified on the surface of the working electrode through Au-S bonds; and/or, the working electrode is a carbon electrode; and/or the working electrode is a screen printing electrode; and/or, directly reducing chloroauric acid into nanogold by adopting a constant potential deposition method, modifying the nanogold on the working electrode, and then bonding the nanogold and the working electrode with the DNA of the regular tetrahedron nanostructure.
9. The electrochemical biosensor of claim 7, wherein the regular tetrahedral nanostructure DNA comprises a1、B1、C1And D1Four chains, the nucleotide sequences of which are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4; and/or the inverted tetrahedral nano-structured DNA comprises A2、B2、C2And D2Four chains, the nucleotide sequences of which are respectively shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8; and/or the nucleotide sequence of the biotin-modified aptamer is shown as SEQ ID NO. 9.
10. Use of a signaling probe according to any of claims 1 to 3 or an electrochemical biosensor according to any of claims 7 to 9 for detecting circulating tumor cells.
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