CN112500850B - Three-functional magnetic aptamer trinucleotide nanoprobe - Google Patents
Three-functional magnetic aptamer trinucleotide nanoprobe Download PDFInfo
- Publication number
- CN112500850B CN112500850B CN202110169824.1A CN202110169824A CN112500850B CN 112500850 B CN112500850 B CN 112500850B CN 202110169824 A CN202110169824 A CN 202110169824A CN 112500850 B CN112500850 B CN 112500850B
- Authority
- CN
- China
- Prior art keywords
- trinucleotide
- aptamer
- stranded dna
- magnetic
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
- C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Nanotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Materials Engineering (AREA)
- Optics & Photonics (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Crystallography & Structural Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Zoology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a three-functional magnetic aptamer three-nucleic acid nano probe, which comprises: (1) magnetic beads, (2) aptamer trinucleotide framework chains, and (3) single-stranded DNA targets. The invention constructs the magnetic aptamer trinucleotide DNA nanoprobe with triple functions by taking an aptamer sequence as an identification element, trinucleotide as a molecular conversion element and magnetic beads as a separation and purification element, thereby realizing the equivalent conversion of the ligand and single-stranded DNA. The ligand which is difficult to detect is converted into the single-stranded DNA target which is easy to determine, so that the problem that the ligand is difficult to directly, qualitatively, quantitatively and simultaneously determine at present is solved.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a three-functional magnetic aptamer trinucleotide nanoprobe.
Background
Trinuclear acids belong to one class of functional nucleic acids and are one of the tools for constructing novel biosensors. Trinucleotides are triplex nucleotides that bind the third strand in the major groove of the double-stranded nucleotide duplex structure in the form of either a huster hydrogen bond or a trans-huster hydrogen bond. The formation of trinucleotide is closely related to conditions such as nucleic acid sequence, base composition, ionic environment, pH value and the like. Due to the reversibility of their structures, trinuclear acids can act as "switches" in biosensors, often used for target substance conversion.
The detection method of exosome or other ligands commonly applied at present is difficult to realize simultaneous qualitative and quantitative detection of ligand substances, and the detection technology of nucleic acid is mature, so that the conversion of ligands which are difficult to detect into single-stranded nucleic acid targets which are easy to detect is of great significance.
Disclosure of Invention
The invention aims to provide a trifunctional magnetic aptamer trinucleotide nanoprobe.
Another object of the present invention is to provide a method for converting an equivalent amount of a ligand species to a single stranded nucleic acid target.
In order to achieve the purpose of the invention, the inventor designs an aptamer trinucleotide backbone chain containing an aptamer sequence in the middle according to the specific aptamer of a ligand substance and the special requirement of formation of trinucleotide, forms a stem-loop structure through self-assembly with a single-stranded DNA target, and fixes magnetic beads at the tail end of the stem-loop structure through interaction, thereby constructing a magnetic aptamer trinucleotide probe with triple functions.
In a first aspect, the present invention provides a trifunctional magnetic aptamer trinucleotide nanoprobe, comprising: (1) magnetic beads, (2) aptamer trinucleotide framework chains, and (3) single-stranded DNA targets.
The probe has triple functions of target recognition, molecular conversion, separation and purification.
Wherein the aptamer trinucleotide backbone chain comprises: 5 'end nucleic acid sequence A, aptamer sequence B and 3' end nucleic acid sequence C; the single-stranded DNA target includes: a single-stranded nucleic acid sequence D; the aptamer sequence B is positioned between the 5 'end nucleic acid sequence A and the 3' end nucleic acid sequence C; the aptamer sequence B comprises a nucleic acid sequence which can be specifically combined with a ligand substance; the single-stranded nucleic acid sequence D can self-assemble with the 5 'end nucleic acid sequence A and the 3' end nucleic acid sequence C to form a trinucleotide.
The aptamer trinucleotide framework chain and the single-stranded DNA target are self-assembled to form a stem-loop structure, the loop part is an aptamer sequence B, and the stem part is trinucleotide.
The magnetic beads are fixed at the 5' end of the aptamer trinucleotide framework chain in the stem-loop structure through interaction.
When the ligand exists, the ring part of the probe can recognize and combine the ligand, the conformation of the aptamer trinucleotide framework chain is changed, the trinucleotide structure of the stem part is disintegrated, and the single-stranded DNA target is released. Separating the single-stranded DNA target by an external magnetic field, wherein the amount of the single-stranded DNA target and the ligand in the supernatant are positively correlated.
Specifically, the probe further comprises at least one of the following 1) to 3):
1) the aptamer trinucleotide backbone chain comprises: the SEQ ID No: 3 and SEQ ID No: 5 is expressed by the nucleotide sequence shown in SEQ ID No: 4 to obtain a nucleic acid chain;
2) the aptamer trinucleotide backbone chain comprises: the SEQ ID No:1 is substituted and/or deleted and/or added by one or more nucleotides and has the nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID No:1 has the same function;
3) the single-stranded DNA target includes: the SEQ ID No: 2 is substituted and/or deleted and/or added by one or more nucleotides and has the nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID No: 2 has the same function.
The A, B, C, D is used only to distinguish between different sequences and not for sorting.
The invention also provides the use of the aforementioned probe for the conversion of a ligand species to a single stranded nucleic acid target, which conversion may be manifested as an equivalence conversion.
In a second aspect, the present invention provides a method for converting a ligand substance into a single-stranded nucleic acid target in equal amounts using the probe, comprising the steps of:
s1, mixing and incubating the aptamer trinucleotide backbone chain and a single-stranded DNA target to obtain aptamer trinucleotide nanoprobe stock solution;
s2, coupling the magnetic beads with the aptamer trinucleotide probes to obtain magnetic aptamer trinucleotide probes;
and S3, mixing and incubating the obtained magnetic aptamer trinucleotide nanoprobe and the ligand, and carrying out magnetic separation to obtain a converted single-stranded DNA target solution.
S1 is specifically as follows: and (3) mixing the aptamer trinucleotide framework chain and the single-stranded DNA target according to the ratio of 1: 1-1.5, incubating for 0.5-1.5h under the conditions of light shielding and 35-40 ℃ to obtain aptamer trinucleotide nanoprobe stock solution.
Specifically, the aptamer trinucleotide framework chain and the single-stranded DNA target are arranged in a mode of 1: 1-1.1, incubating for 1h under the conditions of keeping out of the sun and 37 ℃ to obtain aptamer trinucleotide nanoprobe stock solution.
S2 is specifically as follows: and adding 80-120 mu L of aptamer trinucleotide nano-probe stock solution into the magnetic beads, uniformly mixing, and incubating for 0.5-1.5h at room temperature under the condition of rotation and slow oscillation to obtain the magnetic aptamer trinucleotide nano-probe.
And concretely, adding 100 mu L of aptamer trinucleotide nano-probe stock solution into the magnetic beads, uniformly mixing, and incubating for 1h at room temperature under the conditions of rotation and slow oscillation to obtain the magnetic aptamer trinucleotide nano-probe.
S3 is specifically as follows: and taking 40-60 muL of the obtained magnetic aptamer trinucleotide nano-probe solution, adding 10-20 muL of the ligand solution, incubating for 1-3h at 35-40 ℃ under a light-tight condition, and performing magnetic separation to obtain a supernatant solution containing the free single-stranded DNA target.
And specifically, taking 50 muL of the obtained magnetic aptamer trinucleotide nanoprobe solution, adding 15 muL of the ligand solution, incubating for 2h at 37 ℃ under a light-tight condition, and performing magnetic separation to obtain a supernatant solution containing the free single-stranded DNA target.
The invention provides a method for equivalently converting a ligand substance into a single-stranded nucleic acid target, which comprises the following steps:
firstly, an aptamer trinucleotide framework chain and a single-stranded DNA target are mixed according to the proportion of 1: 1-1.5, incubating for 0.5-1.5h under the conditions of light shielding and 35-40 ℃ to obtain aptamer trinucleotide nanoprobe stock solution. And adding 80-120 mu L of aptamer trinucleotide nano-probe stock solution into the magnetic beads, uniformly mixing, and incubating for 0.5-1.5h at room temperature under the condition of rotation and slow oscillation to obtain the magnetic aptamer trinucleotide nano-probe. And taking 40-60 muL of the obtained magnetic aptamer trinucleotide nano-probe solution, adding 10-20 muL of the ligand solution, incubating for 1-3h at 35-40 ℃ under a light-tight condition, and performing magnetic separation to obtain a supernatant solution containing the free single-stranded DNA target, wherein the amount of the free single-stranded DNA target is the same as that of the ligand.
The aptamer trinucleotide backbone chain comprises: 5 'end nucleic acid sequence A, aptamer sequence B and 3' end nucleic acid sequence C; the single-stranded DNA target can self-assemble with the 5 'end nucleic acid sequence a and the 3' end nucleic acid sequence C of the aptamer trinucleotide backbone chain to form a trinucleotide; the aptamer is specifically bound to its ligand substance.
Preferably, the T-A: T ratio of the trinucleotide is 50%.
Preferably, in the above method, the aptamer trinucleotide backbone chain and single-stranded DNA target base sequences are as follows:
aptamer trinucleotide backbone chain: 5' -TTTTTCTCTCCCTTTCACCCCACCTCGCTCCCGTGACACTAATGC TATTTCCCTCTC-3’
Single-stranded DNA target: 5'-GAGAGAGAGAGAGAGAGGGAAAAGGAAAGG-3'
Wherein the underlined part indicates the aptamer sequence.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention uses an aptamer sequence as an identification element, trinucleotide as a molecular conversion element and magnetic beads as a separation and purification element, constructs a magnetic aptamer trinucleotide nano probe with triple functions, is used for carrying out equivalent conversion on a ligand substance and a single-stranded nucleic acid target, converts a ligand which is difficult to measure into the single-stranded nucleic acid target which is easy to measure, and can overcome the problem that the simultaneous qualitative and quantitative detection of the ligand substance is difficult to realize at present.
The nano probe is of a stem-loop structure, and the loop part is an aptamer sequence and can be used for recognizing and combining a ligand substance; the stem part is trinucleotide, can fix a single-stranded DNA target and is used as a molecular switch to convert target molecules; the terminal magnetic beads can be separated and purified under the action of an external magnetic field; the quantity of the single-stranded DNA target and the ligand substance in the obtained supernatant is in positive correlation, and the single-stranded DNA target and the ligand substance can be directly used for subsequent detection.
The probe is simple and universal in construction, and conversion between various ligand substances and single-stranded DNA targets can be realized only by replacing specific aptamer sequences in aptamer trinucleotide chains.
And (III) converting the ligand substance which is difficult to measure into a single-stranded nucleic acid target which is easy to detect, so that the simultaneous qualitative and quantitative measurement of the ligand substance is facilitated.
Drawings
FIG. 1 shows the principle of the target transformation of the magnetic aptamer trinucleotide nanoprobe of the invention
FIG. 2 shows the result of the successful assembly of the aptamer trinucleotide nanoprobes verified by the fluorescence method in example 1 of the present invention; wherein, the black curve represents that the system only contains an aptamer three-nucleic acid skeleton chain of which the 5 'end and the 3' end are respectively marked with a fluorescent group and a quenching group, and the red curve represents that the system simultaneously contains the skeleton chain and a single-stranded DNA target.
FIG. 3 shows the results of verifying the target transition function of the aptamer trinucleotide nanoprobe by fluorescence in example 1 of the present invention; wherein, the red curve represents that the system only contains the stem-loop structured aptamer three-nucleic acid nano probe, and the blue curve represents that exosome is added into the stem-loop structured aptamer three-nucleic acid nano probe system.
FIG. 4 is a Zeta potential representation of the successful assembly of magnetic aptamer trinucleotide nanoprobes in example 1 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
In the invention, the formula of the working buffer solution is as follows: 10mM HEPES, 150mM NaCl, pH 5.5.
Example 1 construction of a Tri-functional magnetic aptamer trinucleotide nanoprobe
1. Experimental Material
HEPES and sodium chloride were purchased from Aladdin, and magnetic beads were purchased from Zhongke thunder (Beijing) technologies, Inc. The experimental water was obtained from a Milli-Q pure water system.
The sequence was designed as follows (SEQ ID NOS: 1-2):
note: the aptamer sequence of the exosome surface CD63 protein is underlined;
the bold part indicates the sequence forming the trinucleotide.
2. Construction and characterization of aptamer trinucleotide nanoprobes
The aptamer three-nucleic acid skeleton chain with the 5 'end and the 3' end respectively marked with a fluorescent group and a quenching group and a single-stranded DNA target are mixed according to the proportion of 1: 1.1, and incubating for 1h under the conditions of keeping out of the sun and 37 ℃ to obtain aptamer trinucleotide nanoprobe stock solution with the concentration of 200 nM; the fluorescence intensity was measured with a microplate reader under conditions of 488nm for excitation light and 500-600nm for emission light. The single aptamer trinucleotide backbone chain solution has strong fluorescence, and after the single-stranded DNA target is added, due to the formation of a trinucleotide stem-loop structure, a fluorescent group is close to a quenching group, fluorescence quenching occurs, and the fluorescence intensity is obviously reduced; the successful construction of aptamer trinucleotide nanoprobes of stem-loop structures is illustrated (FIG. 2); the fluorescence quenching rate is close to 60%, which shows that the self-assembly rate of the probe is about 60%.
3. Target conversion function verification of aptamer trinucleotide nanoprobe
The aptamer three-nucleic acid skeleton chain with the 5 'end and the 3' end respectively marked with a fluorescent group and a quenching group and a single-stranded DNA target are mixed according to the proportion of 1: 1.1, and incubating for 1h under the conditions of keeping out of the sun and 37 ℃ to obtain aptamer trinucleotide nanoprobe stock solution with the concentration of 200 nM; taking 50 muL of the obtained stock solution, adding 15 muL of exosome solution to serve as an experimental group, taking 50 muL of the obtained stock solution, adding 15 muL of working buffer solution to serve as a control group, and incubating for 2 hours at 37 ℃ under a light-tight condition; the fluorescence intensity was measured with a microplate reader under conditions of 488nm for excitation light and 500-600nm for emission light. After the single aptamer trinucleotide nanoprobe solution is added into an exosome, the aptamer sequence of the loop part is combined with the exosome surface protein CD63, the nucleic acid conformation is changed, the stem-loop structure is disintegrated, the single-stranded DNA target is released, the fluorescent group and the quenching group are far away from each other, and the fluorescence is recovered; the target molecule switching function of the aptamer trinucleotide nanoprobe was demonstrated (figure 3).
4. Assembly characterization of magnetic aptamer trinucleotide nanoprobes
Zeta potential characterization is carried out on the prepared magnetic aptamer trinucleotide Nano probe by adopting a Zetasizer Nano ZS analyzer. Before the DNA is not modified, the surface potential of the magnetic bead is-2.68 mV, when the DNA is bound on the surface of the magnetic bead, the potential is obviously reduced, and the potential of the binding of the aptamer trinucleotide probe is reduced more than that of the binding of the aptamer trinucleotide chain, which indicates the successful assembly of the magnetic aptamer trinucleotide probe (FIG. 4).
Example 2 validation of effective target conversion efficiency
The aptamer three-nucleic acid skeleton chain with the 5 'end and the 3' end respectively marked with a fluorescent group and a quenching group and a single-stranded DNA target are mixed according to the proportion of 1: 1.1, and incubating for 1h under the conditions of keeping out of the sun and 37 ℃ to obtain aptamer trinucleotide nanoprobe stock solution with the concentration of 200 nM; detecting the fluorescence intensity by using a microplate reader under the conditions of 488nm of exciting light and 500-600nm of emitted light, storing and recording, recording the fluorescence intensity at 532nm of maximum emitted light as I1, recording the fluorescence intensity of a control group as I2, and calculating the fluorescence quenching rate Q, wherein the calculation formula is as follows: q = (I2-I1)/I2 × 100%.
Taking 50 muL of the obtained stock solution, and adding 15 muL of exosome solution as an experimental group; taking 50 muL of the obtained stock solution, adding 15 muL of working buffer solution as a control group, and incubating for 2h at 37 ℃ under a dark condition; and (3) detecting the fluorescence intensity of the obtained experimental group and control group solution under the conditions of 488nm exciting light and 500-600nm emitting light by using an enzyme-labeling instrument, storing and recording, recording the fluorescence intensity of the experimental group when the maximum emitting light is 532nm as I3, recording the fluorescence intensity of the control group as I4, and calculating the fluorescence enhancement rate F, wherein the calculation formula is as follows: f = (I3-I4)/I3 × 100%.
The Q is 79.21%, the F is 78.65%, and the conversion rate of the effective target (the product of Q and F) reaches 62.30%.
Sequence listing
<110> university of agriculture in China
<120> a three-functional magnetic aptamer trinucleotide nanoprobe
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 57
<212> DNA
<213> Artificial Sequence
<400> 1
tttttctctc cctttcaccc cacctcgctc ccgtgacact aatgctattt ccctctc 57
<210> 2
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 2
gagagagaga gagagaggga aaaggaaagg 30
<210> 3
<211> 15
<212> DNA
<213> Artificial Sequence
<400> 3
tttttctctc ccttt 15
<210> 4
<211> 32
<212> DNA
<213> Artificial Sequence
<400> 4
caccccacct cgctcccgtg acactaatgc ta 32
<210> 5
<211> 10
<212> DNA
<213> Artificial Sequence
<400> 5
Claims (5)
1. A trifunctional magnetic aptamer trinucleotide nanoprobe, comprising: (1) magnetic beads, (2) aptamer trinucleotide framework chains, (3) single-stranded DNA targets;
the probe has triple functions of target recognition, molecular conversion, separation and purification;
the 5' end of the aptamer trinucleotide framework chain is fixed on a magnetic bead;
wherein, the aptamer trinucleotide framework chain sequence has the following sequence from 5 'to 3': trinucleotide sequence A, aptamer sequence B and trinucleotide sequence C;
wherein the three nucleic acid sequences A are: 5'-TTTTTCTCTCCCTTT-3', respectively;
the three nucleic acid sequences C are: 5'-TTTCCCTCTC-3', respectively;
the single-stranded DNA target is a single-stranded nucleic acid sequence D which is: 5'-GAGAGAGAGAGAGAGAGGGAAAAGGAAAGG-3' are provided.
2. The probe of claim 1, wherein the aptamer trinucleotide backbone strand self-assembles with the single-stranded DNA target to form a stem-loop structure, the loop portion being the aptamer sequence B and the stem portion being a trinucleotide.
3. The probe of claim 2, wherein the loop portion of the probe recognizes and binds the ligand when the ligand is present, the aptamer trinucleotide backbone chain conformation changes, the trinucleotide structure of the stem portion disintegrates, releasing the single-stranded DNA target; separating the single-stranded DNA target by an external magnetic field, wherein the amount of the single-stranded DNA target and the ligand in the supernatant are positively correlated.
4. A trifunctional magnetic aptamer trinucleotide nanoprobe, comprising: (1) magnetic beads, (2) aptamer trinucleotide framework chains, (3) single-stranded DNA targets;
the aptamer trinucleotide backbone chain is shown as SEQ ID NO: 1;
the single-stranded DNA target is SEQ ID NO: 2;
the probe has triple functions of target recognition, molecular conversion, separation and purification;
the aptamer trinucleotide backbone chain is immobilized at the 5' end on a magnetic bead.
5. Use of the probe of any one of claims 1 to 4 for identification and target switching for non-therapeutic or diagnostic purposes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110169824.1A CN112500850B (en) | 2021-02-08 | 2021-02-08 | Three-functional magnetic aptamer trinucleotide nanoprobe |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110169824.1A CN112500850B (en) | 2021-02-08 | 2021-02-08 | Three-functional magnetic aptamer trinucleotide nanoprobe |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112500850A CN112500850A (en) | 2021-03-16 |
| CN112500850B true CN112500850B (en) | 2021-04-27 |
Family
ID=74952731
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110169824.1A Active CN112500850B (en) | 2021-02-08 | 2021-02-08 | Three-functional magnetic aptamer trinucleotide nanoprobe |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112500850B (en) |
-
2021
- 2021-02-08 CN CN202110169824.1A patent/CN112500850B/en active Active
Non-Patent Citations (4)
| Title |
|---|
| A sensitive SERS assay for detecting proteins and nucleic acids using a triple-helix molecular switch for cascade signal amplification;Sujuan Ye等;《Chem Commun (Camb)》;20140825;第50卷(第60期);图1 * |
| Highly Sensitive Electrochemical Detection of Tumor Exosomes Based on Aptamer Recognition-Induced Multi-DNA Release and Cyclic Enzymatic Amplification;Huilei Dong等;《Anal. Chem.》;20180305;第90卷(第7期);摘要和图1 * |
| Jing Zheng等.Universal Surface-Enhanced Raman Scattering Amplification Detector for Ultrasensitive Detection of Multiple Target Analytes.《Anal. Chem.》.2014,第86卷(第4期),第2205页右栏最后一段和图1. * |
| Universal Surface-Enhanced Raman Scattering Amplification Detector for Ultrasensitive Detection of Multiple Target Analytes;Jing Zheng等;《Anal. Chem.》;20140118;第86卷(第4期);第2205页右栏最后一段和图1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112500850A (en) | 2021-03-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112501353B (en) | Kit for detecting new coronavirus and mutant thereof | |
| KR20080084029A (en) | Detection method for target protein using aptamer and the detection kit | |
| JPH09508268A (en) | Nucleic acid sequencing | |
| CN103436608B (en) | Rapid detection method based on nucleic acid aptamers and kit | |
| CN113604612B (en) | Aronia virus loop-mediated isothermal amplification detection primer set, kit containing primer set and application of kit | |
| CN112626242B (en) | Method for detecting food-borne pathogenic bacteria based on double signals of nucleic acid conformation initiation chain replacing driving DNA Walker | |
| CN108796040B (en) | Bioluminescence detection probe based on transcription activator-like effector and construction method and application thereof | |
| CN112500850B (en) | Three-functional magnetic aptamer trinucleotide nanoprobe | |
| CN108395462B (en) | Novel DNA probe synthesis method for Y-STR locus detection | |
| CN102220417A (en) | Method for detecting food pathogen by electrochemical luminescence gene sensor on basis of magnetic in-situ amplification | |
| Chen et al. | Dual-channel fluorescence detection of antibiotic resistance genes based on DNA-templated silver nanoclusters | |
| CN111855625B (en) | CA125 detection kit based on Cu-MOF and application thereof | |
| CN104178463B (en) | A kind of method for preparing enhanced monomer bacterial luciferase luxAB | |
| CN112609010A (en) | CRISPR-Cas13 nucleic acid detection kit based on lighting RNA aptamer | |
| CN112280829B (en) | Kit, sample marking method and single-cell sequencing method | |
| CN108251428B (en) | Aptamer for identifying various organophosphorus pesticides and application thereof | |
| CN112175958B (en) | Optimized aptamer sequence for specifically recognizing Listeria monocytogenes and application thereof | |
| CN109689681B (en) | Recombinant protein derived from limulus and DNA encoding the same | |
| CN113528412B (en) | Explosive visual biosensor based on escherichia coli cell surface display technology and preparation method and application thereof | |
| CN111876423B (en) | Aptamer of nitrobenzene, aptamer derivative and application of aptamer derivative | |
| CN116297376B (en) | Photoelectric dual-mode biological probe and application thereof | |
| CN102373212A (en) | Chymotrypsin nucleic acid aptamers, preparation method and application thereof | |
| US20140328762A1 (en) | Fluorogenic Peptide Probes and Assays | |
| CN108396029B (en) | A group of oligonucleotide aptamers capable of specifically recognizing Escherichia coli O157H 7 in different growth stages | |
| JP4560285B2 (en) | Hybridization assay of nucleic acid amplification products using nucleic acid precipitants |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |