CN114250271B - Amplification-free single quantum dot biosensor for detecting TET2 at single molecule level and detection method and application thereof - Google Patents

Amplification-free single quantum dot biosensor for detecting TET2 at single molecule level and detection method and application thereof Download PDF

Info

Publication number
CN114250271B
CN114250271B CN202111436593.2A CN202111436593A CN114250271B CN 114250271 B CN114250271 B CN 114250271B CN 202111436593 A CN202111436593 A CN 202111436593A CN 114250271 B CN114250271 B CN 114250271B
Authority
CN
China
Prior art keywords
tet2
dsdna
quantum dot
probe
reporter probe
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
Application number
CN202111436593.2A
Other languages
Chinese (zh)
Other versions
CN114250271A (en
Inventor
张春阳
刘明昊
王传瑞
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shandong Normal University
Original Assignee
Shandong Normal University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Shandong Normal University filed Critical Shandong Normal University
Priority to CN202111436593.2A priority Critical patent/CN114250271B/en
Publication of CN114250271A publication Critical patent/CN114250271A/en
Application granted granted Critical
Publication of CN114250271B publication Critical patent/CN114250271B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90245—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention provides a single quantum dot biosensor without amplification for detecting TET2 at a single molecular level, and a detection method and application thereof, belonging to the technical field of fluorescence detection. The single quantum dot sensor includes at least: reporter probes, capture probes, quantum dots, and free fluorescent dyes. The single quantum dot sensor designed by the invention is based on the formation of biotin-labeled Cy5-dsDNA complex, and the biotin-labeled dsDNA complex is assembled on the surface of 605nm emission quantum dot (605 QD) through biotin-streptavidin interaction, so that 605QDdsDNA-Cy5 nano structure is obtained for detecting TET2. The method can quantitatively monitor the activity of TET2 by simply counting Cy5 molecules under the condition of not involving any signal amplification, and has the advantages of simple and rapid operation and accurate and reliable test result.

Description

Amplification-free single quantum dot biosensor for detecting TET2 at single molecule level and detection method and application thereof
Technical Field
The invention belongs to the technical field of fluorescence detection, and particularly relates to a single quantum dot biosensor without amplification for detecting TET2 at a single molecular level, and a detection method and application thereof.
Background
The information disclosed in the background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an admission or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.
TET2 is a dioxygenase that depends on iron (II) and alpha-ketoglutarate (alpha KG) and is capable of oxidizing 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5 hmC) and then to 5-formylcytosine (5 fC) or 5-carboxycytosine (5 caC). The oxidation products of cytosine can be recognized and cleaved by Thymine DNA Glycosylase (TDG) to produce basic nucleotides that can be substituted with cytosine by DNA Base Excision Repair (BER), thereby initiating an active DNA demethylation process in mammals. Normal activity of TET2 ensures dynamic equilibrium of methylation in organisms. In addition, abnormal TET2 expression is closely related to alzheimer's disease, hematopoietic diseases, asthma, cancer, and the like.
Traditional detection methods for detecting TET2 include chromatin immunoprecipitation (ChIP), enzyme-linked immunosorbent assay (ELISA), western blotting, classical Thin Layer Chromatography (TLC), liquid chromatography-mass spectrometry (LC-MS), fluorescence polarization, electrochemical methods. The chromatin immunoprecipitation method is to form a protein-DNA complex in living cells, divide the complex into chromatin fragments, precipitate the complex by an immunological method, and enrich the DNA fragment bound to a target protein for detection. Enzyme-linked immunosorbent assay (ELISA) is based on covalent binding of an enzyme molecule to an antibody or an anti-antibody molecule, and the presence or absence of a corresponding reaction is determined by measurement with an ELISA detector. Western blotting uses specific antibodies to stain a gel-electrophoretically treated cell or biological tissue sample, and then analyzes the stained location and depth for detection. Classical thin layer chromatography is to uniformly coat a stationary phase on a flat plate with a smooth surface to form a thin layer for separation and detection. Liquid chromatography-mass spectrometry (LC-MS) is a method in which a liquid is used as a mobile phase, and various eluents are added to a chromatographic column to separate various components in the column, and the separated components are introduced into a mass spectrometer and separated and detected by a mass analyzer. The fluorescence polarization method is based on that after fluorescent substances are irradiated by polarized light blue light (wavelength of 485 nm) with a single plane, the fluorescent substances can jump into an excited state, and when the fluorescent substances return to a ground state, energy is released and polarized fluorescence (wavelength of 525 nm) with the single plane is emitted. The measurement is performed by polarized light intensity. Electrochemical methods are based on the electrochemical behavior of proteins on solid electrodes, with changes in electrical parameters being used for detection.
However, the inventors found that these conventional detection methods are too complicated in detection steps and long in analysis time; relates to complicated probe and template designs, has high background signals caused by nonspecific amplification, and has poor specificity and sensitivity.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a single quantum dot sensor for detecting TET2 without amplification at a single molecular level, and a detection method and application thereof. The single quantum dot sensor designed by the invention is based on the formation of biotin-labeled Cy5-dsDNA complex, and the biotin-labeled dsDNA complex is assembled on the surface of 605nm emission quantum dot (605 QD) through biotin-streptavidin interaction, so that 605QDdsDNA-Cy5 nano structure is obtained for detecting TET2. By using the effective Fluorescence Resonance Energy Transfer (FRET) from 605QD to Cy5, the method can quantitatively monitor the TET2 activity by simply counting Cy5 molecules without any signal amplification, and has the advantages of simple and rapid operation and accurate and reliable test result.
In order to achieve the technical purpose, the technical scheme of the invention is as follows:
in a first aspect of the present invention, there is provided a single quantum dot sensor comprising at least:
reporter probes, capture probes, quantum dots, and free fluorescent dyes.
Wherein the reporter probe has a complementary region that specifically hybridizes to the capture probe, so that both can form double-stranded DNA.
The reporter probe is modified with 5-iodocytosine (5-IC) capable of forming 5-vinylcytosine under the action of a palladium complex;
the terminal end of the capture probe is modified with biotin, and further, the 3' -end of the capture probe is modified with biotin.
The Quantum Dots (QDs) have significant advantages of excellent photostability, long fluorescence lifetime and high quantum yield, broad excitation, narrow and symmetrical emission spectrum, and broad absorption spectrum. Can be used as ideal energy transfer donor for manufacturing the biosensor based on Fluorescence Resonance Energy Transfer (FRET).
The fluorescent dye is Cy5; more specifically, the Cy5 is an aminated Cy5 (Cy 5-NH) 2 )。
In a second aspect of the invention there is provided the use of a single quantum dot sensor as described above in the detection of TET2.
In a third aspect of the invention, there is provided a method of detecting TET2, the method comprising detecting using a single quantum dot sensor as described above.
In a fourth aspect of the invention there is provided the use of a single quantum dot sensor and/or detection method as described above in TET 2-related drug screening and/or biological sample TET2 analysis.
The beneficial technical effects of one or more of the technical schemes are as follows:
(1) Without additional amplification steps
Conventional detection related enzymes often incorporate complex amplification steps that often require complex template designs for increased sensitivity, and false positive signals are prone to occur during the amplification process. The single-molecule detection used in the technical scheme has unique advantages in the ultra-sensitive detection aspect, and high sensitivity can be achieved without additional amplification steps.
(2) The FRET biosensor based on the single quantum dot is constructed by the technical scheme
TET2 mediated oxidation of 5-VC to 5-FMC can induce the formation of biotinylated Cy 5-labeled dsDNA complexes that can assemble onto 605QD surfaces, resulting in 605QDdsDNA-Cy5 nanostructures, effectively going from 605QD to Cy5. The biosensor is very simple, and uniform and sensitive detection of TET activity can be realized without any specific antibody.
In conclusion, the preparation and detection methods of the single quantum dot sensor are simple, the detection sensitivity is improved, the detection cost is effectively reduced, and meanwhile, the technical scheme is adopted to react with the pH value, the incubation time of TET2 and Cy5-NH of each reaction condition 2 The proportion of the fluorescent dye to the double-stranded DNA and the concentration of the double-stranded DNA are carefully optimized, so that in the detection process, the detection sensitivity is greatly improved, the occurrence of nonspecific reaction is effectively prevented, and the accurate detection of TET2 in a complex biological sample can be realized.
Experiments prove that the detection limit of the technical scheme on TET2 is 0.042 nanograms per microliter. Compared with an electrochemiluminescence biosensor (0.37 microgram per milliliter) based on gold nano particles, the sensitivity of the sensor is improved by 10 times, so that the sensor has good practical application value.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a schematic diagram of a mechanism of a single quantum dot sensor of the present invention for detecting TET 2;
FIG. 2 is a diagram showing the feasibility of detecting TET2 according to the embodiment of the invention; wherein A is polyacrylamide gel electrophoresis analysis, SYBRgold is used as an indicator, and a reaction product is subjected to PAGE analysis; b is direct excitation Cy5 to carry out PAGE analysis on the reaction product; c is the superposition of the PAGE analysis results of A and B; d is the fluorescence emission spectra of 605QD and Cy5 were measured without TET2 and in the presence of TET 2; e is the fluorescence lifetime curve of 605QDs without TET2 and in the presence of TET2, the lifetime of which was measured in the 605nm emission channel; wherein the TET2 concentration is 10 nanograms per microliter, the double-stranded DNA concentration is 500 nanomoles per liter, and the Cy5-NH concentration is 2 The concentration was 5. Mu. Mol/l.
FIG. 3 is a schematic diagram of a single-molecule imaging detection TET2 imaging by total internal reflection fluorescence microscopy in accordance with an embodiment of the invention; . The 605QD fluorescence signal is represented by green (a, D), the Cy5 fluorescence signal is represented by red (B, E), and the 605QD and Cy5 fluorescence signals coexist is represented by yellow (C, F). TET2 concentration was nanograms per microliter, and the scale was 5 micromolar.
FIG. 4 is a graph of the optimization of different conditions in an embodiment of the invention. Wherein, A is the optimization of pH value, B is the optimization of TeT2 incubation time, and C is Cy5-NH 2 Molar ratio optimization with double-stranded DNA, plot D is double-stranded DNA concentration optimization.
FIG. 5 is a graph showing the correlation between sensitivity and specificity of a single quantum dot sensor to TET2 in the embodiment of the invention, wherein A is the effect of different TET2 concentrations on the Cy5 count corresponding to different concentrations of TET2 measured under the optimal experimental conditions; b is a control comparing 10 nanograms per microliter of TET2, 0.1 units per microliter of Bovine Serum Albumin (BSA), 0.1 units per microliter of Uracil DNA Glycosylase (UDG), 0.1 units per microliter of m.sssi, 0.1 units per microliter of Cy5 cell count generated by HAEA iii, and no target. Error bars represent standard deviation of triplicate experiments.
FIG. 6 is a diagram showing the correlation of inhibitor analysis and the detection of cell samples in the examples of the present invention; wherein A is the influence of NOG with different concentrations on the relative activity of TET 2; b is the effect of different cell numbers of HEK-293T on Cy5 cell counts at a TET2 concentration of 10 nanograms per microliter. The inset shows the linear Cy5 counts versus the log HEK-293T cell numbers. Error bars represent standard deviation of triplicate experiments.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. 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 application 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 example embodiments in accordance with the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The invention will now be further illustrated with reference to specific examples, which are given for the purpose of illustration only and are not intended to be limiting in any way. If experimental details are not specified in the examples, it is usually the case that the conditions are conventional or recommended by the reagent company; reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified.
As described above, the conventional detection method for detecting TET2 has the defects of excessively complex detection steps, long analysis time, complicated probe and template design, high background signal caused by non-specific amplification, and poor specificity and sensitivity.
In view of this, the present invention constructs a single quantum dot sensor comprising at least: reporter probes, capture probes, quantum dots, and free fluorescent dyes.
Wherein the reporter probe has a complementary region that specifically hybridizes to the capture probe, so that both can form double-stranded DNA.
The reporter probe is modified with 5-iodocytosine (5-IC) capable of forming 5-vinylcytosine under the action of a palladium ligand complex;
the terminal end of the capture probe is modified with biotin, and further, the 3' -end of the capture probe is modified with biotin.
In one embodiment of the present invention, the length of the reporter probe is 25nt, and the base sequence of the reporter probe is 5' -CTCCTCCCCCATCTCCTCCCAGTCC-3', underlined base'C"means 5-iodocytosine.
In one specific embodiment of the invention, the length of the capture probe is 25nt, the base sequence of the capture probe is 5' -GGACTGGGAGGAGATGGGGGAGGAG-biotin-3', and the 3' -end of the capture probe is marked with biotin.
The Quantum Dots (QDs) have significant advantages of excellent photostability, long fluorescence lifetime and high quantum yield, broad excitation, narrow and symmetrical emission spectrum, and broad absorption spectrum. Can be used as ideal energy transfer donor for manufacturing the biosensor based on Fluorescence Resonance Energy Transfer (FRET). In a specific embodiment of the present invention, the quantum dot is 605QD, and further, the 605QD is 605QD coated with streptavidin.
In one embodiment of the invention, the fluorescent dye is Cy5; more specifically, the Cy5 is an aminated Cy5 (Cy 5-NH) 2 )。
In the present invention, the 605QD acts as a FRET donor and Cy5 acts as a FRET acceptor, since the crosstalk of 605QD emissions is negligible and there is significant spectral overlap between 605QD emissions and Cy5 absorption spectra. Theoretically, the radius of 605QD coated with streptavidin is 5.0-7.5nm, and the distance between adjacent bases in double-stranded DNA is 0.34nm. The distance between 605QD and Cy5 in the 605QD-dsDNA-Cy5 nanostructure was calculated to be 9.08-11.58nm, and the fluorescence resonance energy was measured in FRET (2R 0 =15.4 nm). So an effective FERT can be generated between 605QD and Cy5.
Specifically, the principle of detecting TET2 by the single quantum dot sensor is as follows:
the invention designs a reporting probe modified by 5-iodo-cytosine (5-IC)A needle and a 3' -end biotin-labeled capture probe. The reporter probe can be paired with the capture probe to form a complementary double-stranded structure by Watson-Crick base pairing. First, a 5-iodocytosine (5-IC) modified reporter probe is subjected to a Suzuki-Miyaura reaction in the presence of a palladium ligand complex to produce a 5-vinylcytosine (5-VC) modified reporter probe, which is then hybridized with a biotinylated capture probe to form a 5-VC modified double stranded DNA. Second, TET2 catalyzes the oxidation of 5-VC in double stranded DNA to form an oxidation product of 5-formylmethylcytosine (5-FMC) modified double stranded DNA. Third, the aldehyde group of 5-formylmethylcytosine and free Cy5-NH 2 Through nucleophilic addition and elimination reactions, a biotin-labeled Cy 5-double stranded DNA complex is formed. Fourth, biotin-labeled double-stranded DNA complexes are assembled onto the surface of 605nm emission quantum dots (605 QDs) by biotin-streptavidin interactions, resulting in 605QDdsDNA-Cy5 nanostructures, resulting in efficient FRET from 605QDs to Cy5. TET2 activity can be quantitatively monitored by simply counting Cy5 molecules. Thus, the method enables TET2 activity and kinetic parameter determination without any signal amplification involved.
Accordingly, in a further embodiment of the present invention there is provided the use of a single quantum dot sensor as described above for detecting TET2.
In yet another embodiment of the present invention, a method of detecting TET2 is provided, the method comprising detecting using the single quantum dot sensor described above.
Specifically, the method comprises the following steps:
s1, incubating a reporter probe modified by 5-vinyl cytosine (5-vC) obtained after pretreatment with a capture probe to obtain double-stranded DNA;
s2, incubating the sample to be detected and the double-stranded DNA obtained in the step S1, inactivating at a high temperature, and then adding fluorescent dye to continue incubation; finally, quantum dots are added.
In the step S1, the specific method for obtaining the 5-vinylcytosine modified reporter probe after pretreatment is as follows: the above-described 5-iodocytosine (5-IC) -modified reporter probe undergoes a Suzuki-Miyaura reaction in the presence of a palladium ligand complex to produce a 5-vinylcytosine (5-VC) -modified reporter probe.
More specifically, the pretreatment method comprises the following steps: adding vinylboric acid MIDA ester, 5-iodo cytosine modified reporter probe, palladium (II) acetate and 2-amino-4, 6-dihydroxypyrimidine disodium into Tris buffer solution, heating at 85-95deg.C (preferably 90deg.C) for 1-3h (preferably 2 h), and centrifuging and purifying.
The co-incubation conditions of the 5-vinylcytosine (5-vC) modified reporter probe and the capture probe are specifically as follows: incubation is carried out for 1-10 minutes (preferably 5 minutes) at 90-100℃ (preferably 95℃).
In the step S2, the high-temperature inactivation after incubating the sample to be tested and the double-stranded DNA obtained in the step S1 specifically includes: incubation is carried out at 30-40deg.C (preferably 37deg.C) for 1-3h (preferably 2 h), the reaction environment is weakly acidic (such as pH6.5-6.8, preferably pH 6.8), and inactivation is carried out at 80-90deg.C (preferably 85deg.C) for 10-60 min (preferably 20 min).
Then adding fluorescent dye to continue incubation specifically as follows: cy5-NH was added 2 Incubating for 6-18h (preferably 12 h) at 30-40 ℃ (preferably 37 ℃) to generate Cy5-dsDNA complex; further, excess Cy5-NH was removed 2 ；
Cy5-NH 2 The molar ratio of dsDNA is 5-10:1, since high concentration of Cy5-NH2 can improve the reaction efficiency and enhance the fluorescence intensity of Cy5, however, an internal filtration effect may be generated, resulting in weakening of the fluorescence signal of Cy5. Cy5-NH 2 Molar ratio to dsDNA from 5:1 to 10: at 1, the F/F0 value gradually increases, exceeding 10: since the F/F0 value decreases at 1, cy5-NH is preferably used 2 The ratio to dsDNA was 10:1.
at the same time, the concentration of dsDNA was optimized in the present application. In the sensor of the present invention, a plurality of Cy5-dsDNA complexes can be assembled onto one 605QD, thereby improving FRET efficiency. As dsDNA concentration increases from 0.1mol/L to 0.5mol/L, the F/F0 value increases gradually, and after exceeding 0.5mol/L, the F/F0 value decreases. Therefore, 0.5mol/LdsDNA is preferably used.
Finally, quantum dots are added into the glass fiber reinforced plastic material, specifically: the Cy 5-double stranded DNA complex is incubated with streptavidin coated 605QDs for 1-30 minutes (preferably 15 minutes) at room temperature to form 605 QD-double stranded DNA-Cy5 nanostructure.
In yet another embodiment of the present invention, the method further comprises performing a fluorescence detection assay on the reaction product (605 QD-dsDNA-Cy5 nanostructure) obtained in step S3. In yet another embodiment of the invention, fluorescence intensity is quantitatively detected using a fluorometer, specifically exciting QDs at 488nm and collecting signals at 605nm and collecting fluorescence signals of Cy5 at 670 nm.
In a fourth aspect of the invention there is provided the use of a single quantum dot sensor and/or detection method as described above in TET 2-related drug screening and/or biological sample TET2 analysis.
Wherein the TET 2-related drugs include TET2 inhibitors and TET2 activators.
The biological samples include, but are not limited to, ex vivo blood, body fluids, tissues, and cells. Experiments prove that the single quantum dot sensor can sensitively detect TET2 expressed on the cellular level, so that the single quantum dot sensor can be effectively applied to biomedical basic research and clinical diagnosis.
It should be noted that, although the present invention is exemplified by the detection of TET2, and provides a related single quantum dot sensor and a detection method, it is obviously also conceivable to replace 5-iodocytosine in the capture probe for detecting other related enzymes based on the concept of the present invention, and therefore, the present invention shall also be covered.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments. In the following examples, the relevant probe sequences were used as follows:
wherein the reporter probeMiddle underlined base'C"means 5-iodocytosine.
Examples
Test method
1. Preparation of a vinylcytosine (5-vC) modified reporter probe product. 5 mg of MIDA vinylborate, 10 nanomoles of a 5-iodocytosine modified reporter probe, 100 micromoles per liter of palladium (II) acetate, 100 micromoles per liter of 2-amino-4, 6-dihydroxypyrimidine disodium are added to a Tris buffer containing 100 micromoles of 50 millimoles per liter of pH 10.9. Heating at 90 ℃ for 2h. Subsequently, the reaction product was centrifuged at 15000rpm for 10 minutes to remove black precipitate. Finally, the resulting supernatant was purified and stored at-20 ℃ for further use.
2. Preparation of double-stranded DNA stock solution. 5. Mu. Moles per liter of 5-vinylcytosine modified reporter probe and 5. Mu. Moles per liter of biotin modified capture probe were added to an annealing buffer containing 10 mM Tris-HCl,5 mM magnesium chloride at pH 8.0. Incubation was carried out at 95℃for 5 minutes, and then the temperature was slowly lowered to room temperature to prepare a double-stranded DNA substrate. The double stranded DNA substrate obtained was stored at-20℃for further use.
Tet2 activity assay. The detection of TET2 activity included four sequential steps. First, 500 nanomoles per liter of double stranded DNA substrate and varying amounts of TET2 were added to 50 microliters of a reaction mixture containing 1 millimole per liter of alpha-ketoglutaric acid, 2 millimoles per liter of ascorbic acid, 100 millimoles per liter of sodium chloride, 1 millimole per liter of dithiothreitol, 75 micromoles per liter of ferrous ions, and 50 millimoles per liter of 4-hydroxyethylpiperazine ethanesulfonic acid at pH6.8, incubated at 37℃for 2 hours, inactivated at 85℃for 20 minutes. Then, 1. Mu.l of 250. Mu.mol per liter of Cy5-NH was added 2 Incubation at 37℃for 12h produced a Cy5-dsDNA complex. UsingA500 centrifuge concentrator removes excess Cy5-NH 2 . Third, cy 5-double stranded DNA complex was coated with 2.5. Mu.l of 0.2. Mu.l streptavidin to a final concentration of 10. Mu.l 605QDs with 3 mM magnesium chloride, 100 mM Tris-HCl,10 mM pH8.0(NH 4 ) 2 SO 4 In 50 microliters of buffer. Incubation was performed for 15 min at room temperature to form 605 QD-double stranded DNA-Cy5 nanostructures.
4. Gel imaging: before mixing the double-stranded DNA-Cy5 product with 605QDs, adding SYBRgold dye to dye the DNA, pouring the mixture into 8% non-denaturing polyacrylamide gel, putting the gel into 1 XTris-boric acid-EDTA buffer solution, and carrying out room temperature electrophoresis for 45 minutes at 110V. Finally, gel imaging is carried out on the enzyme digestion reaction product by a ChemiDocMP imaging system.
5. Fluorescence detection: fluorescence emission spectra and fluorescence lifetime were collected by a fluorescence spectrophotometer at excitation wavelength of 488 nm. SYBRgold signals were analyzed using an EpiBlue (460-490 nm excitation) light source and a 518-546nm filter, and Cy5 signals were analyzed using an EPI-Red (625-650 nm excitation) illumination source and a 675-725nm filter.
6. Single molecule detection and data analysis. Prior to single molecule detection, the reaction products were diluted 1000-fold with a diluent (10 mmol per liter Tris-HCl,50 mmol per liter Potassium chloride, 5 mmol per liter magnesium chloride, 1 mmol per liter pH=8.0 quinine dimethacrylate. Subsequently, 10. Mu.l of the diluent was spotted onto a glass slide. Single molecule imaging was obtained by a total internal reflection fluorescence microscope, 605QDs were excited with 488nm laser light, photons of 605QDs and Cy5 were collected with a 100-fold objective lens. The signals were split into QD channels (573-613 nm filters) and Cy5 channels (661.5-690.5 nm filters) by dichroic mirrors and imaged at an electron multiplying charge coupled camera. Cy5 spots in the 300X 300 pixel region were counted using software imageJ and Cy5 counts were averaged.
7. Inhibitor testing. Different concentrations of N-oxaloglycine (NOG) were placed with a reporter DNA containing 10 nanograms per microliter of TET2,0.5 micromole per liter of 5-vinylcytosine modification, 1 millimole per liter of alpha-ketoglutaric acid, 2 millimoles per liter of ascorbic acid, 100 millimoles per liter of sodium chloride, 1 millimole per liter of dithiothreitol, 75 micromole per liter of ferrous ions, 50 millimoles per liter of 4-hydroxyethylpiperazine ethanesulfonic acid for 2 hours at 37 ℃, followed by inactivation at 85 ℃ for 20 minutes. TET2 activity was detected according to the procedure described above.
8. Cell culture and preparation of cell extracts. Human embryonic kidney 293 cell line (HEK) culture was performed in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin streptomycin. The medium was placed in an incubator containing 5% carbon dioxide at 37℃until the cells were mature. Cells were collected by digestion with pancreatin during exponential growth phase, washed 2 times with ice-cold phosphate buffer (137 mmol per liter of sodium chloride, 2.7 mmol per liter of potassium chloride, 10 mmol per liter of phosphate buffer, pH 7.4), and centrifuged at 800rpm for 5 minutes at 4 ℃. 100. Mu.l of lysis buffer was suspended in a buffer containing 10 mM Tris-HCl,150 mM sodium chloride, 1% (w/v) NP-40,0.25 mM sodium deoxycholate, 1% (w/v) glycerol, 0.1 mM 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, pH8.0, incubated on ice for 30 min, and spun for 30 seconds every 5 min. Finally, the cells were centrifuged at 12000rpm for 20 minutes at 4℃and the resulting supernatant was transferred to a new tube and stored at-80 ℃.
Results analysis and discussion
1. Test for detecting feasibility of TET2
In this study gel electrophoresis analysis was used to analyze whether the experiment was viable. To determine whether TET2 was able to initiate conversion of 5-vc to 5-fmc, 8% non-denaturing polyacrylamide gel electrophoresis (PAGE) analysis was performed with direct excitation of SYBRGold and Cy5, as shown in fig. 2A. Different double stranded DNA bands were observed in the presence of TET2 (fig. 2A, band 2) and in the absence of TET2 (fig. 2A, band 1), but Cy5 signal was only detected in the presence of TET2 (fig. 2B, band 2). In the presence of TET2, the SYBRgold and Cy5 signals co-localize simultaneously (FIG. 2C, lane 2), but only the SYBRgold signal is observed in the absence of TET2 (FIG. 2C, lane 1), indicating that the presence of TET2 can induce 5-vC oxidation to 5-fmC and promote the construction of 605QD-dsDNA-Cy5 nanostructures.
The polyacrylamide gel electrophoresis and fluorescence measurements were consistent (fig. 2), with only 605QD fluorescence signal but no Cy5 fluorescence signal being detected at excitation wavelength of 488nm in the control group without TET2 (fig. 2D). In the presence of TET2, the presence of TET2 resulted in a decrease in 605QD fluorescence signal while Cy5 fluorescence signal appeared/increased (fig. 2D), indicating that efficient FRET occurred between the QD donor and Cy5 acceptor due to TET2 inducing 605QD-dsDNA-Cy5 nanostructure formation.
In addition, the fluorescence lifetime profile of 605QDs was measured (fig. 2E). The average lifetime of 605QDs without TET2 was 29.87ns (fig. 2E) and after TET2 was added, the average lifetime of 605QDs was reduced to 11.62ns (fig. 2E).
To further verify experimental feasibility, single-molecule fluorescence imaging based on Total Internal Reflection Fluorescence (TIRF) was used to detect TET2 activity (fig. 3). In the presence of TET2, both 605QD (fig. 3A) and Cy5 fluorescent signals were observed (fig. 3B). Complete co-localization (fig. 3C) indicates effective FRET from 605QD to Cy5 in the 605QD-dsDNA-Cy5 nanostructure. In contrast, in the absence of TET2, only 605QD fluorescence signal was detected (fig. 3D), but no Cy5 signal was observed (fig. 3E), indicating that no FRET occurred between 605QD to Cy5.
These results clearly demonstrate that the method can be used to accurately detect TET2 activity.
2. Optimizing experimental conditions
To obtain the best experimental result, the pH value of the reaction, the incubation time of TET2 and Cy5-NH are optimized 2 Ratio to double-stranded DNA and concentration of double-stranded DNA.
The pH of the reaction was first optimized. As shown in FIG. 4A, the pH value was 6.5 to 6.8, the F/F0 value was gradually increased as the pH value of the reaction was increased, and the F/F0 value was decreased after the pH exceeded 6.8. Thus, pH6.8 was chosen as the optimal reaction pH. Since the incubation time of TET2 directly determines the yield of 5-fmC in the 5-vC oxidation reaction in double stranded DNA. The incubation time of TET2 is therefore an important factor affecting the detection. As shown in FIG. 4B, the F/F0 value increased with increasing TET2 incubation time, gradually increasing from 0.5h to 2h, and reached plateau at 2h, so 2h was chosen as the TET2 incubation time. Further optimize Cy5-NH 2 Ratio to dsDNA and concentration of dsDNA. High concentration of Cy5-NH 2 The reaction efficiency can be improved, the fluorescence intensity of Cy5 is enhanced, but an internal filtering effect can also be generated, so that the fluorescence signal of Cy5 is weakened. As shown in FIG. 4C, cy5-NH 2 Molar ratio to double-stranded DNA from 5:1 to 10: at 1, the F/F0 value gradually increases, exceeding 10: F/F0 value drop at 1Low, thus Cy5-NH was used in subsequent studies 2 The ratio to double-stranded DNA was 10:1. finally, the concentration of double-stranded DNA was optimized. In this biosensor, a plurality of Cy 5-double stranded DNA complexes can be assembled to one 605QD, thereby improving FRET efficiency. As shown in FIG. 4D, the F/F0 value gradually increased as the double-stranded DNA concentration increased from 0.1 mol/liter to 0.5 mol/liter, and the F/F0 value decreased after exceeding 0.5 mol/liter. Thus, subsequent studies used 0.5mol per liter of double stranded DNA.
Sensitivity detection of tet2 protein.
To investigate the sensitivity of this biosensor we measured Cy5 counts in different concentrations of TET2 response under optimal experimental conditions (fig. 5). As shown in fig. 5A, the Cy5 count gradually increased as the TET2 concentration increased from 0.1ng/μl to 32 ng/μl, reaching plateau at 10ng/μl concentration, and the Cy5 count correlated linearly with TET2 concentration in the range of 0.1ng/μl to 10ng/μl (fig. 4A inset), regression equation was n=126.4 lgc+181.4 (R 2 =0.997), where N is Cy5 count and C is TET2 concentration. The detection limit of the sensor is 0.042 ng/. Mu.L, and the sensitivity of the sensor is improved by 10 times compared with that of an electrochemiluminescence biosensor (0.37. Mu.g/mL) based on gold nanoclusters.
Specificity of TET2 protein
To verify the specificity of this method, cy5 counts of TET2 and interfering proteins including Bovine Serum Albumin (BSA), uracil DNA Glycosylase (UDG), m.sssi methyltransferase (m.sssi mtase) and HaeIII enzyme were measured. As shown in fig. 5B, high Cy5 signal was observed in the presence of TET2, whereas no significant Cy5 signal was detected in the presence of BSA, UDG, m.sssimtase, heaIII, and control. These results indicate that the biosensor has good specificity for TET2.
5. Inhibitor analysis
To verify the inhibitory capacity of the biosensor on TET2, N-oxaloglycine (NOG) was used as an inhibitor. Since NOG has a chemical structure similar to that of α -ketoglutarate (αkg). NOG has the ability to inhibit histone demethylase like JMJD2C, thus potentially effectively inhibiting alpha-KG dependent bis-by competing with alpha-KGActivity of oxygenase TET2. As shown in FIG. 6A, from 0 to 1000. Mu. Mol per liter, the relative activity of TET2 was decreased with increasing NOG concentration, and the IC of NOG was calculated 50 The value was 183.2. Mu. Moles per liter, consistent with the IC50 value (157.1. Mu. Moles per liter) obtained by mass spectrometry. This result suggests that the proposed single QD biosensor may provide an ideal platform for screening of TET2 inhibitors.
6. Cell detection
We used the human embryonic kidney cell line (HEK-293T) as a model to verify the ability of the biosensor to detect cell TET2 activity. As shown in fig. 6B, cy5 count increased as HEK-293T cell numbers increased from 1 to 10000. In the logarithmic range, cy5 counts correlated linearly with HEK-293T cell numbers in the range of 1-1000 cells (see fig. 6B), regression equation n=41.7lgx+24.6 (R 2 =0.988), where N is Cy5 count and X is HEK-293T cell number. The lower limit of detection (LOD) was 1 cell. This result clearly demonstrates the high accuracy and high sensitivity of the biosensor for the detection of cellular TET2 activity.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Shandong university of teachers and students
<120> an amplification-free single quantum dot biosensor for detecting TET2 at a single molecular level, a detection method thereof, and a detection program therefor
Application of
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213> artificial sequence
<400> 1
ctcctccccc atctcctccc agtcc 25
<210> 2
<211> 25
<212> DNA
<213> artificial sequence
<400> 2
ggactgggag gagatggggg aggag 25

Claims (18)

1. A single quantum dot sensor, the single quantum dot sensor comprising at least: reporter probes, capture probes, quantum dots, and free fluorescent dyes;
wherein the reporter probe has a complementary region that specifically hybridizes to the capture probe;
the reporter probe is modified with 5-iodocytosine, which is capable of forming 5-vinylcytosine under the action of a palladium ligand complex;
the tail end of the capture probe is modified with biotin;
the quantum dots are 605 QDs; the 605QD is 605QD coated by streptavidin;
the fluorescent dye is Cy5; the Cy5 is an aminated Cy5;
the base sequence of the reporter probe is 5' -CTC CTC CCC CATCTC CTC CCA GTC C-3', underlined base'C"means 5-iodocytosine;
the base sequence of the capture probe is 5'-GGA CTG GGA GGA T GGG GGA G-biotin-3'.
2. Use of the single quantum dot sensor of claim 1 for detecting TET2.
3. A method of detecting TET2 for non-diagnostic and therapeutic purposes, the method comprising detecting using the single quantum dot sensor of claim 1.
4. A method according to claim 3, wherein the method comprises:
s1, incubating a 5-vinyl cytosine modified reporter probe obtained after pretreatment with a capture probe to obtain double-stranded DNA;
s2, incubating the sample to be detected and the double-stranded DNA obtained in the step S1, inactivating at a high temperature, and then adding fluorescent dye to continue incubation; finally, quantum dots are added.
5. The method of claim 4, wherein,
in the step S1, the specific method for the 5-vinylcytosine modified reporter probe obtained after pretreatment is as follows: the 5-iodocytosine modified reporter probe is subjected to Suzuki-Miyaura reaction in the presence of a palladium ligand complex to generate a 5-vinylcytosine modified reporter probe;
then adding fluorescent dye to continue incubation specifically as follows: cy5-NH was added 2 Incubating 6-18h at 30-40 ℃ to generate Cy5-dsDNA complex; further, excess Cy5-NH was removed 2 ；
Cy5-NH 2 The molar ratio of dsDNA is 5-10:1, a step of;
the concentration of dsDNA is 0.1 mol/L-0.5 mol/L;
finally, quantum dots are added into the glass fiber reinforced plastic material, specifically: the Cy5-dsDNA complex described above was incubated with streptavidin-coated 605QDs for 1-30 minutes at room temperature to form 605QD-dsDNA-Cy5 nanostructures.
6. The method of claim 5, wherein the pretreatment method comprises: adding vinylboric acid MIDA ester, 5-iodo cytosine modified reporter probe, palladium (II) acetate and 2-amino-4, 6-dihydroxypyrimidine disodium into Tris buffer solution, heating at 85-95deg.C for 1-3h, and centrifuging and purifying.
7. The method of claim 6, wherein the preprocessing method comprises: adding vinylboric acid MIDA ester, a 5-iodo cytosine modified reporter probe, palladium (II) acetate and 2-amino-4, 6-dihydroxypyrimidine disodium into Tris buffer solution, heating at 90 ℃ for 2h, centrifuging and purifying to obtain the final product.
8. The method of claim 5, wherein the conditions under which the 5-vinylcytosine-modified reporter probe is co-incubated with the capture probe are specifically: incubating at 90-100deg.C for 1-10 min.
9. The method of claim 8, wherein the conditions under which the 5-vinylcytosine modified reporter probe is co-incubated with the capture probe are specifically: incubate at 95℃for 5 min.
10. The method according to claim 5, wherein in the step S2, the high temperature inactivation after incubating the sample to be tested with the double-stranded DNA obtained in the step S1 is specifically: incubating at 30-40deg.C for 1-3h, reacting at weak acid pH, and inactivating at 80-90deg.C for 10-60 min.
11. The method according to claim 10, wherein in the step S2, the high temperature inactivation after incubating the sample to be tested with the double-stranded DNA obtained in the step S1 is specifically: incubation at 37℃for 2h, reaction environment pH6.5-6.8, inactivation at 85℃for 20min.
12. The method of claim 11, wherein the reaction environment pH is pH6.8.
13. The method according to claim 5, wherein the further incubation after adding the fluorescent dye is: cy5-NH was added 2 Incubation at 37℃for 12h resulted in Cy5-dsDNA complexes.
14. The method of claim 5, wherein Cy5-NH 2 The molar ratio to dsDNA was 10:1.
15. the method of claim 5, wherein the dsDNA concentration is 0.5mol/L.
16. The method of claim 5, wherein the quantum dots are added to the substrate at last: the Cy5-dsDNA complex described above was incubated with streptavidin-coated 605QDs for 15 minutes at room temperature to form 605QD-dsDNA-Cy5 nanostructures.
17. The method of claim 4, further comprising performing a fluorescence detection assay on the reaction product obtained in step S2.
18. Use of the single quantum dot sensor of claim 1 and/or the detection method of non-diagnostic and therapeutic purposes of any one of claims 3-17 in TET 2-related drug screening and/or biological sample TET2 analysis;
the TET2 related drugs include TET2 inhibitors and TET2 activators;
the biological sample includes ex vivo blood, body fluids, tissues and cells.
CN202111436593.2A 2021-11-29 2021-11-29 Amplification-free single quantum dot biosensor for detecting TET2 at single molecule level and detection method and application thereof Active CN114250271B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111436593.2A CN114250271B (en) 2021-11-29 2021-11-29 Amplification-free single quantum dot biosensor for detecting TET2 at single molecule level and detection method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111436593.2A CN114250271B (en) 2021-11-29 2021-11-29 Amplification-free single quantum dot biosensor for detecting TET2 at single molecule level and detection method and application thereof

Publications (2)

Publication Number Publication Date
CN114250271A CN114250271A (en) 2022-03-29
CN114250271B true CN114250271B (en) 2024-03-08

Family

ID=80793540

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111436593.2A Active CN114250271B (en) 2021-11-29 2021-11-29 Amplification-free single quantum dot biosensor for detecting TET2 at single molecule level and detection method and application thereof

Country Status (1)

Country Link
CN (1) CN114250271B (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110057793A (en) * 2019-03-15 2019-07-26 山东师范大学 The method of nano-sensor detection people's alkyl polyglucosides enzyme based on single quantum dot
CN110398481A (en) * 2019-07-25 2019-11-01 山东师范大学 A kind of single quantum dot fluorescence nano-sensor and its preparation method and application based on no enzymatic self assembly
CN110408680A (en) * 2019-06-28 2019-11-05 山东师范大学 For detection of alkaline phosphatase, based on ligase amplified reaction catalysis assembling single quantum dot nano-sensor and application
CN112662777A (en) * 2021-01-19 2021-04-16 山东师范大学 Compound and method for simultaneously detecting multiple long-chain non-coding RNAs

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110057793A (en) * 2019-03-15 2019-07-26 山东师范大学 The method of nano-sensor detection people's alkyl polyglucosides enzyme based on single quantum dot
CN110408680A (en) * 2019-06-28 2019-11-05 山东师范大学 For detection of alkaline phosphatase, based on ligase amplified reaction catalysis assembling single quantum dot nano-sensor and application
CN110398481A (en) * 2019-07-25 2019-11-01 山东师范大学 A kind of single quantum dot fluorescence nano-sensor and its preparation method and application based on no enzymatic self assembly
CN112662777A (en) * 2021-01-19 2021-04-16 山东师范大学 Compound and method for simultaneously detecting multiple long-chain non-coding RNAs

Also Published As

Publication number Publication date
CN114250271A (en) 2022-03-29

Similar Documents

Publication Publication Date Title
CN107723338B (en) Fluorescent chemical sensor for simultaneously detecting multiple DNA glycosylases at single-molecule level and detection method and application thereof
KR102378388B1 (en) Improved assay methods
KR102497054B1 (en) Improved assay methods
KR101029343B1 (en) Immunoassay-based Antigen Detecting Kit and Method
Qin et al. Emerging biosensing and transducing techniques for potential applications in point-of-care diagnostics
ES2659798T3 (en) Detection of immunoglobulin light chain restriction by in situ RNA hybridization
CN109142710B (en) Method for rapidly and sensitively detecting tetrodotoxin TTX
CN113088557B (en) Fluorescent chemical sensor for simultaneously detecting multiple DNA glycosylases and detection method and application thereof
CN112852922B (en) Fluorescent biosensor for detecting DNA methylation, detection method and application
Zhang et al. Catalytic single-molecule Förster resonance energy transfer biosensor for uracil-DNA glycosylase detection and cellular imaging
Li et al. A single quantum dot-based nanosensor with multilayer of multiple acceptors for ultrasensitive detection of human alkyladenine DNA glycosylase
Lin et al. An enzyme-free fluorescent biosensor for highly sensitive detection of carcinoembryonic antigen based on aptamer-induced entropy-driven circuit
Yang et al. High-fidelity and simultaneous sensing of endogenous mutant and wild p53 proteins for precise cancer diagnosis and drug screening
US20230042710A1 (en) Electrochemical proximity assay
CN107083437B (en) Method for simultaneously detecting multiple DNA glycosylases by using inherent fluorescent nucleotide ultrasensitiveness
Song et al. Allosteric aptasensor-initiated target cycling and transcription amplification of light-up RNA aptamer for sensitive detection of protein
CN111855625B (en) CA125 detection kit based on Cu-MOF and application thereof
CN113340863A (en) Enzyme-free circulating amplification aptamer sensor and preparation method and application thereof
CN114250271B (en) Amplification-free single quantum dot biosensor for detecting TET2 at single molecule level and detection method and application thereof
CN112903641A (en) Biosensor for detecting histone modification enzyme and detection method and application thereof
CN110468182A (en) It is a kind of detect Platelet-derived growth factor BB homogeneous bioanalytical method and its application
CN114517225B (en) Single-molecule fluorescent biosensor for detecting alkaline phosphatase and method thereof
Tian et al. Dual Self-Amplification Homogeneous Electrochemiluminescence Biosensor for Terminal Deoxynucleotidyl Transferase Activity Based on Controlling the Surface Morphology and Charge of Reporter Nanoparticles
CN113151402B (en) Nano sensor for detecting DNA hydroxylase TET1, and detection method and application thereof
Ren et al. CRISPR/Cas12a-based fluorescence aptasensor integrated with two-dimensional cobalt oxyhydroxide nanosheets for IFN-γ detection

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