CN114703192A - Application of adjacent base pair enhanced deoxyribozyme and biosensing and biosensor - Google Patents
Application of adjacent base pair enhanced deoxyribozyme and biosensing and biosensor Download PDFInfo
- Publication number
- CN114703192A CN114703192A CN202210544494.4A CN202210544494A CN114703192A CN 114703192 A CN114703192 A CN 114703192A CN 202210544494 A CN202210544494 A CN 202210544494A CN 114703192 A CN114703192 A CN 114703192A
- Authority
- CN
- China
- Prior art keywords
- enhanced
- biosensor
- deoxyribozyme
- dnazyme
- nucleotide 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.)
- Granted
Links
- 108091027757 Deoxyribozyme Proteins 0.000 title claims abstract description 76
- 241000588724 Escherichia coli Species 0.000 claims abstract description 54
- 239000002299 complementary DNA Substances 0.000 claims abstract description 36
- 108020004635 Complementary DNA Proteins 0.000 claims abstract description 34
- 238000010804 cDNA synthesis Methods 0.000 claims abstract description 34
- 238000001514 detection method Methods 0.000 claims abstract description 29
- 229940025294 hemin Drugs 0.000 claims abstract description 25
- 108091023037 Aptamer Proteins 0.000 claims abstract description 21
- BTIJJDXEELBZFS-QDUVMHSLSA-K hemin Chemical compound CC1=C(CCC(O)=O)C(C=C2C(CCC(O)=O)=C(C)\C(N2[Fe](Cl)N23)=C\4)=N\C1=C/C2=C(C)C(C=C)=C3\C=C/1C(C)=C(C=C)C/4=N\1 BTIJJDXEELBZFS-QDUVMHSLSA-K 0.000 claims abstract description 20
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 8
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 3
- 239000002773 nucleotide Substances 0.000 claims description 19
- 125000003729 nucleotide group Chemical group 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 18
- 238000002835 absorbance Methods 0.000 claims description 15
- 230000008859 change Effects 0.000 claims description 15
- 239000000243 solution Substances 0.000 claims description 10
- 102000053602 DNA Human genes 0.000 claims description 7
- 239000007987 MES buffer Substances 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 4
- 239000010453 quartz Substances 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 36
- 230000000295 complement effect Effects 0.000 abstract description 13
- 238000009396 hybridization Methods 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 238000011084 recovery Methods 0.000 abstract description 2
- 230000003197 catalytic effect Effects 0.000 description 20
- 238000004458 analytical method Methods 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 10
- 150000003212 purines Chemical class 0.000 description 8
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical class NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 6
- 238000010586 diagram Methods 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- MWBWWFOAEOYUST-UHFFFAOYSA-N 2-aminopurine Chemical compound NC1=NC=C2N=CNC2=N1 MWBWWFOAEOYUST-UHFFFAOYSA-N 0.000 description 4
- 229940045686 antimetabolites antineoplastic purine analogs Drugs 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 4
- 230000007547 defect Effects 0.000 description 4
- 239000000543 intermediate Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000005502 peroxidation Methods 0.000 description 4
- 230000003595 spectral effect Effects 0.000 description 4
- 239000008399 tap water Substances 0.000 description 4
- 235000020679 tap water Nutrition 0.000 description 4
- KQLXBKWUVBMXEM-UHFFFAOYSA-N 2-amino-3,7-dihydropurin-6-one;7h-purin-6-amine Chemical compound NC1=NC=NC2=C1NC=N2.O=C1NC(N)=NC2=C1NC=N2 KQLXBKWUVBMXEM-UHFFFAOYSA-N 0.000 description 3
- 102000035195 Peptidases Human genes 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 3
- 230000006399 behavior Effects 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
- 241000607142 Salmonella Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000191967 Staphylococcus aureus Species 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 229940104302 cytosine Drugs 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- -1 porphyrin cationic radical Chemical class 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 2
- 208000009304 Acute Kidney Injury Diseases 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- 206010014896 Enterocolitis haemorrhagic Diseases 0.000 description 1
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 description 1
- 208000032759 Hemolytic-Uremic Syndrome Diseases 0.000 description 1
- 101001091385 Homo sapiens Kallikrein-6 Proteins 0.000 description 1
- 102100034866 Kallikrein-6 Human genes 0.000 description 1
- 208000033626 Renal failure acute Diseases 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 201000011040 acute kidney failure Diseases 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000013211 curve analysis Methods 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 244000078673 foodborn pathogen Species 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000012933 kinetic analysis Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
-
- 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
- C12Q1/682—Signal amplification
-
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
- C12N2310/127—DNAzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
-
- 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
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an enhanced deoxyribozyme with adjacent base pairs, a biosensor and application of the biosensor, wherein the enhanced deoxyribozyme has one of nucleotide sequences G4-AA, G4-AG, G4-A-MdA, G4-A-AP and G4-A-dI; the biosensor comprises an enhanced deoxyribozyme, a complementary DNA chain, an Escherichia coli aptamer and hemin. The invention enhances the activity of DNAzymes by changing the base adjacent to two bits of G4 and enabling the base to be complementary with the base adjacent to the second bit by hybridization according to the Watson-Crick base complementary pairing principle. The biosensor based on the enhanced deoxyribozyme can specifically detect escherichia coli and has higher detection sensitivity and recovery rate.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an enhanced dnazyme with adjacent base pairs and application of a biosensing and biosensor.
Background
Escherichia coli is a common food-borne pathogen, and can cause hemolytic uremic syndrome, hemorrhagic colitis, acute kidney injury and even death of human body. Outbreaks of food-borne escherichia coli have been associated with eating contaminated foods such as fresh vegetables, acidic beverages and ground beef. Therefore, the detection of Escherichia coli in food is an important part in food safety detection. The traditional method for detecting escherichia coli is represented by microbial culture, although strains can be identified according to the specificity such as morphological characteristics, growth characteristics and the like, the operation process is complicated, the time consumption is long, generally, the detection time needs several days, and the escherichia coli is easily polluted by microbes in the air. With the attention of researchers to the detection of microorganisms, the application of technologies such as instrument analysis, molecular biology, immunology and the like in the detection of microorganisms appears in the visual field of people, and the problems of long time consumption, easy pollution and the like of the traditional microorganism detection method are solved to a certain extent, but the method has the defects of complex operation, higher cost, low specificity, low sensitivity and the like, so that the detection result is unstable. Therefore, it is important to develop a detection method that is economical, simple, fast and highly sensitive. At present, biosensors are well known due to the advantages of high detection speed, economy and the like, but the existing methods have the defects of low sensitivity, high detection background and the like.
In order to improve the sensitivity of the biosensor in the detection process, an enzyme with a catalytic function is usually added to accelerate the oxidation-reduction reaction and convert the biochemical reaction into a fluorescent, colorimetric and electrochemical signal. Most of enzymes used for catalysis in the prior art are proteinases, such as horseradish peroxidase commonly used in the field of biosensing, and although the proteases have high catalytic activity, the proteases have the defects of high storage requirement, unstable enzyme activity and the like. However, the deoxyribozyme (DNAzyme, which is composed of G (guanine) -rich DNA single strand and hemin) with similar horseradish peroxidase catalytic activity has the advantages of small volume, easy synthesis and the like, and is expected to replace the position of horseradish peroxidase in biosensing. However, in practical application, the activity of the deoxyribozyme is not high, and the activity of the deoxyribozyme cannot reach the activity of horseradish peroxidase. In order to enhance the activity of the deoxyribozyme, high-catalytic-activity deoxyribozyme is screened by means of sequence screening, G4 skeleton modification, activity activator addition, flanking sequence addition and the like, and the methods provide a reference idea for improving the activity of the deoxyribozyme, even if the activity multiple of the deoxyribozyme is far less than that of horseradish peroxidase. Therefore, how to improve the activity of the dnazyme is one of the important factors that must be considered in the application of biosensing to detection.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides an enhanced DNAzyme with adjacent base pairs, constructs a biosensor for detecting Escherichia coli based on the DNAzyme, and is finally applied to Escherichia coli detection.
In order to achieve the above object, the present invention provides an enhanced dnazyme with an adjacent base pair, wherein the enhanced dnazyme has one of the nucleotide sequences G4-AA, G4-AG, G4-a-MdA, G4-a-AP, G4-a-dI;
the nucleotide sequence of G4-AA is as follows: G4-AA-TTTCGCTATGTCTG;
the nucleotide sequence of G4-AG is: G4-AG-TTTCGCTATGTCTG;
the nucleotide sequence of G4-A-MdA is as follows: G4-A-/6-med a/-TTTCGCTATGTCTG; (/6-med a/is N6' methylated adenine);
the nucleotide sequence G4-A-/i2-amp/-TTTCGCTATGTCTG of G4-A-AP; (/ i 2-amp/is 2' aminopurine);
the nucleotide sequence G4-A-/6-oxide i/-TTTCGCTATGTCTG of G4-A-dI. (/ 6-oxoi/is hypoxanthine).
The adjacent base pair enhanced deoxyribozyme is characterized in that the nucleotide sequence of G4 is shown in SEQ ID NO. 1.
The adjacent base pair enhanced deoxyribozymes further comprise topological structures of G4-AA, G4-AG, G4-A-MdA, G4-A-AP and G4-A-dI.
Based on a general technical concept, the present invention also provides a biosensor comprising the proximity base pair enhanced deoxyribozyme of claim 1 or 2, a complementary DNA strand (cDNA), E.coli aptamer (aptamer) and hemin (hemin).
The above biosensor, further, wherein the complementary DNA strand is cDNA,
the nucleotide sequence of the cDNA is shown as SEQ ID NO. 3;
the above biosensor, further, the nucleotide sequence of the E.coli aptamer is shown in SEQ ID NO. 2.
The biosensor further comprises a MES buffer system and ABTS2-And H2O2。
The biosensor as described above, further, the concentration of the adjacent base pair-enhanced dnazyme is 200 nM: the concentration of the complementary DNA chain is 400nM, and the concentration of the hemin is 200 nM; the ABTS2-Is 1 mM; said H2O2Was 2 mM.
Based on a general technical concept, the invention also provides an application of the biosensor in detecting escherichia coli.
The above application, further, the method of the application is:
(1) adding the liquid to be tested into MES buffer solution containing the aptamer of the escherichia coli for incubation to obtain a system I;
(2) adding the adjacent base pair enhanced deoxyribozyme, hemin and cDNA chain into the system I for hatching to obtain a system II;
(3) transferring the system II into a quartz cuvette, and adding H2O2And ABTS2-Shaking up to obtain a system III;
(4) and measuring the change value of the absorbance of the system III at 420nm along with the time by using an ultraviolet spectrophotometer.
Compared with the prior art, the invention has the advantages that:
(1) the invention provides an adjacent base pair enhanced deoxyribozyme, which can enhance the activity of DNAzyme by changing the base of G4 adjacent to two bits and hybridizing and complementing the adjacent second bit according to Watson-Crick base complementary pairing principle.
(2) The invention provides a biosensor which has the advantages of simple, rapid and specific detection of escherichia coli and the like. The experimental result shows that the catalytic activity of DNAzyme can be obviously further enhanced by hybridization of G4 tail end base pair. Under the optimal condition, the enhanced DNAzyme biosensor with adjacent base pairs can specifically detect Escherichia coli within the detection range of 2 × 102-2×107CFU/mL, the detection limit is 16CFU/mL, and the recovery rate and the detection sensitivity are higher.
(3) The invention provides an application of a biosensor in detecting escherichia coli, which regulates the DNAzyme catalytic activity phenomenon based on adjacent base pairs, namely DNAzyme formed by combining a G4 core part at the 5' end of G4-BIO with hemin (hemin), wherein the DNAzyme activity is inhibited due to the existence of A: T base pairs at the tail end of G4-BIO. When cDNA exists (the circular part of G4-BIO can be opened), not only the catalytic activity of the deoxyribozyme is recovered, but also the catalytic activity of the DNAzyme is further enhanced because the A base at the +2 position of the tail end of G4 is complemented by T. Aptamers recognizing specifically E.coli were introduced into buffer solution at concentrations of complementary DNA (cDNA) and G4-BIO that resulted in binding of complementary DNA (cDNA), so that G4-BIO did not express high activity. When escherichia coli in a targeted G4-BIO, cDNA and aptamer solution exists, the escherichia coli can be preferentially combined with the escherichia coli aptamer, free cDNA can be combined with G4-BIO, and a G4-BIO stem part is opened, so that the enzyme activity of G4 DNAzyme is recovered, and the aim of detecting the escherichia coli is fulfilled.
Drawings
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.
FIG. 1 is a schematic diagram of a base pair-adjacent enhanced deoxyribozyme according to example 1 of the present invention.
FIG. 2 shows the time-dependent absorbance change of different bases hybridized with purine and pyrimidine in the first experiment of the present invention.
FIG. 3 is a schematic diagram showing the structures of four purine analogues in example 2 of the present invention.
FIG. 4 shows the results of the change of absorbance with time after the influence of different purine analogues on the catalytic activity in experiment two of the present invention.
FIG. 5 shows the results of the peroxidation behavior studies of G4-AA: O and G4-AA: T in experiment III of the present invention. A is at H2O2(2mM) absorbance decay kinetics analysis of G4 DNAzymes at 404 nm; b is G4-AA, T-hemin and H2O2(2mM) spectral change of the visible region with time during the reaction; c is G4-AA, O-hemin and H2O2(2mM) spectral change of the visible region with time during the reaction.
FIG. 6 is a schematic diagram of the mechanism of competition analysis based on free complementary DNA in example 4 of the present invention.
FIG. 7 shows the result of specificity analysis in experiment four of the present invention.
FIG. 8 shows the analysis result of the working curve in the fifth experiment of the present invention.
Detailed Description
The invention is further described below with reference to specific preferred embodiments, without thereby limiting the scope of protection of the invention.
The materials and equipment used in the following examples are commercially available.
The nucleic acid sequences used are detailed in Table 1.
TABLE 1 nucleotide sequence
Example 1
A dnazyme, whose nucleotide sequence has the following structure:
Changing the base types of near two bits of the tail end of G4, and adding the complementary strand to hybridize the second residual base sequence adjacent to the G4 strand according to the Watson-Crick base complementary pairing principle to form 16 types of deoxyribozymes, which specifically comprise: G4-AA, G4-AC, G4-AG, G4-AT, G4-CA, G4-CC, G4-CG, G4-CT, G4-GA, G4-GC, G4-GG, G4-GT, G4-TA, G4-TC, G4-TG, G4-TT.
G4-AA：GGGTTGGGTGGGTTGGGAATTTCGCTATGTCTG；
G4-AC：GGGTTGGGTGGGTTGGGACTTTCGCTATGTCTG；
G4-AG：GGGTTGGGTGGGTTGGGAGTTTCGCTATGTCTG；
G4-AT：GGGTTGGGTGGGTTGGGATTTTCGCTATGTCTG；
G4-CA：GGGTTGGGTGGGTTGGGCATTTCGCTATGTCTG；
G4-CC：GGGTTGGGTGGGTTGGGCCTTTCGCTATGTCTG；
G4-CG：GGGTTGGGTGGGTTGGGCGTTTCGCTATGTCTG；
G4-CT：GGGTTGGGTGGGTTGGGCTTTTCGCTATGTCTG；
G4-GA：GGGTTGGGTGGGTTGGGGATTTCGCTATGTCTG；
G4-GC：GGGTTGGGTGGGTTGGGGCTTTCGCTATGTCTG；
G4-GG：GGGTTGGGTGGGTTGGGGGTTTCGCTATGTCTG；
G4-GT：GGGTTGGGTGGGTTGGGGTTTTCGCTATGTCTG；
G4-TA：GGGTTGGGTGGGTTGGGTATTTCGCTATGTCTG；
G4-TC：GGGTTGGGTGGGTTGGGTCTTTCGCTATGTCTG；
G4-TG：GGGTTGGGTGGGTTGGGTGTTTCGCTATGTCTG；
G4-TT：GGGTTGGGTGGGTTGGGTTTTTCGCTATGTCTG。
In the present invention, bases adjacent to two positions of G4 are altered and hybridized and complemented adjacent to the second position according to Watson-Crick base complementary pairing rules, and it was found that if bases flanking two adjacent positions of the G4(DNAzyme core sequence) sequence are adenine, such purine hybridization with pyrimidine can enhance the activity of DNAzyme.
Based on the idea that the hybridization of adjacent base pairs at the tail end of G4 inhibits the activity of the deoxyribozyme, the base type of the near two bits at the tail end of G4 is changed, and the complementary strand is added to hybridize the base sequence remained at the second bit adjacent to the G4 chain to form 16 types of the deoxyribozyme. FIG. 1 shows that the enhancement of the activity of the original DNAzyme occurs when the +1 position of the tail end of G4 is A and the +2 base pairs A: T or G: C.
Experiment one: examination of the Activity of 16 deoxyribozymes
FIG. 2 shows the results of absorbance changes with time for different bases hybridized with purine: pyrimidine. Table 2 shows the catalytic performance parameters of various G4-DNAzymes.
Table 2: catalytic performance parameters of the various G4-DNAzymes.
V and TOF represent catalytic rate and switching frequency, respectively.
As can be seen from the results of FIGS. 2 and 2 (Table 2 shows the catalytic rate and turnover frequency of DNAzyme in FIG. 2): in the above 16 dnazymes, when the vicinal bases of G4 at the tail end are AA (two adenines) and the +2 position A hybridizes with the T base of the corresponding complementary strand (i.e., G4-AA: T), the dnazyme activity is enhanced, which can be further increased to about 3.0 times based on the original G4-ADA zyme, as shown in A in FIG. 2. This also demonstrates that hemin is stacked on the G4 plane of G4. In addition, similar situation occurs when the adjacent base at the tail end of G4 is AG (adenine-guanine) and the corresponding complementary strand hybridization position is C (cytosine), as shown in the C diagram of FIG. 2. However, the B, D-P diagram in FIG. 2 shows no enhancement effect of the deoxyribozyme. The above phenomenon is referred to as enhancement of the deoxyribozyme activity by adjacent base pairing.
Example 2: the possibility of enhancing the deoxyribozyme phenomenon at purine analogues by adjacent base pairs was investigated.
For the study, different purine analogs were selected, such as: methylated adenine (MdA), 2-Aminopurine (AP) and hypoxanthine (dI) form G4 DNAzyme. The method specifically comprises the following steps:
G4-A-MdA：GGGTTGGGTGGGTTGGGA/6-med a/TTTCGCTATGTCTG；
G4-A-AP：GGGTTGGGTGGGTTGGGA/i2-amp/TTTCGCTATGTCTG；
G4-A-dI：GGGTTGGGTGGGTTGGGA/ide oxy i/TTTCGCTATGTCTG。
experiment two: the activities of the three G4 DNAzymes G4-A-MdA, G4-A-AP and G4-A-dI were examined.
FIG. 3 is an inset schematic showing the structure of a purine analog. FIG. 4 is a graph of the change in absorbance over time following the effect of different purine analogs on catalytic activity.
As can be seen from the figure: when these purine analogs were placed second in the position ortho to the tail end of G4 and hybridized to the corresponding bases, the resulting signal differences were analyzed. MdA and AP have a very similar effect on peroxidation activity to that of G4-AA, and DNA-T can significantly enhance DNAzyme activity in the presence of adenine analogs supplemented with a "T" base. For dI, the base complement of either T (thymine) or C (cytosine) enhances the activity of the DNA-dI DNAzyme to a similar extent, since it can supplement almost any base. This phenomenon also suggests that adenine analogs can produce a proximal base pair enhancing effect.
The purine analogues were also found to work effectively by replacing the first base with the other non-four common bases.
Experiment three, the peroxidation behaviors of G4-AA: O and G4-AA: T are studied:
to further elucidate the function of neighboring AA: T, the peroxidation behavior of G4-AA: O and G4-AA: T was studied extensively. Catalytic cycling of DNAzymes begins with G4/hemin being H2O2Oxidation to an intermediate (compound I) comprising one fe (iv) ═ O and one porphyrin cationic radical; fe (iv) ═ O can be reduced to hemin (fe (iii)), thereby producing two free single electrons that oxidize ABTS2-And the catalytic process is completed.
(1) Analysis of decay kinetics: G4-AA (5. mu.M) and its complementary DNA strand (10. mu.M), and hemin (5. mu.M) were added to a 2 XMES buffer system. Incubation was performed at room temperature for 30min to form a complex. Transferring the solution into a quartz cuvette, monitoring the absorbance change at 404nm in a kinetic analysis mode, and adding H after 50s2O2(2mM), kinetics were observed continuously.
(2) Spectral analysis: the mixture synthesis procedure was as above. The above mixture was placed in a cuvette and UV-Vis spectra were collected using a UV spectrophotometer in the range of 450-700nm and repeated at intervals of 30 s.
In FIG. 5A is at H2O2(2mM) absorbance decay kinetics analysis of G4 DNAzymes at 404 nm. As can be seen from the figure, the decay rate of the Soret band of G4-AA: T is significantly faster than that of G4-AA: O.
At the same time, the absorbance of the DNAzyme in the visible region was also monitored over time to observe the amount of signal released on intermediates of the DNAzyme. B in FIG. 5 is G4-AA, T-hemin and H2O2(2mM) spectral change of visible region with time during the reaction, C in FIG. 5 is G4-AA: O-hemin and H2O2(2mM) spectral change of the visible region with time during the reaction. The formation of intermediates of class I was explored and the direction of change in absorbance is indicated by the arrow.
As can be seen from the figure: T-G4-AA having a slight absorbance at the E-band (500 nm) and D-band (630 nm)The changes, 550-600nm and 650-700nm, increase slightly (B). However, G4-AA: O showed that the E band and D band disappeared rapidly within 0.5min after the start of the reaction (C). Thus, this phenomenon indicates that the original intermediate of the G4-hemin complex has been rapidly generated. The bulk attenuation at 450-700nm occurred after 0.5min, indicating that hemin degradation is by H2O2Is caused by the continuous reaction of (2). The phenomenon of enhancement of adjacent base pairs is attributed to the accelerated formation of adjacent AA: T (fe (iv) ═ O), and the generation of porphyrin cationic radicals.
Example 3
A biosensor for detecting Escherichia coli, which uses an Escherichia coli aptamer as a recognition element and the higher catalytic deoxyribozyme of example 1 as a signal amplification element. Specifically comprises Escherichia coli aptamer, 200nM G4 DNA, 400nM DNA strand complementary to the aptamer, 200nM hemin, 2 XMES buffer system, and 1mM ATBTS2-And 2mM H2O2。
The nucleotide sequence aptamer of the escherichia coli aptamer is shown as SEQ ID NO.2, and specifically comprises the following steps:
GACGGTGGCAGGGAAAGGGGTCGGGCATATGGCGGAGGGG。
the complementary DNA sequence cDNA is shown as SEQ ID NO.3, and specifically comprises:
TATGCCCGACCCCTTTCCAGT。
FIG. 6 is a schematic diagram of the mechanism of competition assay based on free complementary DNA.
In the figure, line (1) is a DNAzyme formed by binding hemin to the G4 core moiety at the 5' end of G4-BIO, which is a phenomenon of inhibiting DNAzyme catalytic activity based on the adjacent bases, and DNAzyme activity is inhibited. When cDNA exists (the circular part of G4-BIO can be opened), not only the catalytic activity of the deoxyribozyme is recovered, but also the catalytic activity of the DNAzyme is further enhanced because the A base at the +2 position of the tail end of G4 is complemented by T.
In the figure, line (2) is an aptamer specifically recognizing Escherichia coli introduced into a buffer solution, and the concentrations of complementary DNA (cDNA) and G4-BIO are such that the G4-BIO does not express high activity due to the binding of complementary DNA (cDNA).
In the figure, line (3) is when Escherichia coli in a solution of targeting G4-BIO, cDNA and aptamer exists, the Escherichia coli can be preferentially combined with the Escherichia coli aptamer, and free cDNA can be combined with G4-BIO, so that a G4-BIO stem part is opened, and the enzyme activity of G4 DNAzyme is recovered.
Experiment five, investigating the specificity of the biosensor:
specific study: taking Escherichia coli, Staphylococcus aureus, Salmonella and Pseudomonas at a concentration of 2 × 108CFU/mL, adding the bacterial liquid into MES buffer solution containing 200nM Escherichia coli aptamer (the sequence of Escherichia coli aptamer is shown in Table 3-1), incubating for 10min, adding 200nM G4-BIO (containing G4 sequence), 200nM hemin and 200nM cDNA into the system, incubating for 30min, transferring the above solution into a cuvette, adding H2O2(2mM) and ABTS2-(1mM), shaking up. And measuring the absorbance change value at 420nm by using an ultraviolet-visible spectrophotometer. A sample without the addition of the bacterial suspension was used as a blank control.
FIG. 7 shows the results of the concentration of Escherichia coli at 2X 108And (5) analyzing the result of the specificity under the CFU/mL condition. It can be seen from the figure that the sensor based on the enhanced deoxyribozyme only has response to Escherichia coli serving as a target, and the response values of staphylococcus aureus, salmonella, pseudomonas and no target are approximately the same, and the sensor shows a lower absorbance change value at 420 nm. Therefore, the enhanced deoxyribozyme sensor based on adjacent base pairs has more remarkable specificity.
And sixthly, inspecting the detection limit of the biosensor:
7 concentrations (2X 10) were prepared separately1CFU/mL、2×102CFU/mL、2×103CFU/mL、2×104CFU/mL、2×105CFU/mL、2×106CFU/mL、2×107CFU/mL), the sensor of example 2 was examined for the above concentration of e.coli, and a working curve was plotted.
FIG. 8 shows the results of kinetic analyses of different E.coli concentrations. In the graph, A shows that the absorbance change region of 75s at 420nm increases with the increasing concentration of Escherichia coli. From B in the figure, it is found that the detection range of the sensor based on the enhanced deoxyribozyme of the adjacent base pair is 2X 102-2×107CFU/mL shows good linear correlation, the correlation coefficient is 0.998, the calibration equation is that y is-0.192 +0.177x, and the detection limit is about 16 CFU/mL.
Example 4
An application of the biosensor in example 3 in detection of escherichia coli in a natural environment, the application method comprising:
placing tap water in a conical flask, placing in a high temperature sterilizing pot, and sterilizing at 121 deg.C for 15 min. Adding escherichia coli with different concentrations into sterilized and cooled water in a clean bench, and mixing uniformly to prepare a sample. The subsequent operating steps refer to example 3.
In order to ensure the practical application significance of the detection method for detecting the Escherichia coli based on the enhanced deoxyribozyme constructed by adjacent base pairs in the practical sample. In this chapter, standard addition method was used for further verification and evaluation, and different concentrations of E.coli were added to high-temperature sterilized tap water, and a water sample without E.coli was used as a control. Samples with and without added E.coli were added and E.coli concentration was determined by conventional plate counting.
Table 3 shows the results of detection of Escherichia coli in tap water using the biosensor of example 2.
Table 3: detection of E.coli in tap water based on an adjacent base pair-enhanced DNAzyme
As shown in Table 3, the addition of different final concentrations of E.coli (0CFU/mL, 2X 10)2CFU/mL,2×103CFU/mL,2×104CFU/mL,2×105CFU/mL,2×106CFU/mL) between 87% and 96%, and the Relative Standard Deviation (RSD) of the experiment is small and within 7%. Therefore, the method is feasible and reliable for detecting the Escherichia coli in real samples based on the construction of the biosensor based on the enhanced deoxyribozyme, and the method is also suitable for other practical samplesAnd (4) detecting and analyzing escherichia coli.
And (4) conclusion: the invention changes the last two bases of the G4 tail end, and adds the complementary DNA single strand, thereby forming 16 different G4 DNAzyme types. It was found that the catalytic activity of the deoxyribozyme was further improved when the base adjacent to the G4 chain was AA and the complementary base was T, and this phenomenon was a phenomenon in which the DNAzyme activity was enhanced by the adjacent base pairs. The same phenomenon occurs when the base adjacent to the G4 chain is AG and the base complementary to the G4 chain is C. Subsequently, the application conditions of the G4-AA T DNAzyme in various buffers, reaction substrates and pH are analyzed, and the results show that the adjacent base pair enhanced deoxyribozyme effect has universality; the enhancement effect of adjacent base pairs is obtained through ultraviolet spectrum analysis and does not influence the original structure of the deoxyribozyme; analysis of the distance between AA at the tail end of G4 and different base analogs shows that the phenomenon exists only at the position adjacent to G4, and the adenine analogs also have the effect. A simple and rapid escherichia coli detection system is constructed based on the enhancement of the deoxyribozyme effect by the adjacent base pairs, and the specificity and the working curve analysis of the detection system prove that the sensor has excellent performances of high specificity and lower detection limit. The method can also be applied to the detection of Escherichia coli in water sources.
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make many possible variations and modifications to the disclosed embodiments, or equivalent modifications, without departing from the spirit and scope of the invention, using the methods and techniques disclosed above. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention.
Sequence listing
<110> technical university of the middle and south forestry
<120> adjacent base pair enhanced deoxyribozyme and biosensor and application of biosensor
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gggttgggtg ggttggg 17
<210> 2
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gacggtggca gggaaagggg tcgggcatat ggcggagggg 40
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tatgcccgac ccctttccag t 21
Claims (9)
1. An enhanced dnazyme with adjacent base pairs, which is characterized in that the enhanced dnazyme has one of nucleotide sequences G4-AA, G4-AG, G4-A-MdA, G4-A-AP and G4-A-dI;
the nucleotide sequence of G4-AA is as follows: G4-AA-TTTCGCTATGTCTG;
the nucleotide sequence of G4-AG is: G4-AG-TTTCGCTATGTCTG;
wherein the nucleotide sequence of G4-A-MdA is: G4-A-/6-med a/-TTTCGCTATGTCTG;
the nucleotide sequence G4-A-/i2-amp/-TTTCGCTATGTCTG of G4-A-AP;
the nucleotide sequence G4-A-/6-oxoi/-TTTCGCTATGTCTG of G4-A-dI.
2. The adjacent base pair enhanced deoxyribozyme according to claim 1, wherein the nucleotide sequence of G4 is shown in SEQ ID No. 1.
3. A biosensor comprising the proximity base pair-enhanced deoxyribozyme according to claim 1 or 2, a complementary DNA strand, an E.coli aptamer and hemin.
4. The biosensor of claim 3, wherein the complementary DNA strand is cDNA,
the nucleotide sequence of the cDNA is shown in SEQ ID NO. 3.
5. The biosensor of claim 3, wherein the nucleotide sequence of the E.coli aptamer is shown as SEQ ID No. 2.
6. The biosensor of any one of claims 3 to 5, further comprising MES buffer system, ABTS2-And H2O2。
7. The biosensor according to claim 6, wherein the concentration of the enhanced dnazyme is 200 nM: the concentration of the complementary DNA chain is 400nM, and the concentration of the hemin is 200 nM; the ABTS2-Is 1 mM; said H2O2Was 2 mM.
8. Use of a biosensor according to any one of claims 3 to 7 for the detection of E.
9. The application according to claim 8, wherein the method of application is:
(1) adding the liquid to be tested into MES buffer solution containing the aptamer of the escherichia coli for incubation to obtain a system I;
(2) adding the adjacent base pair enhanced deoxyribozyme, hemin and complementary DNA chain of claim 1 or 2 into the system I for incubation to obtain a system II;
(3) transferring the system II into a quartz cuvette, and adding H2O2And ABTS2-Shaking up to obtain a system III;
(4) and measuring the absorbance change value at 420nm of the third system by using an ultraviolet spectrophotometer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210544494.4A CN114703192B (en) | 2022-05-18 | 2022-05-18 | Proximity base pair enhanced deoxyribozymes and biosensing and use of biosensing |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210544494.4A CN114703192B (en) | 2022-05-18 | 2022-05-18 | Proximity base pair enhanced deoxyribozymes and biosensing and use of biosensing |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114703192A true CN114703192A (en) | 2022-07-05 |
| CN114703192B CN114703192B (en) | 2024-06-14 |
Family
ID=82176139
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210544494.4A Active CN114703192B (en) | 2022-05-18 | 2022-05-18 | Proximity base pair enhanced deoxyribozymes and biosensing and use of biosensing |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114703192B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104845969A (en) * | 2015-05-08 | 2015-08-19 | 首都师范大学 | Method for controlling and improving DNAzyme catalytic activity |
| CN107119031A (en) * | 2017-06-21 | 2017-09-01 | 南京大学 | A kind of new thermophilic serobila DNA enzymatics of quaternary G tetra- |
| CN111763673A (en) * | 2020-07-09 | 2020-10-13 | 南方科技大学 | C-quadruplex deoxyribozyme and preparation method and application thereof |
| CN112063691A (en) * | 2020-09-18 | 2020-12-11 | 湖北医药学院 | Method for detecting single-chain target nucleic acid sequence based on G4-heme DNase system |
| CN113730593A (en) * | 2021-08-02 | 2021-12-03 | 成都凌泰氪生物技术有限公司 | Method for enhancing sustained release capacity of nucleic acid drug |
| CN114113267A (en) * | 2021-12-28 | 2022-03-01 | 郑州大学 | Construction method and application of aptamer sensor based on TdT and G4/hemin mimic enzyme amplification technology |
-
2022
- 2022-05-18 CN CN202210544494.4A patent/CN114703192B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104845969A (en) * | 2015-05-08 | 2015-08-19 | 首都师范大学 | Method for controlling and improving DNAzyme catalytic activity |
| CN107119031A (en) * | 2017-06-21 | 2017-09-01 | 南京大学 | A kind of new thermophilic serobila DNA enzymatics of quaternary G tetra- |
| CN111763673A (en) * | 2020-07-09 | 2020-10-13 | 南方科技大学 | C-quadruplex deoxyribozyme and preparation method and application thereof |
| CN112063691A (en) * | 2020-09-18 | 2020-12-11 | 湖北医药学院 | Method for detecting single-chain target nucleic acid sequence based on G4-heme DNase system |
| CN113730593A (en) * | 2021-08-02 | 2021-12-03 | 成都凌泰氪生物技术有限公司 | Method for enhancing sustained release capacity of nucleic acid drug |
| CN114113267A (en) * | 2021-12-28 | 2022-03-01 | 郑州大学 | Construction method and application of aptamer sensor based on TdT and G4/hemin mimic enzyme amplification technology |
Non-Patent Citations (3)
| Title |
|---|
| WANG LI等: "Insight into G-quadruplex-hemin DNAzyme/RNAzyme: adjacent adenine as the intramolecular species for remarkable enhancement of enzymatic activity", NUCLEIC ACIDS RESEARCH, 15 July 2016 (2016-07-15), pages 7373 * |
| 刘卓靓;李旺;黄燕;姚守拙;: "基于DNAzyme的单链核酸酶活性研究", 分析科学学报, no. 05, 20 October 2009 (2009-10-20), pages 11 - 15 * |
| 杨华林;吴庆华;苏东晓;王允;李利;: "G-四联体结构在生物传感器中的应用进展", 化学试剂, no. 05, 15 May 2017 (2017-05-15), pages 487 - 492 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114703192B (en) | 2024-06-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zeng et al. | Advances and challenges in viability detection of foodborne pathogens | |
| Calvo-Bado et al. | Spatial and temporal analysis of the microbial community in slow sand filters used for treating horticultural irrigation water | |
| Jennings et al. | Determination of deoxynivalenol and nivalenol chemotypes of Fusarium culmorum isolates from England and Wales by PCR assay | |
| Chen et al. | Characterization of mixed-species biofilm formed by Vibrio parahaemolyticus and Listeria monocytogenes | |
| Ehara et al. | Antimicrobial action of achacin is mediated by L-amino acid oxidase activity | |
| Amaro et al. | Functional GFP-metallothionein fusion protein from Tetrahymena thermophila: a potential whole-cell biosensor for monitoring heavy metal pollution and a cell model to study metallothionein overproduction effects | |
| Churruca et al. | Detection of Campylobacter jejuni and Campylobacter coli in chicken meat samples by real-time nucleic acid sequence-based amplification with molecular beacons | |
| Rakshith et al. | Isolation and characterization of antimicrobial metabolite producing endophytic Phomopsis sp. from Ficus pumila Linn.(Moraceae) | |
| Pérez‐Sierra et al. | High vegetative compatibility diversity of Cryphonectria parasitica infecting sweet chestnut (Castanea sativa) in Britain indicates multiple pathogen introductions | |
| CN102676664B (en) | Fluorescent quantitative polymerase chain reaction (PCR) primers and probes for detecting pathogenic bacteria of multiple aquatic products simultaneously and detection method | |
| Yang et al. | CRISPR-based nucleic acid diagnostics for pathogens | |
| Aridgides et al. | Multiplex PCR allows simultaneous detection of pathogens in ships' ballast water | |
| Fu et al. | Responses of phosphate transporter gene and alkaline phosphatase in Thalassiosira pseudonana to phosphine | |
| Srivastava et al. | Detection of siderophore production from different cultural variables by CAS-agar plate assay | |
| Kraiselburd et al. | The LOV protein of Xanthomonas citri subsp. citri plays a significant role in the counteraction of plant immune responses during citrus canker | |
| Labra et al. | Toxic and genotoxic effects of potassium dichromate in Pseudokirchneriella subcapitata detected by microscopy and AFLP marker analysis | |
| Hua et al. | Alteration of microbial properties and community structure in soils exposed to napropamide | |
| CN114703192A (en) | Application of adjacent base pair enhanced deoxyribozyme and biosensing and biosensor | |
| Ishfaq et al. | Molecular and Biochemical Screening of Local Aspergillus niger Strains Efficient in Catalase and Laccase Enzyme Production. | |
| Abulreesh et al. | Recovery of thermophilic campylobacters from pond water and sediment and the problem of interference by background bacteria in enrichment culture | |
| Liu | Handbook of nucleic acid purification | |
| Okada et al. | Two-round treatment with propidium monoazide completely inhibits the detection of dead Campylobacter spp. cells by quantitative PCR | |
| Liu et al. | Rapid and Accurate Detection of Bacteriophage Activity against Escherichia coli O157: H7 by Propidium Monoazide Real‐Time PCR | |
| Cotto et al. | Quantitative polymerase chain reaction for microbial growth kinetics of mixed culture system | |
| CN110358811B (en) | Method for optimizing loop-mediated isothermal amplification reaction |
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 |