CN113049656A - Electrochemical fast-scan voltammetry method with high-order repeatability and reproducibility and analysis application thereof - Google Patents
Electrochemical fast-scan voltammetry method with high-order repeatability and reproducibility and analysis application thereof Download PDFInfo
- Publication number
- CN113049656A CN113049656A CN202110167690.XA CN202110167690A CN113049656A CN 113049656 A CN113049656 A CN 113049656A CN 202110167690 A CN202110167690 A CN 202110167690A CN 113049656 A CN113049656 A CN 113049656A
- Authority
- CN
- China
- Prior art keywords
- tdt
- repeatability
- electrochemical
- reproducibility
- analysis
- 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
- 238000004458 analytical method Methods 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 31
- 238000004832 voltammetry Methods 0.000 title claims abstract description 18
- 102100033215 DNA nucleotidylexotransferase Human genes 0.000 claims abstract description 80
- 238000001514 detection method Methods 0.000 claims abstract description 36
- 239000003112 inhibitor Substances 0.000 claims abstract description 24
- 230000004044 response Effects 0.000 claims abstract description 19
- 230000000694 effects Effects 0.000 claims abstract description 16
- 230000035945 sensitivity Effects 0.000 claims abstract description 12
- 238000002360 preparation method Methods 0.000 claims abstract description 9
- 230000008901 benefit Effects 0.000 claims abstract description 7
- 239000010931 gold Substances 0.000 claims description 11
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 10
- 229910052737 gold Inorganic materials 0.000 claims description 10
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 8
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 claims description 7
- 150000003384 small molecules Chemical class 0.000 claims description 7
- 238000006116 polymerization reaction Methods 0.000 claims description 2
- 238000009585 enzyme analysis Methods 0.000 claims 1
- 238000003944 fast scan cyclic voltammetry Methods 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 13
- 238000010408 sweeping Methods 0.000 abstract description 3
- 238000012360 testing method Methods 0.000 abstract description 2
- 108010008286 DNA nucleotidylexotransferase Proteins 0.000 description 69
- 239000000243 solution Substances 0.000 description 20
- 108020004414 DNA Proteins 0.000 description 19
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 11
- 235000011180 diphosphates Nutrition 0.000 description 11
- 238000005516 engineering process Methods 0.000 description 8
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 8
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 7
- 238000002484 cyclic voltammetry Methods 0.000 description 5
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 5
- 102000012440 Acetylcholinesterase Human genes 0.000 description 4
- 108010022752 Acetylcholinesterase Proteins 0.000 description 4
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 4
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 4
- 108010089254 Cholesterol oxidase Proteins 0.000 description 4
- 108090000526 Papain Proteins 0.000 description 4
- 239000004365 Protease Substances 0.000 description 4
- 229940022698 acetylcholinesterase Drugs 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 239000012154 double-distilled water Substances 0.000 description 4
- 229940055729 papain Drugs 0.000 description 4
- 235000019834 papain Nutrition 0.000 description 4
- 239000011541 reaction mixture Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 102000008130 Cyclic AMP-Dependent Protein Kinases Human genes 0.000 description 3
- 108010049894 Cyclic AMP-Dependent Protein Kinases Proteins 0.000 description 3
- 229910021607 Silver chloride Inorganic materials 0.000 description 3
- 238000007664 blowing Methods 0.000 description 3
- 239000008151 electrolyte solution Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 3
- 150000003573 thiols Chemical class 0.000 description 3
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 2
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 2
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000000840 electrochemical analysis Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- 238000005498 polishing Methods 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- QHGUCRYDKWKLMG-QMMMGPOBSA-N (R)-octopamine Chemical compound NC[C@H](O)C1=CC=C(O)C=C1 QHGUCRYDKWKLMG-QMMMGPOBSA-N 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 229920000049 Carbon (fiber) Polymers 0.000 description 1
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 1
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- YJPIGAIKUZMOQA-UHFFFAOYSA-N Melatonin Natural products COC1=CC=C2N(C(C)=O)C=C(CCN)C2=C1 YJPIGAIKUZMOQA-UHFFFAOYSA-N 0.000 description 1
- QHGUCRYDKWKLMG-MRVPVSSYSA-N Octopamine Natural products NC[C@@H](O)C1=CC=C(O)C=C1 QHGUCRYDKWKLMG-MRVPVSSYSA-N 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000010256 biochemical assay Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000004917 carbon fiber Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 229960003638 dopamine Drugs 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 229940029575 guanosine Drugs 0.000 description 1
- 229960001340 histamine Drugs 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- DRLFMBDRBRZALE-UHFFFAOYSA-N melatonin Chemical compound COC1=CC=C2NC=C(CCNC(C)=O)C2=C1 DRLFMBDRBRZALE-UHFFFAOYSA-N 0.000 description 1
- 229960003987 melatonin Drugs 0.000 description 1
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000001722 neurochemical effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 229960001576 octopamine Drugs 0.000 description 1
- 230000033116 oxidation-reduction process Effects 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 229940076279 serotonin Drugs 0.000 description 1
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 description 1
- 229940048086 sodium pyrophosphate Drugs 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 235000019818 tetrasodium diphosphate Nutrition 0.000 description 1
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses an electrochemical fast-scan voltammetry method with high-order repeatability and reproducibility and analysis application thereof. By changing the final concentration of TdT (0.1-190U/mL) or PP (0-9 mM) in a mixed reaction solution, testing a sensor by adopting FSCV (free space constant) and setting the initial potential to be 0V, the termination potential to be 1V and the sweeping speed to be 400V/s, the fast sweeping method is applied to the analysis and detection of TdT and a small molecular inhibitor thereof, has the advantages of high sensitivity, strong specificity, short analysis time, short response speed, simple preparation and high-order repeatability and reproducibility of electrochemical signals, can well realize the analysis of the TdT activity with lower concentration, and has the detection limits of (respectively) (0.067U/mL), (0.1U/mL), (0.067U/mL) and (0.1U/mL), and the inhibitor detection IC (integrated circuit) detects50The values are 1 respectively.7mM, 1.6mM, 1.5mM and 1.7mM, and has good application prospect.
Description
Technical Field
The present invention relates to two fields: an electrochemical analysis technology and biosensing, in particular to an electrochemical fast-scan voltammetry method with high-order repeatability, which is applied to biosensing analysis of terminal deoxynucleotidyl transferase and small molecular inhibitors thereof.
Background
Terminal deoxynucleotidyl transferase (TdT), unlike other DNA polymerases, has the specific ability to incorporate nucleotides into the 3' -OH end of single-stranded DNA in a template-independent manner. The expression of the TdT gene is normally inhibited in most human cells, but is abnormally high in acute lymphocytic leukemia cells and some other leukemia-related cells. Notably, acute lymphoblastic leukemia accounts for about 25% of the most common cancers in childhood, and as such, detection of TdT is critical for the development of cancer-related drugs and basic biochemical studies. Although TdT is biologically functional, the traditional TdT activity detection methods, which are very classical, include gel electrophoresis combined with radiolabelling, biochemical assays and immunoassays, and often suffer from inherent limitations including harmful radiation, low sensitivity or complex procedures, which are very time consuming. In recent years, several new methods have been developed for the analysis of TdT and its small molecule inhibitors, including fluorescence, colorimetry, and electrochemiluminescence. However, these methods still have some limitations, such as complicated chemical reaction, insufficient sensitivity, complicated material synthesis, etc., resulting in poor reproducibility and reproducibility. Therefore, development of new methods for TdT analysis that are simple, fast, sensitive, and have high reproducibility and reproducibility is still a goal pursued by researchers.
The Fast Sweep Voltammetry (FSV) technique is an electrochemical technique with a sweep rate higher than 10V/s, retains the advantages of simple operation, fast response, high sensitivity and the like of a common electrochemical method, has a larger spatial and sub-second time measurement resolution and can obviously improve the sensitivity of analyte detection. Background-subtracted Fast Scanning Cyclic Voltammetry (FSCV) in combination with carbon fiber microelectrodes has been widely used to probe in vivo neurochemical kinetics, and is currently mainly applied to applications including dopamine, melatonin, serotonin, histamine, octopamine, adenosine, guanosine, and the like. The FSCV equipment used by the invention comprises a Tektronix AFG1022 function generator, a Tektronix TBS1102 oscilloscope and a group of self-development circuits which can compensate the ohmic drop of the solution on line based on a positive feedback technology and a microcontroller technology. The device can actively compensate the ohmic drop of the whole reaction system at different sweeping speeds, ensure the output volt-ampere signal to be real and effective and obtain accurate electrochemical information. In the FSCV map, we can clearly see the characteristic behavior and retention time of each electrochemical peak, and due to the high scan speed, we can obtain the sub-second reversible electrochemical information that is not available on the ordinary electrochemical workstation: under the high sweep rate provided by the equipment, the output electrochemical signal presents high-order repeatability and reproducibility, and compared with a commercialized electrochemical workstation (the sweep rate is generally 0.01-0.5V/s), the analysis method with the high-order repeatability and reproducibility can avoid the contingency of experimental results, improve the accuracy of measured data, reduce the error between each measured data, and most importantly, can obviously reduce the dispersion degree of the measured results of the same method after a period of time. The FSCV signal can be stable from the beginning of the experiment to the end of the experiment, even though the randomly intercepted data period shows a high degree of consistency. Under each oxidation-reduction period, the voltammetric signal output by FSCV excitation is clear and stable, and the electrochemical technology is applied to the field of analytical sensing and is expected to help solve other biological problems.
The invention designs an FSCV with high-order repeatability and reproducibility, and applies the technology to TdT activity analysis. The method introduces sulfydryl DNA into the surface of a gold electrode through the interaction of Au and sulfydryl, continuously extends dTTP to the 3' -OH end of the sulfydryl DNA by utilizing the polymerization and extension of TdT to form a poly-T DNA long chain, and the long chain passes through T-Hg2+-T action on Hg2+Has strong affinity, and a certain amount of Hg is enriched on the surface of the electrode2+And outputting electrochemical signals through a built rapid scanning device. Hg is known to be capable of electron withdrawing at high scan rates due to its better ability to gain and lose electrons2+When first reduced to Hg, does not pass from T-Hg2+the-T structure is broken off and can be oxidized again, which cannot be realized on a common electrochemical workstation, and moreover, the electrochemical signal output by us,and the high-order repeatability and reproducibility state are presented, and the defects of other analysis sensing methods applied to TdT activity analysis are overcome. The fast scanning method with high-order repeatability and reproducibility provides a new visual angle for the development of an electrochemical analysis technology and opens up a new path for biosensing analysis.
Disclosure of Invention
The invention aims to provide a novel analysis means for detecting TdT and a small molecule inhibitor thereof, which has the advantages of good specificity, high detection speed, high sensitivity, accurate and reliable result, low cost and high-order repeatability and reproducibility.
The technical scheme adopted by the invention for solving the technical problems is as follows: an electrochemical fast-scan voltammetry method with high-order repeatability and reproducibility and an analysis application thereof are disclosed, which comprises the following specific steps:
(1) pretreatment of electrodes
Gold electrode with a diameter of 2mm was coated with 1 μm, 0.3 μm and 0.05 μm of Al in this order2O3Polishing the powder into a mirror surface, then ultrasonically washing the mirror surface by water and absolute ethyl alcohol for 2 to 10min in sequence, drying the mirror surface by nitrogen, and then placing the gold electrode at 0.5M H2SO4The solution was scanned to stability using Cyclic Voltammetry (CV). And finally, blowing the mixture by using nitrogen for standby.
(2) Preparation of electrochemical biosensor
Electrode 1: dripping 2-10 mu L of sulfhydryl DNA with the concentration of 3-10 mu M on the surface of the treated gold electrode, and placing the gold electrode in a refrigerator at 4 ℃ overnight. After the incubation is finished, the surface of the electrode is lightly rinsed to remove the unbound sulfhydryl DNA, and then the electrode is immersed in 2-Mercaptoethanol (MCH) with the concentration of 0.5-2 mM for 0.5-3 h to block the active sites of the unbound sulfhydryl DNA.
Electrode 2: preparing a TdT reaction mixture: 0.05-0.3 muL of TdT enzyme solution with the concentration of 0.01-20U/muL, 0.5-2 muL of dTTP solution with the concentration of 10mM, 2-10 muL of 5 xTdT buffer (1M potassium carbonate, 0.125M Tris, 0.05% (v/v) Triton X-100, 5mM cobalt chloride (pH 7.2at 25 ℃)) and 1-5 muL of double distilled water are mixed uniformly and dripped on an electrode 1, the electrode is placed at 37 ℃ for reaction for 0.5-3 h to form a poly-T DNA long chain, and residual TdT reaction reagent is washed by water.
Electrode 3: placing the prepared electrode 2 in 100-200 mul with the concentration of 10-5M Hg2+Placing the electrode in a solution centrifuge tube at 37 ℃ for 0.5-1 h to ensure that Hg is contained in the solution2+Inducing poly-T DNA long chain to form T-Hg2+-a T structure.
(3) TdT activity and inhibitor assays
Based on the reaction solution dripped to the electrode 2, the TdT activity is detected by changing the concentration of TdT (0.01U/mL-20U/muL) and other steps and experimental conditions are the same as those of the electrode preparation step.
Based on the reaction liquid dripped to the electrode 2, sodium pyrophosphate (PP) with different concentrations is mixed with the reaction liquid while the concentration of TdT is fixed, and the rest steps and experimental conditions are the same as the electrode manufacturing steps, so that the analysis and detection of the TdT inhibitor are realized.
(4) FSCV testing
The electrode 3 obtained in the process is used as a working electrode, a platinum wire electrode is used as a counter electrode, an Ag/AgCl electrode is used as a reference electrode, the counter electrode is placed in PBS electrolyte solution with pH of 7, a rapid scanning cyclic voltammetry (FSCV) is adopted, the initial potential is set to be-0.5-0V, the final potential is set to be 0.9-1.2V, and the potential scanning speed is 50-400V/s; and measuring voltage peak values corresponding to different TdT concentrations and different inhibitor concentrations (fixed TdT concentrations), establishing a quantitative relation between voltage response and TdT/PP, and determining the TdT/PP content in the unknown actual sample according to the quantitative relation between the voltage response and the TdT/PP.
The invention principle is as follows: the invention relates to an electrochemical fast-scanning voltammetry method with high-order repeatability and reproducibility and analysis application thereof, and the technology is successfully applied to the analysis and detection of TdT and small molecule inhibitors thereof. Firstly, firmly fixing sulfhydryl DNA on a gold electrode by utilizing Au-S bond, taking a 3' -OH end as a TdT action site, adding a TdT reaction mixed solution, adding dTTP to the tail part of the sulfhydryl DNA by the enzyme to form a poly-T DNA long chain, and utilizing T and Hg2+Induce the long chain to form T-Hg2+-a T structure. First, Hg is fixed2+By varying the concentration of TdTThe final concentration of TdT in the reaction mixture is within a certain range, and the higher the concentration is, the longer the poly-TDNA long chain which can be extended is, and therefore Hg which can be bound is2+The more the electrochemical response signal is, the stronger the electrochemical response signal is, and the detection of TdT activity can be realized; the concentration of TdT is then fixed, and by varying the final concentration of PP in the mixture of TdT reactions, the higher the final concentration of PP, the higher the degree of inhibition of the TdT enzyme activity, and the shorter the length of poly-T DNA chain that can be extended, the bound Hg is2+The less the electrochemical response signal, the lower the analytical detection of TdT inhibitors. Compared with the ordinary electrochemical Cyclic Voltammetry (CV), due to the ultrahigh scanning rate of the FSCV, according to the electrochemical principle, the higher the scanning speed V provided by the electrode in the adsorption state, the larger the current i value, and the direct ratio relationship between the current i value and the current i value, and in the same system, the resistance is certain, so the output voltage (V) is higher. FSV can significantly improve the sensitivity of detection compared to classical voltammetric analysis. Further, Hg2+As signal output, under the high scanning speed, the in-situ reduction-oxidation reaction can be realized, namely, the electrochemical output signal is not reduced along with the lengthening of the analysis time, so that the change of TdT activity along with the time is clearly visible, and the detection of the activity and the inhibitor is realized. Based on the method, the method for analyzing the FSCV of the TdT and the inhibitor is easy to operate, high in response speed, high in sensitivity and excellent in selectivity.
Compared with the prior art, the invention has the advantages that:
(1) high sensitivity. The long TdT extended poly-T DNA chain can capture more Hg2+Realizing the first-stage amplification of the electric signal; the FSCV provides a high scanning rate, can generate larger electrochemical signal feedback, realizes the secondary amplification of an electric signal, has detection limits of 0.067U/mL, 0.1U/mL, 0.067U/mL and 0.1U/mL respectively for two selected periods of fast-scanning voltammetry data, and shows ultrahigh sensitivity.
(2) High specificity. Detection of TdT: control substances such as alkaline phosphatase (ALP), cholesterol oxidase (ChOx), Protein Kinase A (PKA), acetylcholinesterase (AChE) and papain (papain) did not interfere with this system.
(3) The analysis time is short, and the response speed is high. The FSCV has larger spatial and sub-second time measurement resolution, so that the analysis time can be greatly shortened, and the time cost is saved.
(4) The preparation method is simple, the reagent dosage is less, and the cost is low. The sensor can be built up only by consuming a small amount of reagent consumables, and the analysis and detection of TdT and the inhibitor thereof are realized.
(5) High repeatability and reproducibility. The self-assembled online compensation ohmic drop fast-scan voltammetric device is benefited, and the electrochemical voltammetric signal with high repeatability and reproducibility can be output while the data is real and effective by utilizing the advantage of high scan speed. The two period data obtained at random are analyzed to show that the linear equations are similar, and are within the error tolerance range: the linear range of detection of TdT activity is: 0.5-150U/mL, wherein the detection limit of each period is (0.067U/mL), (0.1U/mL), (0.067U/mL) and (0.1U/mL); data per cycle of TdT inhibitor was calculated as IC501.7mM, 1.6mM, 1.5mM and 1.7mM respectively,
the data are similar and all accord with the report.
In conclusion, the electrochemical fast sweep voltammetry method with high-order repeatability and reproducibility, which is constructed by the invention, is applied to the analysis and detection of TdT and small molecule inhibitors thereof, has the advantages of high sensitivity, strong specificity, short analysis time, high response speed, simple preparation, capability of outputting electrochemical signals with high-order repeatability and reproducibility and the like, can well realize the analysis of TdT activity with lower concentration, and has good application prospect.
Drawings
FIG. 1 is a high level reproducibility and repeatability experimental plot presented by the FSCV technique of the present invention;
FIG. 2 is a graph of the feasibility of the sensor of the present invention for the analytical detection of TdT and inhibitors;
FIG. 3 is a graph of voltage response versus concentration for different concentrations TdT, PP in accordance with the present invention;
FIG. 4 is a diagram showing the specificity of the sensor of the present invention for TdT.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Example 1 preparation of an electrochemical biosensor comprising the following steps:
bare electrode: gold electrode with a diameter of 2mm was coated with 1 μm, 0.3 μm and 0.05 μm of Al in this order2O3Polishing the powder to obtain mirror surface, ultrasonic washing with water and anhydrous ethanol for 5min, blowing with nitrogen gas, and drying the gold electrode at 0.5M H2SO4The solution was scanned by Cyclic Voltammetry (CV) until the signal stabilized. And finally, blowing the mixture by using nitrogen for standby.
Electrode 1: mu.L of thiol DNA at a concentration of 10. mu.M was dropped onto the surface of the treated gold electrode, and the resulting solution was placed in a refrigerator at 4 ℃ overnight. After the incubation was completed, the electrode surface was gently rinsed to remove unbound thiol DNA, and thereafter, the electrode was immersed in 2-Mercaptoethanol (MCH) at a concentration of 2mM for 1h to block the active site of unbound thiol DNA.
Electrode 2: preparing a TdT reaction mixture: mu.L of 20U/. mu.L TdT enzyme solution, 1. mu.L of 10mM dTTP solution, 2. mu.L of 5 XTdT buffer and 2. mu.L of double distilled water were mixed uniformly and added dropwise to the electrode 1, the electrode was left at 37 ℃ to react for 3 hours to form a long poly-T DNA chain, and the residual TdT reaction reagent was washed away with water.
Electrode 3: the electrode 2 prepared above was placed at a concentration of 10 in 200. mu.L-5M Hg2+The electrode was placed in a centrifuge tube of solution at 37 ℃ for 1h to allow Hg to flow2+Inducing poly-T DNA long chain to form T-Hg2+-a T structure.
Example 2 feasibility analysis, comprising the following steps:
the procedure of the electrochemical biosensor in example 1 was followed, and the TdT mixed reaction solution dropped to the electrode 2 was composed of 0.2. mu.L of TdT solution at concentrations of 0 and 100U/mL, 1. mu.L of dTTP solution at a concentration of 10mM, 2. mu.L of 5 XTdT buffer and 2. mu.L of double distilled water. Other steps and experimental conditions are the same as the electrode preparation steps and are used for feasibility detection of the invention.
Adopting FSCV detection, using the prepared electrode 3 as a working electrode, a platinum electrode as a counter electrode, AThe g/AgCl electrode was a reference electrode, placed in a PBS electrolyte solution of pH 7, set at an initial potential of 0V, a termination potential of 1V, and a scanning speed of 400V/s. The results are shown in FIG. 1 (C)TdT100U/mL), the data form presented by the method has obvious high-order reproducibility and repeatability characteristics, and the data results in hundreds of periods are highly consistent, which indicates that the fast scanning technology is successfully applied to TdT analysis.
All the data that follow we do random intercept for two cycles, as in fig. 2A, when TdT concentration is 0, the sensor has no electrochemical response; while for TdT at a concentration of 100U/mL, FSCV measurements show a good electrochemical response signal, indicating that the sensor can be applied to the detection of TdT activity.
Next, the TdT mixed reaction solution added dropwise to the electrode 2, including 0.2. mu.L of TdT solution with a concentration of 140U/mL, PP (0, 1, 5mM) with different final concentrations, 1. mu.L of dTTP solution with a concentration of 10mM, 2. mu.L of 5 XTdT buffer cobalt chloride and 2. mu.L of double distilled water, was mixed and added dropwise to the reaction solution of the electrode 2 in accordance with the procedure for preparing the electrochemical biosensor in example 1, and the remaining steps and experimental conditions were the same as those in the electrode-preparing step.
Using FSCV detection, the prepared electrode 3 as a working electrode, a platinum electrode as a counter electrode, and an Ag/AgCl electrode as a reference electrode were placed in a PBS electrolyte solution with pH of 7, and an initial potential of 0V, a final potential of 1V, and a scanning speed of 400V/s were set. The results are shown in fig. 2B, and it can be seen that as the final concentration of PP increases, the electrochemical response signal decreases, and the sensor can be applied to the analytical detection of TdT inhibitors.
Example 3 detection of TdT enzymatic Activity and inhibitors
The electrochemical response of the sensor was tested by FSCV by varying the final concentration of TdT (0.1-190U/mL) or PP (0-9 mM) in the mixed reaction solution according to the procedure of example 1 and example 2, setting the initial potential to 0V, the final potential to 1V, and the sweep rate to 400V/s. As shown in fig. 3A, the voltage response of the sensor to TdT is in good linear relation with the concentration, and the linear correlation equation of the voltage response of the sensor to TdT concentration is that (y) is 0.0009x +0.050, and R2＝0.984、②y＝-0.001x-0.110，R2＝0.991，③y＝0.0009x+0.048，R20.986 and y-0.001 x-0.115, R20.994. The linear equations of the two selected periodic data are similar, the detection linear range is 0.5-150U/mL within the error allowable range, and the detection limits are (0.067U/mL), (0.1U/mL), (0.067U/mL) and (0.1U/mL) respectively. As shown in FIG. 3B, from the TdT inhibitor PP concentration vs. voltage response fitted curve, two cycles of data were analyzed for IC501.7mM, 1.6mM, 1.5mM and 1.7mM respectively, and the results are similar and accord with the existing report. The reason for the slight difference in data per cycle may be derived from the difference in the redox potential of Hg. The fast-scan voltammetry constructed by the invention is applied to the analysis and detection of terminal transferase and small molecule inhibitors, the obtained high-order repeatability and reproducibility data are reliable, the stability of the measured data is greatly improved, the error between the data is reduced, and the dispersion degree of the measured result is reduced.
Example 4 specific assay
To verify whether the sensor prepared by the present invention has specificity and anti-interference capability, other active enzymes having the same concentration as TdT were added to the TdT reaction mixture according to the preparation steps and detection methods of examples 1, 2, and 3 above: such as alkaline phosphatase (ALP), cholesterol oxidase (ChOx), protein kinase a (pka), acetylcholinesterase (AChE), and papain (papain), the FSCV parameter settings were the same as in the examples, and the selectivity of the sensor for TdT was examined. The results are shown in fig. 4, indicating that the sensor invention has good specificity for TdT detection.
Of course, the above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Variations, modifications, additions and substitutions which may occur to those skilled in the art and which fall within the spirit and scope of the invention are also considered to be within the scope of the invention.
Claims (4)
1. An electrochemical fast-scan voltammetry method with high-order repeatability and reproducibility and analysis application thereof are successfully applied to TdT analysis, and are characterized by comprising the following steps: firstly, sulfhydryl DNA is introduced to the surface of gold electrode, so as toThe dTTP is continuously extended to the 3' -OH end of the sulfhydryl DNA by the unique polymerization extension of TdT, so that the sulfhydryl DNA is extended into a poly-T DNA long chain. Second, a fixed concentration of Hg is introduced2+Solutions based on T bases and Hg2+Strong affinity between the two to form T-Hg2+-T specific structure, whereby a certain amount of Hg is enriched at the electrode surface2+And successfully preparing the sensor. Electrochemical signal output is carried out by a built rapid scanning device, and Hg is utilized2+The self is used as a signal molecule, and the activity of the self is detected by adding TdT solutions with different final concentrations; and fixing the concentration of TdT, and adding PP with different concentrations to realize the analysis of the TdT inhibitor. Based on this, the electrochemical fast sweep voltammetry method with high-order repeatability and reproducibility constructed by the invention is applied to the analysis and detection of TdT and small molecule inhibitors thereof, has the advantages of high sensitivity, strong specificity, short analysis time, fast response speed, simple preparation, capability of outputting electrochemical signals with high-order repeatability and reproducibility and the like, can well realize the analysis of TdT activity with lower concentration, and has good application prospect.
2. The electrochemical fast-scan voltammetry with high-order repeatability and reproducibility and its analytical application of claim 1, wherein: using FSCV provides a higher scan rate, under which condition Hg is output as a signal2+The electrochemical fast-scan voltammetry method with high-order repeatability and reproducibility is constructed on the basis that the electrochemical signal output with continuous stability and high repeatability can be used as feedback in the detection period, which cannot be realized in classical voltammetry.
3. An electrochemical fast-scan voltammetry with high order repeatability and reproducibility and its analytical application according to claims 1-2, wherein: the electrochemical fast-scan voltammetry method with high-order repeatability and reproducibility is applied to the analysis of TdT activity and small molecule inhibitors thereof for the first time, and has excellent selectivity.
4. An electrochemical fast-scan voltammetry with high order repeatability and reproducibility and its analytical application according to claims 1-3, wherein: the sensor prepared by the invention realizes high-sensitivity detection of TdT, the detection limits are respectively (0.067U/mL), (0.1U/mL), (0.067U/mL) and (0.1U/mL), and the inhibitor detection IC50The values are 1.7mM, 1.6mM, 1.5mM and 1.7mM respectively, and the invention can open up a new idea for an enzyme analysis method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110167690.XA CN113049656B (en) | 2021-01-25 | 2021-01-25 | Electrochemical fast sweep voltammetry with high-order repeatability and reproducibility and analysis application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110167690.XA CN113049656B (en) | 2021-01-25 | 2021-01-25 | Electrochemical fast sweep voltammetry with high-order repeatability and reproducibility and analysis application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113049656A true CN113049656A (en) | 2021-06-29 |
| CN113049656B CN113049656B (en) | 2024-03-01 |
Family
ID=76508729
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110167690.XA Active CN113049656B (en) | 2021-01-25 | 2021-01-25 | Electrochemical fast sweep voltammetry with high-order repeatability and reproducibility and analysis application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113049656B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114609210A (en) * | 2022-03-01 | 2022-06-10 | 宁波大学 | Rapid scanning sensing method based on copper ion in-situ electrochemical signal output and biological application thereof |
| CN114609210B (en) * | 2022-03-01 | 2024-07-12 | 江苏跨境数据科技有限公司 | Rapid scanning sensing method based on copper ion in-situ electrochemical signal output and biological application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102375020A (en) * | 2010-08-19 | 2012-03-14 | 华东师范大学 | Method for detecting mercury ions through gold electrode modified by polythymidine DNA |
| CN109536577A (en) * | 2018-11-21 | 2019-03-29 | 暨南大学 | A kind of measuring method and application of terminal deoxy nuclease vigor |
| CN109576342A (en) * | 2018-11-21 | 2019-04-05 | 暨南大学 | A kind of fluorescence chemical method for detection of alkaline phosphatase and application |
| CN109856212A (en) * | 2019-01-21 | 2019-06-07 | 宁波大学 | A kind of preparation method and applications of electrochemica biological sensor that is while detecting HAT and TdT |
| CN109856211A (en) * | 2019-01-21 | 2019-06-07 | 宁波大学 | A kind of preparation method and applications of electrochemica biological sensor that is while detecting Exo I and TdT |
-
2021
- 2021-01-25 CN CN202110167690.XA patent/CN113049656B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102375020A (en) * | 2010-08-19 | 2012-03-14 | 华东师范大学 | Method for detecting mercury ions through gold electrode modified by polythymidine DNA |
| CN109536577A (en) * | 2018-11-21 | 2019-03-29 | 暨南大学 | A kind of measuring method and application of terminal deoxy nuclease vigor |
| CN109576342A (en) * | 2018-11-21 | 2019-04-05 | 暨南大学 | A kind of fluorescence chemical method for detection of alkaline phosphatase and application |
| CN109856212A (en) * | 2019-01-21 | 2019-06-07 | 宁波大学 | A kind of preparation method and applications of electrochemica biological sensor that is while detecting HAT and TdT |
| CN109856211A (en) * | 2019-01-21 | 2019-06-07 | 宁波大学 | A kind of preparation method and applications of electrochemica biological sensor that is while detecting Exo I and TdT |
Non-Patent Citations (8)
| Title |
|---|
| FAN YANG ET AL.: "Fast-Scan Anodic Stripping Voltammetry for Detection of Pb(II) at Picomolar Level", 《RUSSIAN JOURNAL OF ELECTROCHEMISTRY 》 * |
| SHAOHUA MA ET AL.: "Adenine/Au complex-dependent versatile electrochemical platform for ultrasensitive DNA-related enzyme activity assay", 《SENSORS AND ACTUATORS B: CHEMICAL》 * |
| 孟维强: "基于快速伏安技术超痕量检测重金属离子的研究", 《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》 * |
| 徐宁: "汞离子检测的电化学DNA传感器研究", 《中国优秀硕士学位论文全文数据库信息科技辑》 * |
| 杨丽 等: "基于poly-T的末端脱氧核苷酸转移酶的检测方法", 《中国化学会第十三届全国分析化学年会论文集（二）》 * |
| 杨帆: "水体系中重金属离子的快速检测", 《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》 * |
| 王静茹 等: "基于T-HgⅡ-T结构和TdT功能介导的检测碱性磷酸酶的荧光化学检测方法", 《第十一届全国化学生物学学术会议论文摘要（第三卷）》 * |
| 胡宇芳 等: "末端脱氧核苷酸转移酶活性检测的电化学传感研究及应用", 《中国化学会第30届学术年会摘要集-第四分会：生物分析和生物传感》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114609210A (en) * | 2022-03-01 | 2022-06-10 | 宁波大学 | Rapid scanning sensing method based on copper ion in-situ electrochemical signal output and biological application thereof |
| CN114609210B (en) * | 2022-03-01 | 2024-07-12 | 江苏跨境数据科技有限公司 | Rapid scanning sensing method based on copper ion in-situ electrochemical signal output and biological application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113049656B (en) | 2024-03-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kueng et al. | Amperometric ATP biosensor based on polymer entrapped enzymes | |
| Wang et al. | Electrochemical sensor for simultaneous determination of uric acid, xanthine and hypoxanthine based on poly (bromocresol purple) modified glassy carbon electrode | |
| CN101849180B (en) | Multizone analyte testing sensor | |
| Yang et al. | Covalent immobilization of an enzyme (glucose oxidase) onto a carbon sol–gel silicate composite surface as a biosensing platform | |
| Brahman et al. | An electrochemical sensing platform for trace recognition and detection of an anti-prostate cancer drug flutamide in biological samples | |
| Bagheri et al. | Gold nanoparticles deposited on fluorine-doped tin oxide surface as an effective platform for fabricating a highly sensitive and specific digoxin aptasensor | |
| KR20100008260A (en) | Protein measurement apparatus by using biosensor | |
| Chen et al. | Electrocatalytic Oxidation and Determination of Dopamine in the Presence of Ascorbic Acid and Uric Acid at a Poly (4‐(2‐Pyridylazo)‐Resorcinol) Modified Glassy Carbon Electrode | |
| Zen et al. | Voltammetric determination of serotonin in human blood using a chemically modified electrode | |
| Dehghanzade et al. | Voltammetric determination of diazepam using a bismuth modified pencil graphite electrode | |
| Josypčuk et al. | Flow electrochemical biosensors based on enzymatic porous reactor and tubular detector of silver solid amalgam | |
| Qijin et al. | Voltammetric behavior of vitamin B2 on the gold electrode modified with a self-assembled monolayer of L-cysteine and its application for the determination of vitamin B2 using linear sweep stripping voltammetry | |
| Morkvenaite‐Vilkonciene et al. | Evaluation of enzymatic kinetics of GOx‐based electrodes by scanning electrochemical microscopy at redox competition mode | |
| CN114441616B (en) | Method for modifying new coronavirus biological probe on electrochemical biosensor | |
| CN102667475A (en) | Method for measuring analyte concentration in a liquid sample | |
| Pemberton et al. | An assay for the enzyme N-acetyl-β-D-glucosaminidase (NAGase) based on electrochemical detection using screen-printed carbon electrodes (SPCEs) | |
| González-López et al. | Electrochemical micropipette-tip for low-cost environmental applications: Determination of anionic surfactants through their interaction with methylene blue | |
| CN113049656B (en) | Electrochemical fast sweep voltammetry with high-order repeatability and reproducibility and analysis application thereof | |
| Wu et al. | Voltammetric Investigation of Vitamin B~ 6 at a Glassy Carbon Electrode and Its Application in Determination | |
| Guo et al. | A miniaturized electrochemical biosensor based on poly (L-threonine) modified pencil graphite electrodes and its application for trace-level determination of uric acid, xanthine and hypoxanthine | |
| Li et al. | Sensitive determination of urapidil at an electrochemically pretreated glassy carbon electrode by linear sweep voltammetry | |
| Zhang et al. | Electrochemical Determination of Ascorbic Acid at γ-MnO 2 Modified Carbon Black Microelectrodes | |
| JPH04279854A (en) | Platinum coated carbon fiber electrode and enzymatic film sensor using same | |
| Dou et al. | Electrochimical determination of uric acid, xanthine and hypoxanthine by poly (xylitol) modified glassy carbon electrode | |
| Niu et al. | Electrochemical behavior of uric acid at a penicillamine self-assembled gold electrode |
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 | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20231215 Address after: 518000 1002, Building A, Zhiyun Industrial Park, No. 13, Huaxing Road, Henglang Community, Longhua District, Shenzhen, Guangdong Province Applicant after: Shenzhen Wanzhida Technology Co.,Ltd. Address before: 315211 Ningbo University, 818 Fenghua Road, Jiangbei District, Ningbo, Zhejiang Applicant before: Ningbo University |
|
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20240130 Address after: Room 301, Building 3, No. 28, Juhai Second Road, Qujiang District, Quzhou City, Zhejiang Province 324000 Applicant after: Zhejiang fengneng Pharmaceutical Technology Co.,Ltd. Country or region after: China Address before: 518000 1002, Building A, Zhiyun Industrial Park, No. 13, Huaxing Road, Henglang Community, Longhua District, Shenzhen, Guangdong Province Applicant before: Shenzhen Wanzhida Technology Co.,Ltd. Country or region before: China |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |