CN1459635A - Enzyme substrate chip and its preparation method and application - Google Patents
Enzyme substrate chip and its preparation method and application Download PDFInfo
- Publication number
- CN1459635A CN1459635A CN 02113760 CN02113760A CN1459635A CN 1459635 A CN1459635 A CN 1459635A CN 02113760 CN02113760 CN 02113760 CN 02113760 A CN02113760 A CN 02113760A CN 1459635 A CN1459635 A CN 1459635A
- Authority
- CN
- China
- Prior art keywords
- chip
- enzyme
- substrate
- various
- probe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
An enzyme substrate chip for qualitatively and quantitatively analyzing activity of different enzymes is prepared through immobilizing a great deal of enzyme substrates onto probe carrier orderly and densely. It can be used for screening medicines and discriminating biologic tissue.
Description
The invention belongs to field of biology, relate to a kind of enzyme substrate chip and preparation method and application.
Enzyme is a kind of biocatalyst, often will carry out the vitality test of enzyme in scientific research, industrial and agricultural production and medical practice.The activity level of enzyme can detect by enzyme (urging) reaction velocity of measuring particular system.Can adopt dual mode when carrying out this mensuration: a kind of is end-point method; Another kind is a KINETIC METHOD.End-point method also claims the achromic point method, and it is meant is determining under the condition, allows a certain amount of substrate of enzyme effect, and the detection reaction system reaches the needed time of a certain index then, and estimates a kind of method of enzyme activity according to the length of time.For example, the commercial production AMS adopts this method to measure with regard to normal.A development of end-point method is exactly to adopt detecting device that Continuous Tracking is carried out in reaction, and when reaction reaches a certain degree, needed time of record automatically accurately, the so-called fixed concentration method during Here it is enzyme is analysis automated.Another development of end-point method is to make easy " the enzyme detection scraps of paper ".KINETIC METHOD is the most frequently used method of enzyme activity determination.The principle that KINETIC METHOD is measured is: under certain conditions, enzyme reaction rate and enzyme concentration are directly proportional, and therefore, measure enzyme reaction rate and just can try to achieve enzyme concentration.But there are following shortcoming and defect in said method: the major defect as end-point method is an out of true, is bordering on sxemiquantitative, and can not observe overall process, therefore, seldom uses.It is big that chemical method in the KINETIC METHOD exists workload, itself comprises certain error.Also have in addition: the determination of light absorption method of optical method requires reaction product and substrate on a certain wavelength or a certain wave band, has obvious characteristics to absorb difference; Electrochemical determination requires (Chen Shigen, Zhou Runqi write, " zymetology " that publishing house of Fudan University publishes, 221-231 page or leaf) such as certain instrument and equipments.
The objective of the invention is to overcome deficiency of the prior art and a kind of enzyme substrate chip is provided, the enzyme substrate chip by its preparation method produces can be used for the vitality test that extensive high flux ground carries out enzyme, and method is simple, weak point consuming time, and systematic error is little.
Enzyme substrate chip of the present invention is to be integrated with thousands of informational molecules relevant with life by micro fabrication on small chip, and with the substrate of super amount specific enzyme classes, high density is solidificated on the probe carrier in an orderly manner.Enzyme classes can be redox enzymes, transferase, hydrolase, lyases, isomerase; Substrate can be albumen, polypeptide, amino acid, nucleic acid, lipid, carbohydrate and various organic compound; Probe carrier can be metal, glass or organic material, and its profile can be a different shape, and it can be plane and other shaped faces that the probe of carrier solidifies face.
The method for preparing enzyme substrate chip comprises the step of following order: (1) according to the characteristic of various enzymes in all kinds of biology enzymes and the every class with the specific reaction substrate reactions, design prepares and has fluorescently-labeled various enzyme-specific substrate probe; (2) utilize selected carrier characteristics, with various zymolyte probe high density in an orderly manner point sample, drying, be solidificated on the carrier, according to different purposes, form the enzyme substrate chip of different model; (3) according to different detected objects, select the chip of different model for use, preparation chip reactant liquor and chip reaction, reacted chip scans through chip scanner again, handle by the specific software data analysis, just can draw the result who treats the qualitative, quantitative of the activity of relevant enzyme in the test sample.
The application of enzyme substrate chip of the present invention has three aspects: (1) is directly used in the qualitative and quantitative analysis of various enzymatic activitys and measures.(2) be used for the in-vitro screening various kinds of drug.(3) get the application that evaluation detects in biological tissue.
The present invention compared with prior art has following advantage:
1. enzyme substrate chip can overcome the existing deficiency of measuring the enzymatic activity technology, utilizes highly sensitive, the high special compatible reaction of enzyme-to-substrate to detect the activity of corresponding enzyme, thereby realizes high flux, the short activity that detects enzyme classes highly sensitive, consuming time.
2. enzyme substrate chip utilizes various diseases all relevant with the active height of some special relevant enzyme in the body, uses the sample that will extract in human or animal's blood or the tissue to carry out the said determination analysis, can make things convenient for, the sensitive detection that applies to various relevant diseases.
3. enzyme substrate chip can make things convenient for, screen efficiently various kinds of drug.Disease is one to be finally the manifesting of biochemical reaction in the routine body, and nearly all biochemical reaction all just can carry out under the catalytic action of various special relevant enzymes.If specificity has suppressed the wherein activity of certain enzyme, thereby blocked above-mentioned pathological biochemistry reaction, then should disease also be under control or the screening of the specific inhibitor of the enzyme of treatment and disease association is called as drug screening, so utilize above-mentioned enzyme substrate chip to combine with the relevant enzyme of various diseases, can set up the medicament sifting motion system of various diseases, screen various kinds of drug conveniently, efficiently.
The present invention is described in further detail below with reference to embodiment:
1. 1 kinds of enzyme substrate chips of embodiment and preparation thereof: be used for the various zymolyte probes of connection, absorption or embedding, make its solid phase material be referred to as carrier with water-insoluble state functionating, on its curing face through surface activation process, just by chemical reaction with various activating reagent various reactive groups on carrier surface (curing face) bonding, so that be connected with various zymolyte probe molecules.Therefore, being integrated with thousands of informational molecules relevant with life on small chip, is the substrate with super amount specific enzyme classes, high density in an orderly manner point sample, drying, be solidificated on the carrier.Selecting carrier material is glass, can be soda-lime glass, potash glass, lithium glass or other unorganic glasses are because all there is the silicate hydroxyl on these carriers, its surface hydroxyl is made surperficial activation processing, then energy coupling nucleic acid, polypeptide, albumen and other biological and organic molecule.
The preparation of zymolyte probe: on the specific substrate molecule of certain selected enzyme, make amino, diazanyl or sulfydryl modification, can not do to modify as nucleic acid, polypeptide, protein etc. as the existing above-mentioned group of zymolyte molecule self, its purpose is to make the substrate molecule energy coupling on carrier.The zymolyte molecule of modifying or need not to modify is done uranium again
3+(Eu
3+) mark, its labeling method is to use the bifunctional group sequestrant.(1) many carboxylic acids chela and agent comprise thiocyanic acid phenyl-EDTA, isothiocyanic acid phenyl-DTTA, DTPA acid anhydride; (2) beta-diketon class sequestrant comprises β-NTA, PTA. (3) W-1174. (4) BCPDA.Eu
3+The probe of mark through ultraviolet excitation, just can produce distinctive fluorescence, and its principal character is: (1) fluorescence decay long half time (～1ms), for the 103-106 of conventional fluorescent doubly.(2) exciting light (340nm) is big with the stokes displacement of emission light (613nm).(3) excitation wavelength wide ranges, and emission light wave peak wide ranges.(4) fluorescence intensity height.
The zymolyte molecule is the mark of probe: inhomogeneity substrate and different functional groups substrate can be selected different bifunctional group sequestrant marks for use, as L-glutamic acid, the available beta-diketon class sequestrant of acetone, the polypeptide class can be selected W-1174 for use, and albumen, lipid, carbohydrate, oligonucleotide class can be selected DTPA acid anhydride etc. for use.
The making of enzyme substrate chip: with XYZ3000 type three-dimensional chip point sample instrument, adopt the some contact method at the carrier-pellet of handling well, with the plurality of enzymes substrate probe for preparing, point sample is coupling on carrier-pellet.This method can be at 3.6cm
2Carrier side on 10000 probe dot matrix of point, after the chip point sample was finished, in constant temperature (45-50 ℃) exsiccator, coupling was solidified (drying) 1.5 hours, can finish with it.
The selection of enzyme and specific substrate: present embodiment selective oxidation reduction enzyme, as: lactic dehydrogenase, L-glutamte dehydrogenase, its specific substrate are respectively pyruvic acid (organism), L-glutamic acid (amino acid).
2. 1 kinds of enzyme substrate chips of embodiment and preparation thereof: principle is similar to embodiment 1 to method.The carrier material of selecting for use is a metal.Can plate the thick golden film of 2 μ m on the surface of quartz glass plate or other metals (silver, copper, aluminium etc.) with gold-plated technology.This gold face need not activation processing, and the zymolyte probe that can directly (HS-probe) probe be contained the HS-base is solidificated on the golden film, because HS-combines with golden film specificity.The selection of enzyme and specific substrate: present embodiment is selected transferase, as: hexokinase, aspartate aminotransferase, its specific substrate are respectively glucose (sugar), L-aspartic acid (amino acid).
3. 1 kinds of enzyme substrate chips of embodiment and preparation thereof: principle is similar to embodiment 1 to method.The carrier material of selecting for use is an organic high molecular polymer, the membranization of carboxylic acid celluosic resin is carried out on the surface of the sheet that can make with polyvinyl chloride, on the membranization face that forms a large amount of carboxyls are arranged, the surface is become active ester with EDC and NHS, and then with the coupling of various zymolyte probe molecule.
The selection of enzyme and specific substrate: present embodiment is selected hydrolase, as: AMS, lipase, elastoser, the pancreas nuclease, its specific substrate is respectively soluble starch, triglyceride contains the polypeptide of serine, contains the oligonucleotide of pyrimidine nucleotide.
4. 1 kinds of enzyme substrate chips of embodiment and preparation thereof: principle is similar to embodiment 1 to method.The carrier material of selecting for use is an organic high molecular polymer, the membranization of carboxylic acid celluosic resin is carried out on the surface of the sheet that can make with organic glass, on the membranization face that forms great amount of hydroxy group is arranged, the surface is become active ester with EDC and NHS, and then with the coupling of various zymolyte probe molecule.
The selection of enzyme and specific substrate: present embodiment is selected the cracking fermentoid, as: citrate synthase, fructosediphosphate aldolase, its specific substrate is respectively acetyl-CoA, D-fructose 1,6-diphosphonic acid.
5. 1 kinds of enzyme substrate chips of embodiment and preparation thereof: principle is similar to embodiment 1 to method.The carrier material of selecting for use is that the membranization of carboxylic acid celluosic resin is carried out on the surface of the sheet that can make with teflon of organic high molecular polymer, on the membranization face that forms a large amount of carboxyls are arranged, with EDC and NHS the surface is become active ester, and then with the coupling of various zymolyte probe molecule.
The selection of enzyme and specific substrate: present embodiment is selected isomerase, as: phosphotriose isomerase, glucose phosphate isomerase, its specific substrate are respectively L-triose 3-phosphoric acid, D-glucose G-phosphoric acid.
The application of 6. 1 kinds of enzyme substrate chips of embodiment: the qualitative and quantitative analysis that is directly used in various enzymatic activitys is measured.As measure lipase, citrate synthase etc.Select for use this chip that the chip of the specific substrate probe of above-mentioned enzyme is arranged.Get testing sample 3ml, add 5ml BaffII (0.4M MgSO
4: 0.3M o-phenylenediamine: pH5.0 citrate buffer solution; 2: 4: 6), shake up standby.1ml takes out with minim pipette with reserve liquid, drops in the chip reaction chamber.The chip that will be added with sample is again put in 30-35 ℃ the constant temperature oven reaction into and is taken out after 2-3 hour, takes off reaction chamber, with pH5.0 citrate buffer solution flushing chip reaction surface 2 times, with Millipore water flushing 3 times, uses N again
2Dry up.Detect: the chip after will drying up, to put into AFFYMETT438 type enzyme substrate chip scanner and scan, the image of scanning uses the Lmagene image analysis software of Phargentech Inc that the information of the every bit of scan image is carried out the digitizing conversion.Scanning is judged: can be according to the pairing point of certain enzyme in the above-mentioned testing sample, and as A5.15 point (lipase), the size of the CPS value of A4.17 point (citrate synthetase) can be judged the amount that whether contains or contain lipase and citrate synthase in the liquid to be measured.
The application of 7. 1 kinds of enzyme substrate chips of embodiment: be used for biological fluid is detected evaluation.The tyrosinase shortage causes that albinism, phenylalanine hydroxylase deficiency cause spiritual primitivization in the human body.As: the detection of spiritual primitivization disease: get detected person's blood 3ml, add anti-coagulants, centrifugal, separate, get serum, add BafflI1.5ml, select the chip that contains phenylalanine hydroxylase substrate probe for use, application of sample, detection are with embodiment 6.The result judges: as CPS value 〉=3.6 of the B6.17 point (phenylalanine hydroxylase) of chip, then the tested person suffers from possibility 〉=85% of spiritual primitivization disease.The application of 8. 1 kinds of enzyme substrate chips of embodiment: be used for the in-vitro screening various kinds of drug.As: the screening of cancer therapy drug, (1) the principle malignant tumour is because of due to a large amount of malignant proliferations of human body cell, in the S phase (DNA synthesis phase) of malignant proliferation, need synthetic DNA under the catalysis of plurality of enzymes such as ribonucleotide reductase, dihyrofolate reductase, if testing compound can be obviously and specificity suppress one of above-mentioned two kinds of enzymes, but then in the anticancer DNA synthetic, and then suppress malignant proliferation, so, this kind compound just might become a kind of new cancer therapy drug.(2) method is waited to sieve compound liquid and is mixed with Baffl III-5 certain density, the ribonucleotide reductase and the dihyrofolate reductase that contain specific concentrations among the Baffl III-5, select the chip that contains ribonucleotide reductase and dihyrofolate reductase substrate probe for use, reaction detection is with embodiment 6, the result judges: the CPS value of A4.1 point (ribonucleotide reductase corresponding point) the A6.12 point (dihyrofolate reductase corresponding point) in the chip: when CPS value 〉=0.26, to enzyme unrestraint effect.As CPS value 0.27-0.56, weak inhibiting effect is arranged.As CPS value 0.57-1.5, inhibiting effect is arranged.As CPS value 1.6-2.6, strong inhibiting effect is arranged.When CPS value 〉=2.6, strong inhibiting effect is arranged.
Claims (5)
1. enzyme substrate chip is to be integrated with thousands of the informational molecules relevant with life by micro fabrication on small chip, it is characterized in that: it is that high density is solidificated on the probe carrier in an orderly manner with the substrate of super amount specific enzyme classes:
1.1 enzyme classes can be redox enzymes, transferase, hydrolase, lyases, isomerase;
1.2 substrate can be albumen, polypeptide, amino acid, nucleic acid, lipid, carbohydrate and various organic compound;
1.3 probe carrier can be metal, glass or organic material, its profile can be a different shape, and it can be plane and other shaped faces that the probe of carrier solidifies face;
2. method for preparing enzyme substrate chip comprises the step of following order:
2.1 according to the characteristic of various enzymes and specific reaction substrate reactions in all kinds of biology enzymes and the every class, the design preparation has fluorescently-labeled various enzyme-specific substrate probe;
2.2 utilize selected carrier characteristics, with various zymolyte probes, high density in an orderly manner point sample, drying, be solidificated on the carrier, according to different purposes, form the enzyme substrate chip of different model;
2.3 according to different determination objects, select the chip of different model for use, preparation chip reactant liquor and chip reaction, chip scanning, data processing, the result judges.
3. the application of an enzyme substrate chip: it is characterized in that: the qualitative and quantitative analysis that is directly used in various enzymatic activitys is measured.
4. the application of an enzyme substrate chip: it is characterized in that: be used for the in-vitro screening various kinds of drug.
5. the application of an enzyme substrate chip: it is characterized in that: the detection that is used for biological tissue is identified.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02113760 CN1459635A (en) | 2002-05-20 | 2002-05-20 | Enzyme substrate chip and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02113760 CN1459635A (en) | 2002-05-20 | 2002-05-20 | Enzyme substrate chip and its preparation method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1459635A true CN1459635A (en) | 2003-12-03 |
Family
ID=29426441
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 02113760 Pending CN1459635A (en) | 2002-05-20 | 2002-05-20 | Enzyme substrate chip and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1459635A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703574A (en) * | 2012-06-08 | 2012-10-03 | 厦门大学 | Method for screening coenzyme dependant type oxidation-reduction enzyme |
| CN101088009B (en) * | 2004-12-28 | 2013-05-22 | 株式会社先端生命科学研究所 | Blocked enzyme probe complex |
| CN104419746A (en) * | 2013-08-23 | 2015-03-18 | 中国科学院大连化学物理研究所 | Protease substrate screening method based on solid-loaded mixed protein as screening database |
| CN106146642A (en) * | 2016-06-27 | 2016-11-23 | 浙江星博生物科技股份有限公司 | A kind of for protein microsphere conjugate detecting sperm acrosin activities in assessing and its preparation method and application |
| CN114308158A (en) * | 2021-12-30 | 2022-04-12 | 广东昭宇生物科技有限公司 | Preparation method of biological enzyme chip with flora balance and repair functions |
-
2002
- 2002-05-20 CN CN 02113760 patent/CN1459635A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101088009B (en) * | 2004-12-28 | 2013-05-22 | 株式会社先端生命科学研究所 | Blocked enzyme probe complex |
| CN102703574A (en) * | 2012-06-08 | 2012-10-03 | 厦门大学 | Method for screening coenzyme dependant type oxidation-reduction enzyme |
| CN102703574B (en) * | 2012-06-08 | 2015-05-13 | 厦门大学 | Method for screening coenzyme dependant type oxidation-reduction enzyme |
| CN104419746A (en) * | 2013-08-23 | 2015-03-18 | 中国科学院大连化学物理研究所 | Protease substrate screening method based on solid-loaded mixed protein as screening database |
| CN104419746B (en) * | 2013-08-23 | 2016-08-10 | 中国科学院大连化学物理研究所 | Based on the protease substrate screening technique that immobilized mixed protein is screening storehouse |
| CN106146642A (en) * | 2016-06-27 | 2016-11-23 | 浙江星博生物科技股份有限公司 | A kind of for protein microsphere conjugate detecting sperm acrosin activities in assessing and its preparation method and application |
| CN114308158A (en) * | 2021-12-30 | 2022-04-12 | 广东昭宇生物科技有限公司 | Preparation method of biological enzyme chip with flora balance and repair functions |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sharma et al. | Biomolecules for development of biosensors and their applications | |
| US6780647B2 (en) | Sensor material, sensor and detection method for bio-substance | |
| Tipton | Principles of enzyme assay and kinetic studies | |
| Marazuela et al. | Free cholesterol fiber-optic biosensor for serum samples with simplex optimization | |
| Lin et al. | Colorimetric assay for S-adenosylhomocysteine hydrolase activity and inhibition using fluorosurfactant-capped gold nanoparticles | |
| CN109856212B (en) | Preparation method and application of electrochemical biosensor for simultaneously detecting HAT and TdT | |
| Kaur et al. | Enzyme-based biosensors | |
| Scheper et al. | A fiber optic biosensor based on fluorometric detection using confined macromolecular nicotinamide adenine dinucleotide derivatives | |
| CN101538615B (en) | Detecting method of nucleotide sequence for detecting mircoRNA content in samples | |
| CN102042975B (en) | Research and application of molecular identification-based fluorescence nanocrystal quartz fluorescent sensor for high-selectivity multicomponent saccharide detection | |
| Marzouk et al. | Prototype amperometric biosensor for sialic acid determination | |
| CN1459635A (en) | Enzyme substrate chip and its preparation method and application | |
| Kivirand et al. | Calibrating biosensors in flow-through set-ups: studies with glucose optrodes | |
| Mattiasson et al. | [25] Assay procedures for immobilized enzymes | |
| CN109613096A (en) | DNA relevant enzyme electrochemica biological sensor and its application based on the building of DNA- copper nano-cluster | |
| Wittstock et al. | SECM feedback imaging of enzymatic activity on agglomerated microbeads | |
| JPH02113897A (en) | Method and sensor for detecting concentration of enzyme substrate | |
| Noori et al. | A Review of Biosensors; Definition, Classification, Properties, and Applications | |
| EP1767931B1 (en) | Method for electrochemically measuring phosphoric acid and/or phosphate | |
| Singh et al. | Plants and plant-derived materials used for biosensor development | |
| Newman et al. | Historical perspective of biosensor and biochip development | |
| Mishra et al. | Overview of enzyme based biosensors and their applications | |
| Prajapati et al. | Bioreceptors for Enzymatic Interactions | |
| Yang et al. | Screening platform based on robolid microplate for immobilized enzyme-based assays | |
| JP6187259B2 (en) | Method of stirring the solution |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |