CN107941766A - Glutathione kit and its detection method are measured using nucleic acid aptamer fluorescence probe - Google Patents

Glutathione kit and its detection method are measured using nucleic acid aptamer fluorescence probe Download PDF

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CN107941766A
CN107941766A CN201711180343.0A CN201711180343A CN107941766A CN 107941766 A CN107941766 A CN 107941766A CN 201711180343 A CN201711180343 A CN 201711180343A CN 107941766 A CN107941766 A CN 107941766A
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glutathione
nucleic acid
fluorescence probe
acid aptamer
fluorescence
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王颖皓
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label

Abstract

The present invention relates to a kind of glutathione GSH kits based on nucleic acid aptamer fluorescence probe, while method, measure reagent composition and component the invention further relates to measure glutathione GSH concentration, belong to medical test determination techniques field.The kit main component of the present invention includes：Erythrocyte cracked liquid, phosphate buffer PBS, glutathione GSH standard items, glutathione GSH nucleic acid aptamer fluorescence probes；Cracked by blood sample, mix ovum and educate processing, detected with reference to sepectrophotofluorometer, so as to calculate the concentration of glutathione GSH.The present invention has the advantages that sample treatment is simple, and easy to operate, detection time is short, detection high specificity, high sensitivity, testing result repeatability is high.

Description

Glutathione kit and its detection method are measured using nucleic acid aptamer fluorescence probe
Technical field
The invention belongs to medical test determination techniques field, more particularly to the gluathione based on nucleic acid aptamer fluorescence probe Peptide GSH kits and its detection method.
Background technology
Glutathione (glutathione, r-glutamyl cysteingl+glycine, GSH) is that one kind contains γ-acyl The tripeptides of amine key and sulfydryl, is made of glutamic acid, cysteine and glycine.It is present in each cell of almost body.Paddy The sweet peptide of Guang can assist in keeping the function of normal immune system, and with antioxidation and integrate detoxication, cysteine On sulfydryl be its active group（Therefore often it is abbreviated as G-SH）, easily and some drugs（Such as paracetamol）, toxin（As free radical, The heavy metal such as iodoacetic acid, mustard gas, lead, mercury, arsenic）Deng combination, and with integration detoxication.Glutathione detoxifies with wide spectrum Effect, cannot be only used for medicine, more can be as the base-material of functional food, in the work(such as anti-aging, strengthen immunity, antitumor Can property food extensive use.
Chromatography：Gas-chromatography-mass spectrometric determination glutathione.The method can measure cysteine, methionine, Guang at the same time The many kinds of substance such as thioether and methylglycine.Chromatography high sensitivity, specificity are good, but sample treatment, separation condition, chromatographic column All multi-Varis are prepared, make it be difficult to standardize.And hplc device is expensive, technical conditions require height, needs special maintenance Personnel, make its Difficulty.
Immunological Method：The anti-S- adenosines glutathione monoclonal technigue of the method application specific, using fluorescence polarization method or Immunization measures.Glutathione is converted into S adenosine gluathiones in the presence of S adenosine glutathione hydrolases and excessive adenosine Peptide;The fluorescence S adenosine glutathione tracers of prediluted mixture, anti-S- adenosines glutathione monoclonal antibody and mark Together it is incubated, instrument detects the change of polarised light automatically, you can measure the total glutathione level of sample.Antibody fluorescence analytic approach and Turbidimetry cannot directly detect Homocysteine, need just go out as a result, complex for operation step for more than one hour.
Enzymatic cycling；It is to utilize zymolyte characteristic, amplifies target substance（Measured object）Detection method.Blood plasma oxidized form paddy Guang Sweet peptide is reduced into sequestered glutathione through three second carboxyethyl phosphines (TCEP), based on glutathione transmethylase, S adenosine paddy Guangs The circulation enzyme system and the principle of dehydrogenase coenzyme system that sweet peptidohydrolase is formed, catabolite colour developing, with automatic biochemical point The content of glutathione of the analyzer in 600nm wavelength survey OD is worth blood plasma.
Currently without the antibody and other molecular probes for being capable of Direct Recognition glutathione.Therefore a kind of height is designed to set The molecular probe of glutathione, and establish simple and quick and cheap detection method based on this and will improve and paddy is equal to cardiovascular and cerebrovascular The monitoring of the relevant disease of the sweet peptide of Guang.
Aptamer is a kind of new identification point that developed recently gets up, and is with referring to high specific and high-affinity The single stranded nucleotide acid molecule of domain target molecule knot.Aptamer can pass through ligand index concentration phyletic evolution technology （Systematic Evolution of Ligands by ExponentialEnrichment, SELEX）Screening obtains. SELEX technologies refer to the random oligonucleotide of applied chemistry method synthesis large capacity (by the fixed sequence program at both ends and middle random Sequence forms) library, by apply selection pressure (with reference to target, elutriation and the mistake of target high special binding fragment Journey), and Amplification Technologies are combined, the circulation selection enrichment through excessively taking turns, obtains the widow combined with target substance high special Nucleic acid molecule, can be that RNA can also be DNA, length is generally 25 ~ 60 nucleotide.Aptamer mainly passes through Generation adaptability folds, and is interacted by hydrogen bond, hydrophobic sedimentation, Van der Waals force and target molecule, with insertion or coated Target molecule forms stable three-D space structure.When aptamer is not combined with target molecule, aptamer is more open Structure, 5' and 3' both ends are mutually free.When being combined with target molecule, target molecule induction aptamer structure changes, nucleic acid The 5' ends and 3' ends of aptamer will all participate in and target molecule specific region（Similar to the antigen site with antibody binding）It is mutual Effect, causes 5' and 3' both ends close to each other.This feature makes aptamer be designed suitable for molecular beacon probe.One of which Molecular beacon design make use of fluorescent dye pyrene molecule.When an excitation state pyrene molecule and another ground state pyrene molecule closely meet with Excitation state dimer can be formed when chance, a photon can be discharged in the position longer than pyrene monomer wavelength.Pyrene excitation state two Aggressiveness emission peak arrives 500nm 480, and the emission peak of pyrene monomer 370 between 400nm.In addition, pyrene molecule is glimmering The light service life than the non-specific excitation fluorescence in biological sample long lifespan, during detection can after non-specific excitation fluorescence disappears, The fluorescence signal of excitation state dimer is collected again, greatly improves specificity and the sensitivity of detection.Identify the core of glutathione Sour aptamer can be applied to the clinical detection of glutathione, improve detection efficiency, reduce testing cost.
Aptamer is combined presented hypersensitivity and high specific with target substance, it is had in medical diagnosis on disease Good application prospect, although clinical practice report ripe at present is less, the research of application aptamer detection target protein It is on the increase, the new detecting technique based on aptamer also continuously emerges.Aptamer becomes due to its unique chemical antibody characteristic A new generation is directed to the molecular medicine or targeting vector of specific protein, and for detecting the target molecule material of its specific recognition. But the glutathione GSH diagnostic reagents that are directed to for being currently based on aptamer also lack very much, and it is directed to glutathione GSH's The exploitation of nucleic acid aptamer fluorescence probe detection kit is there is not yet report.
The content of the invention
The purpose of the present invention is be also easy to produce that operating technology error, the not good enough, disturbing factor of repeatability be very much, behaviour for existing Make the deficiencies of cumbersome, testing cost is high, there is provided a kind of glutathione GSH kits and its inspection based on nucleic acid aptamer fluorescence probe Survey method.
The solution of the present invention is realized in this way：
A kind of glutathione kit based on nucleic acid aptamer fluorescence probe, main component include：Containing NH4Cl、Tris、EDTA- Na2Erythrocyte cracked liquid；Containing MgCl20.2M phosphate buffers；Glutathione standard items；Glutathione aptamer fluorescence Probe；The glutathione nucleic acid aptamer fluorescence probe distinguishes the nucleotide list of mark fluorescent pyrene molecule monomer for 5' and 3' both ends Chain, the nucleotide single-chain sequence are：ggcactgtac ggctaagttc ccggatagtg gattggaact ggcataaccc. The sequence designations are GSH3.
A kind of above-described glutathione kit based on nucleic acid aptamer fluorescence probe, it is described to contain NH4Cl、Tris、 EDTA-Na2Erythrocyte cracked liquid to contain 1 ~ 280 mmol/L NH4Cl, 1~34 mmol/L Tris,1~2mmol/ LEDTA-Na2, pH 7.0~7.2；It is described to contain MgCl20.2M phosphate buffers to contain 1 ~ 10mmol/L MgCl2。
A kind of glutathione kit based on nucleic acid aptamer fluorescence probe described in any of the above, it is described to contain NH4Cl、 Tris、EDTA-Na2Erythrocyte cracked liquid, containing MgCl20.2M phosphate buffers, glutathione standard items, glutathione core Sour aptamer fluorescence probe is to prepare the liquid reagent directly used or use the preceding dry powder that need to be dissolved in water.
A kind of above-described glutathione kit based on nucleic acid aptamer fluorescence probe, the kit are used to detect liver Glutathione concentrations in plain anticoagulated whole blood or peripheral blood.
A kind of glutathione kit described in any of the above based on nucleic acid aptamer fluorescence probe detects glutathione The method of concentration, method and step include：
（1）Blood sample cracks：By blood sample and containing NH4Cl、Tris、EDTA-Na2Erythrocyte cracked liquid press 1:0.5 ~ 5 volume ratio is mixed It is even, 5 ~ 30min, then 5 ~ 10min of medium-speed centrifuge are stood, is collected supernatant；
（2）Mixing ovum is educated：Mixing ovum is educated：Take 20 ~ 100 μ l steps 1）Obtained supernatant and the use of 30 ~ 50ul contains MgCl2's 0.2M phosphate buffers, the glutathione nucleic acid aptamer fluorescence probe reagent that dissolving glutathione nucleic acid aptamer fluorescence probe obtains Mixing, ovum educates 5 ~ 15min at room temperature, glutathione nucleic acid aptamer fluorescence probe is fully combined with blood sample, obtains test fluid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Obtained test fluid 50ul, after fluorescence detector fluorescence excitation, Read fluorescence excitation after 20 to wavelength in the 100ns periods 480 ~ 500nm fluorescent value；
（4）As a result calculate：Using biomedical mapping software, the standard curve made with reference to glutathione standard sample, with reference to Step 3）In the fluorescent value that detects calculate the concentration of blood sample GSH-PX activity.
Above-described biomedicine software is Sigma plot softwares, in being commercially available on the market.
It is above-described a kind of dense to detect glutathione with the glutathione kit based on nucleic acid aptamer fluorescence probe The method of degree, the glutathione nucleic acid aptamer fluorescence probe reagent GSH-PX activity nucleic acid aptamer fluorescence probe concentration for 200 ~ 400 nmol/L, its characteristic also reside in, and the glutathione nucleic acid aptamer fluorescence probe distinguishes mark fluorescent for 5' and 3' both ends The nucleotide single-chain of pyrene molecule monomer, the nucleotide single-chain sequence are：ggcactgtac ggctaagttc ccggatagtg gattggaact ggcataaccc。
When glutathione nucleic acid aptamer fluorescence probe is not combined with glutathione, aptamer is in more open knot Structure, the pyrene molecule monomer at 5' and 3' both ends is mutually free, and launch wavelength is between 370 ~ 400nm after fluorescence excitation；Paddy Guang When sweet peptide aptamers fluorescence probe is combined with glutathione, glutathione induce its recurring structure change, aptamer 5' and The pyrene molecule monomer at 3' both ends is close to each other, forms excitation state dimer, and excitation state dimer launch wavelength exists after fluorescence excitation 480 between 500nm.
It is above-described a kind of dense to detect glutathione with the glutathione kit based on nucleic acid aptamer fluorescence probe The method of degree, the blood sample are anticoagulant heparin whole blood or peripheral blood, described to contain NH4Cl、Tris、EDTA-Na2Red blood cell split Solution liquid is to contain 1 ~ 280 mmol/L NH4Cl, 1~34 mmol/L Tris,1~2mmol/LEDTA-Na2, pH 7.0~7.2； It is described to contain MgCl20.2M phosphate buffers to contain 1 ~ 10mmol/L MgCl2；The fluorescence detector is with the time point Distinguish fluorimetric fluorescence detector.
It is above-described a kind of dense to detect glutathione with the glutathione kit based on nucleic acid aptamer fluorescence probe The method of degree, the detection method are used to detect the glutathione concentrations in anticoagulant heparin whole blood or peripheral blood.
The principle of the present invention is：When not with glutathione, aptamer is in more open structure, 5' and 3' two The pyrene molecule monomer at end is mutually free, and fluorescence emission wavelengths are 370 between 400nm；When being combined with glutathione, paddy The sweet inducing peptide aptamer structure of Guang changes, and the pyrene molecule monomer at the 5' and 3' both ends of aptamer is close to each other, is formed Dimer, pyrene excited state emission wavelength dimer is 480 between 500nm.Pyrene excitation state dimer fluorescence lifetime has length Up to 100 ns, than the biological sample autofluorescence service life（About 5 ns）It is long, by detecting glutathione aptamer probe and sample The mixed fluorescence intensity of product and fluorescence lifetime, calculate the concentration of sample GSH-PX activity.
The present invention substantive distinguishing features and marked improvement be：
（1）Detect easy to operate quick, process and separate without complex sample, after aptamer probe is directly added into cracking Blood sample liquid, with the fluorescent value that can be detected in the sepectrophotofluorometer short time at 480 ~ 500nm；
（2）This kit and its detection method have a high sensitivity, and testing result repeatability is high, sample detection error 0.01 ~ Between 0.1%, high specificity, when glutathione nucleic acid aptamer fluorescence probe is not combined with glutathione, aptamer, which is in, to be compared Loose structure, the pyrene molecule monomer at 5' and 3' both ends is mutually free, after fluorescence excitation launch wavelength 370 ~ 400nm it Between；When it is combined with glutathione, glutathione induces it to change, the pyrene molecule single phase at aptamer 5' and 3' both ends It is mutually close, dimer is formed, dimer launch wavelength is between 480 ~ 500nm after fluorescence excitation.
（3）Reagent used can make to prepare the liquid reagent that can be used directly or using being used after being preceding dissolved in water Dry powder, detection reagent can be with storage at normal temperature, convenient transportation.
Embodiment
Below in conjunction with table 1 and embodiment description the present invention using nucleic acid aptamer fluorescence probe measure glutathione kit and Its detection method.
Kit reagent component composition in 1. embodiment of table
Embodiment 1
After agent formulations dissolving prepares needed for embodiment 1 in table 1, bottle is distributed into, is freeze-dried, dry powder examination is made Agent；Before use, adding ultra-pure water, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows：
（1）Blood sample cracks：By anticoagulant heparin whole blood, erythrocyte cracked liquid by volume 1:0.5 mixes, and stands 30min, then middling speed 7min is centrifuged, collects supernatant；
（2）Mixing ovum is educated：Take 20 μ l steps 1）Obtained supernatant 45ul's dissolves glutathione core with 0.2M phosphate buffers The glutathione nucleic acid aptamer fluorescence probe reagent mixing that sour aptamer fluorescence probe obtains, ovum educates 5min at room temperature, is tested Liquid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Obtained test fluid 50ul, after fluorescence detector fluorescence excitation, Read fluorescence excitation after 20 to wavelength in the 100ns periods 480 ~ 500nm fluorescent value；
（4）As a result calculate：Using biomedical mapping software, the standard curve made with reference to glutathione standard sample, with reference to Step 3）In the fluorescent value that detects calculate the concentration of blood sample GSH-PX activity.
3 parallel determination errors of sample are 0.05 ± 0.01%.
Embodiment 2
After agent formulations dissolving prepares needed for embodiment 2 in table 1, bottle is distributed into, is freeze-dried, dry powder examination is made Agent；Before use, adding ultra-pure water, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows：
（1）Blood sample cracks：By peripheral blood, erythrocyte cracked liquid by volume 1:2.5 mix, and stand 5min, then medium-speed centrifuge 6min, collects supernatant；
（2）Mixing ovum is educated：Take 50 μ l steps 1）Obtained supernatant 30ul's
The glutathione aptamer fluorescence obtained with 0.2M phosphate buffers dissolving glutathione nucleic acid aptamer fluorescence probe is visited Pin reagent mixes, and ovum educates 6min at room temperature, obtains test fluid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Obtained test fluid 50ul, after fluorescence detector fluorescence excitation, Read fluorescence excitation after 20 to wavelength in the 100ns periods 480 ~ 500nm fluorescent value；
（4）As a result calculate：Using biomedical mapping software, the standard curve made with reference to glutathione standard sample, with reference to Step 3）In the fluorescent value that detects calculate the concentration of blood sample GSH-PX activity.3 parallel determination errors of sample are 0.04 ±0.01%。
Embodiment 3
After agent formulations dissolving prepares needed for embodiment 3 in table 1, bottle is distributed into, is freeze-dried, dry powder examination is made Agent；Before use, adding ultra-pure water, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows：
（1）Blood sample cracks：By peripheral blood, erythrocyte cracked liquid by volume 1:3.5 mix, and stand 10min, then medium-speed centrifuge 5min, collects supernatant；
（2）Mixing ovum is educated：Take 75 μ l steps 1）Obtained supernatant 45ul's
The glutathione aptamer fluorescence obtained with 0.2M phosphate buffers dissolving glutathione nucleic acid aptamer fluorescence probe is visited Pin reagent mixes, and ovum educates 7min at room temperature, obtains test fluid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Obtained test fluid 50ul, after fluorescence detector fluorescence excitation, Read fluorescence excitation after 20 to wavelength in the 100ns periods 480 ~ 500nm fluorescent value；
（4）As a result calculate：Using biomedical mapping software, the standard curve made with reference to glutathione standard sample, with reference to Step 3）In the fluorescent value that detects calculate the concentration of blood sample GSH-PX activity.
3 parallel determination errors of sample are 0.02 ± 0.01%.
Embodiment 4
After agent formulations dissolving prepares needed for embodiment 4 in table 1, bottle is distributed into, is freeze-dried, dry powder examination is made Agent；Before use, adding ultra-pure water, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows：
（1）Blood sample cracks：By anticoagulant heparin whole blood, erythrocyte cracked liquid by volume 1:0.5 mixes, and stands 25min, then middling speed 8min is centrifuged, collects supernatant；
（2）Mixing ovum is educated：Take 100 μ l steps 1）Obtained supernatant 50ul's dissolves glutathione core with 0.2M phosphate buffers The glutathione nucleic acid aptamer fluorescence probe reagent mixing that sour aptamer fluorescence probe obtains, ovum educates 8min at room temperature, is tested Liquid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Obtained test fluid 50ul, after fluorescence detector fluorescence excitation, Read fluorescence excitation after 20 to wavelength in the 100ns periods 480 ~ 500nm fluorescent value；
（4）As a result calculate：Using biomedical mapping software, the standard curve made with reference to glutathione standard sample, with reference to Step 3）In the fluorescent value that detects calculate the concentration of blood sample GSH-PX activity.
3 parallel determination errors of sample are 0.03 ± 0.01%.
Embodiment 5
After agent formulations dissolving prepares needed for embodiment 5 in table 1, bottle is distributed into, is freeze-dried, dry powder examination is made Agent；Before use, adding ultra-pure water, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows：
（1）Blood sample cracks：By anticoagulant heparin whole blood, erythrocyte cracked liquid by volume 1:1.0 mix, and stand 20min, then middling speed 9min is centrifuged, collects supernatant；
（2）Mixing ovum is educated：Take 80 μ l steps 1）Obtained supernatant 30ul's dissolves glutathione core with 0.2M phosphate buffers The glutathione nucleic acid aptamer fluorescence probe reagent mixing that sour aptamer fluorescence probe obtains, ovum educates 9min at room temperature, is tested Liquid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Obtained test fluid 50ul, after fluorescence detector fluorescence excitation, Read fluorescence excitation after 20 to wavelength in the 100ns periods 480 ~ 500nm fluorescent value；
（4）As a result calculate：Using biomedical mapping software, the standard curve made with reference to glutathione standard sample, with reference to Step 3）In the fluorescent value that detects calculate the concentration of blood sample GSH-PX activity.
3 parallel determination errors of sample are 0.04 ± 0.01%.
Embodiment 6
After agent formulations dissolving prepares needed for embodiment 6 in table 1, bottle is distributed into, is freeze-dried, dry powder examination is made Agent；Before use, adding ultra-pure water, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows：
（1）Blood sample cracks：By anticoagulant heparin whole blood, erythrocyte cracked liquid by volume 1:4.5 mix, and stand 15min, then middling speed 10min is centrifuged, collects supernatant；
（2）Mixing ovum is educated：Take 30 μ l steps 1）Obtained supernatant 40ul's dissolves glutathione core with 0.2M phosphate buffers The glutathione nucleic acid aptamer fluorescence probe reagent mixing that sour aptamer fluorescence probe obtains, ovum educates 10min at room temperature, is tested Liquid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Obtained test fluid 50ul, after fluorescence detector fluorescence excitation, Read fluorescence excitation after 20 to wavelength in the 100ns periods 480 ~ 500nm fluorescent value；
（4）As a result calculate：Using biomedical mapping software, the standard curve made with reference to glutathione standard sample, with reference to Step 3）In the fluorescent value that detects calculate the concentration of blood sample GSH-PX activity.
3 parallel determination errors of sample are 0.05 ± 0.01%.
Embodiment 7
After agent formulations dissolving prepares needed for embodiment 7 in table 1, bottle is distributed into, is freeze-dried, dry powder examination is made Agent；Before use, adding ultra-pure water, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows：
（1）Blood sample cracks：By peripheral blood, erythrocyte cracked liquid by volume 1:3.0 mix, and stand 18min, then medium-speed centrifuge 8min, collects supernatant；
（2）Mixing ovum is educated：Take 50 μ l steps 1）Obtained supernatant 20ul's dissolves glutathione core with 0.2M phosphate buffers The glutathione nucleic acid aptamer fluorescence probe reagent mixing that sour aptamer fluorescence probe obtains, ovum educates 12min at room temperature, is tested Liquid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Obtained test fluid 50ul, after fluorescence detector fluorescence excitation, Read fluorescence excitation after 20 to wavelength in the 100ns periods 480 ~ 500nm fluorescent value；
（4）As a result calculate：Using biomedical mapping software, the standard curve made with reference to glutathione standard sample, with reference to Step 3）In the fluorescent value that detects calculate the concentration of blood sample GSH-PX activity.
3 parallel determination errors of sample are 0.08 ± 0.01%.
Embodiment 8
After agent formulations dissolving prepares needed for embodiment 8 in table 1, bottle is distributed into, is freeze-dried, dry powder examination is made Agent；Before use, adding ultra-pure water, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows：
（1）Blood sample cracks：By peripheral blood, erythrocyte cracked liquid by volume 1:2.0 mix, and stand 26min, then medium-speed centrifuge 6min, collects supernatant；
（2）Mixing ovum is educated：Take 60 μ l steps 1）Obtained supernatant 25ul's dissolves glutathione core with 0.2M phosphate buffers The glutathione nucleic acid aptamer fluorescence probe reagent mixing that sour aptamer fluorescence probe obtains, ovum educates 13min at room temperature, is tested Liquid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Obtained test fluid 50ul, after fluorescence detector fluorescence excitation, Read fluorescence excitation after 20 to wavelength in the 100ns periods 480 ~ 500nm fluorescent value；
（4）As a result calculate：Using biomedical mapping software, the standard curve made with reference to glutathione standard sample, with reference to Step 3）In the fluorescent value that detects calculate the concentration of blood sample GSH-PX activity.
3 parallel determination errors of sample are 0.02 ± 0.01%.
Embodiment 9
After agent formulations dissolving prepares needed for embodiment 9 in table 1, bottle is distributed into, is freeze-dried, dry powder examination is made Agent；Before use, adding ultra-pure water, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows：
（1）Blood sample cracks：By peripheral blood, erythrocyte cracked liquid by volume 1:4.0 mix, and stand 8min, then medium-speed centrifuge 7min, collects supernatant；
（2）Mixing ovum is educated：Take 40 μ l steps 1）Obtained supernatant 35ul's dissolves glutathione core with 0.2M phosphate buffers The glutathione nucleic acid aptamer fluorescence probe reagent mixing that sour aptamer fluorescence probe obtains, ovum educates 15min at room temperature, is tested Liquid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Obtained test fluid 50ul, after fluorescence detector fluorescence excitation, Read fluorescence excitation after 20 to wavelength in the 100ns periods 480 ~ 500nm fluorescent value；
（4）As a result calculate：Using biomedical mapping software, the standard curve made with reference to glutathione standard sample, with reference to Step 3）In the fluorescent value that detects calculate the concentration of blood sample GSH-PX activity.
3 parallel determination errors of sample are 0.01 ± 0.01%.
Sequence table
<110>Wang Yinghao
<120>Glutathione kit and its detection method are measured using nucleic acid aptamer fluorescence probe
<130> 2017
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 28
<212> DNA
<213>Artificial sequence
<400> 1
gtacacttga agggtaagga ctcccgta 28

Claims (9)

1. one kind utilizes nucleic acid aptamer fluorescence probe measure glutathione kit, it is characterised in that main component includes：Contain NH4Cl、Tris、EDTA-Na2Erythrocyte cracked liquid；Containing MgCl20.2M phosphate buffers；Glutathione standard items；Paddy Guang Sweet peptide aptamers fluorescence probe；The glutathione nucleic acid aptamer fluorescence probe distinguishes mark fluorescent pyrene point for 5' and 3' both ends The nucleotide single-chain of sub- monomer, the nucleotide single-chain sequence are SEQ ID NO:1~SEQ ID NO:Any one DNA piece in 5 Duan Xulie, its sequence are：gtacacttga agggtaagga ctcccgta.
2. one kind according to claim 1 is existed using nucleic acid aptamer fluorescence probe measure glutathione kit, its feature In described to contain NH4Cl、Tris、EDTA-Na2Erythrocyte cracked liquid to contain 1~280 mmol/L NH4Cl, 1~34 mmol/L Tris,1~2mmol/LEDTA-Na2, pH 7.0~7.2；It is described to contain MgCl20.2M phosphate buffers be containing 1 ~ 10mmol/L MgCl2。
3. one kind according to claim 1 or 2 utilizes nucleic acid aptamer fluorescence probe measure glutathione kit, its feature It is, it is described to contain NH4Cl、Tris、EDTA-Na2Erythrocyte cracked liquid, containing MgCl20.2M phosphate buffers, glutathione Standard items, glutathione nucleic acid aptamer fluorescence probe are to prepare the liquid reagent directly used or need to be dissolved in water before using Dry powder.
4. one kind according to claim 1 or 2 utilizes nucleic acid aptamer fluorescence probe measure glutathione kit, its feature It is, which is used to detect the glutathione concentrations in anticoagulant heparin whole blood or peripheral blood.
5. one kind described detects gluathione with right 1 ~ 3 is any using nucleic acid aptamer fluorescence probe measure glutathione kit The method of peptide concentration, it is characterised in that method and step includes：
（1）Blood sample cracks：By blood sample and containing NH4Cl、Tris、EDTA-Na2Erythrocyte cracked liquid press 1:0.5 ~ 5 volume ratio is mixed It is even, 5 ~ 30min, then 5 ~ 10min of medium-speed centrifuge are stood, is collected supernatant；
（2）Mixing ovum is educated：Take 20 ~ 100 μ l steps 1）Obtained supernatant and the use of 30 ~ 50ul contains MgCl20.2M phosphoric acid delay Fliud flushing, the glutathione nucleic acid aptamer fluorescence probe reagent mixing that dissolving glutathione nucleic acid aptamer fluorescence probe obtains, room temperature Lower ovum educates 5 ~ 15min, glutathione nucleic acid aptamer fluorescence probe is fully combined with blood sample, obtains test fluid；
（3）Fluoroscopic examination：Fluorescence detector detecting step 2）Obtained test fluid 50ul, after fluorescence detector fluorescence excitation, Read fluorescence excitation after 20 to wavelength in the 100ns periods 480 ~ 500nm fluorescent value；
（4）As a result calculate：Using biomedical mapping software, the standard curve made with reference to glutathione standard sample, with reference to Step 3）In the fluorescent value that detects calculate the concentration of blood sample GSH-PX activity.
6. one kind according to claim 5 measures glutathione kit to detect paddy Guang using nucleic acid aptamer fluorescence probe The method of sweet peptide concentration, it is characterised in that the glutathione nucleic acid aptamer fluorescence probe reagent Glutathione peptide aptamers Fluorescence probe concentration is 200 ~ 400 nmol/L, and the glutathione nucleic acid aptamer fluorescence probe marks respectively for 5' and 3' both ends The nucleotide single-chain of fluorescence pyrene molecule monomer, the nucleotide single-chain sequence are：gtacacttga agggtaagga ctcccgta.
7. one kind according to claim 6 measures glutathione kit to detect paddy Guang using nucleic acid aptamer fluorescence probe The method of sweet peptide concentration, it is characterised in that when the glutathione nucleic acid aptamer fluorescence probe is not combined with glutathione, core Sour aptamer is in more open structure, and the pyrene molecule monomer at 5' and 3' both ends is mutually free, and launch wavelength exists after fluorescence excitation Between 370 ~ 400nm；When the glutathione nucleic acid aptamer fluorescence probe is combined with glutathione, glutathione induces it Recurring structure changes, and the pyrene molecule monomer at aptamer 5' and 3' both ends is close to each other, forms excitation state dimer, fluorescence excitation Afterwards excitation state dimer launch wavelength 480 between 500nm.
8. one kind according to claim 5 measures glutathione kit to detect paddy Guang using nucleic acid aptamer fluorescence probe The method of sweet peptide concentration, it is characterised in that the blood sample is anticoagulant heparin whole blood or peripheral blood, described to contain NH4Cl、Tris、 EDTA-Na2Erythrocyte cracked liquid to contain 1 ~ 280 mmol/L NH4Cl, 1~34 mmol/L Tris,1~2mmol/ LEDTA-Na2, pH 7.0~7.2；It is described to contain MgCl20.2M phosphate buffers to contain 1 ~ 10mmol/L MgCl2；It is described Fluorescence detector is the fluorescence detector with time-resolved fluorometry.
9. glutathione kit is measured to detect paddy using nucleic acid aptamer fluorescence probe according to one kind described in claim 5 ~ 7 The method of the sweet peptide concentration of Guang, it is characterised in that the detection method is used to detect the gluathione in anticoagulant heparin whole blood or peripheral blood Peptide concentration.
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