CN107907515A - One kind detection glutathione probe reagent box and its detection method - Google Patents
One kind detection glutathione probe reagent box and its detection method Download PDFInfo
- Publication number
- CN107907515A CN107907515A CN201711180319.7A CN201711180319A CN107907515A CN 107907515 A CN107907515 A CN 107907515A CN 201711180319 A CN201711180319 A CN 201711180319A CN 107907515 A CN107907515 A CN 107907515A
- Authority
- CN
- China
- Prior art keywords
- glutathione
- fluorescence
- detection
- nucleic acid
- 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.)
- Withdrawn
Links
Classifications
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
Abstract
The present invention relates to one kind to detect glutathione probe reagent box, while method, measure reagent composition and component the invention further relates to measure glutathione GSH concentration, belongs 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
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：
One kind detection glutathione probe reagent box, main component include：Containing NH4Cl、Tris、EDTA-Na2Erythrocyte splitting
Liquid；Containing MgCl20.2M phosphate buffers；Glutathione standard items；Glutathione nucleic acid aptamer fluorescence probe；The paddy Guang
Sweet peptide aptamers fluorescence probe distinguishes the nucleotide single-chain of mark fluorescent pyrene molecule monomer, the nucleotide list for 5' and 3' both ends
Chain-ordering is：ggtggaggca agagatcggc cgaggttttc.The sequence designations are GSH1.
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 characterised in that institute
State and contain NH4Cl、Tris、EDTA-Na2Erythrocyte cracked liquid, containing MgCl20.2M phosphate buffers, glutathione standard items,
Glutathione nucleic acid aptamer fluorescence probe is to prepare the liquid reagent directly used or use the preceding dry powder that need to be dissolved in water
State.
A kind of above-described glutathione kit based on nucleic acid aptamer fluorescence probe, it is characterised in that the reagent
Box is used to detect the glutathione concentrations in anticoagulant heparin whole blood or peripheral blood.
It is a kind of that glutathione probe reagent box is detected described in any of the above to detect the method for glutathione concentrations, method
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：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 to detect the method for glutathione concentrations, the paddy with detection glutathione probe reagent box
The sweet peptide aptamers fluorescence probe reagent GSH-PX activity nucleic acid aptamer fluorescence probe concentration of Guang is 200 ~ 400 nmol/L, it is special
Property also reside in, the glutathione nucleic acid aptamer fluorescence probe for 5' and 3' both ends distinguish mark fluorescent pyrene molecule monomer nucleosides
Acid is single-stranded, which is：ggtggaggca agagatcggc cgaggttttc.
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 it is a kind of detect the method for glutathione concentrations with detection glutathione probe reagent box, it is described
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 MgCl2's
0.2M phosphate buffers are to contain 1 ~ 10mmol/L MgCl2；The fluorescence detector is with the glimmering of time-resolved fluorometry
Optical detector.
It is above-described it is a kind of detect the method for glutathione concentrations with detection glutathione probe reagent box, the detection
Method is 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 present invention detection glutathione probe reagent box 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 0.03
±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 0.03 ± 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 0.06 ± 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 0.02 ± 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 0.1 ± 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>One kind detection glutathione probe reagent box and its detection method
<130> 2017
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213>Artificial sequence ()
<400> 1
ggtggaggca agagatcggc cgaggttttc 30
Claims (9)
1. one kind detection glutathione probe reagent box, it is characterised in that main component includes：Containing NH4Cl、Tris、EDTA-Na2
Erythrocyte cracked liquid；Containing MgCl20.2M phosphate buffers；Glutathione standard items；Glutathione aptamer fluorescence is visited
Pin；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：ggtggaggca agagatcggc cgaggttttc.
2. detection glutathione probe reagent box according to claim 1, it is characterised in that 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。
3. detection glutathione probe reagent box according to claim 1 or 2, it is characterised in that 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.
4. detection glutathione probe reagent box according to claim 1 or 2, it is characterised in that the kit is used to examine
Survey the glutathione concentrations in anticoagulant heparin whole blood or peripheral blood.
5. a kind of detect the method for glutathione concentrations with any detection glutathione probe reagent box of right 1 ~ 3, its
It is characterized 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. a kind of the side of glutathione concentrations is detected according to claim 5 with detection glutathione probe reagent box
Method, it is characterised in that the glutathione nucleic acid aptamer fluorescence probe reagent GSH-PX activity nucleic acid aptamer fluorescence probe concentration
For 200 ~ 400 nmol/L, the glutathione nucleic acid aptamer fluorescence probe distinguishes mark fluorescent pyrene molecule list for 5' and 3' both ends
The nucleotide single-chain of body, the nucleotide single-chain sequence are：ggtggaggca agagatcggc cgaggttttc.
7. a kind of the side of glutathione concentrations is detected according to claim 6 with detection glutathione probe reagent box
Method, it is characterised in that when the glutathione nucleic acid aptamer fluorescence probe is not combined with glutathione, aptamer be in than
Structure loosely, the pyrene molecule monomer at 5' and 3' both ends is mutually free, and launch wavelength is in 370 ~ 400nm after fluorescence excitation
Between；When the glutathione nucleic acid aptamer fluorescence probe is combined with glutathione, glutathione induces its recurring structure to change
Become, the pyrene molecule monomer at aptamer 5' and 3' both ends is close to each other, formation excitation state dimer, excitation state two after fluorescence excitation
Aggressiveness launch wavelength is 480 between 500nm.
8. a kind of the side of glutathione concentrations is detected according to claim 5 with detection glutathione probe reagent box
Method, it is characterised in that the blood sample is anticoagulant heparin whole blood or peripheral blood, described to contain NH4Cl、Tris、EDTA-Na2It is red thin
Cellular lysate 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 be with when
Between resolved fluorometric measure fluorescence detector.
9. the side of glutathione concentrations is detected with detection glutathione probe reagent box according to a kind of described in claim 5 ~ 7
Method, it is characterised in that the detection method is used to detect the glutathione concentrations in anticoagulant heparin whole blood or peripheral blood.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711180319.7A CN107907515A (en) | 2017-11-23 | 2017-11-23 | One kind detection glutathione probe reagent box and its detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711180319.7A CN107907515A (en) | 2017-11-23 | 2017-11-23 | One kind detection glutathione probe reagent box and its detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107907515A true CN107907515A (en) | 2018-04-13 |
Family
ID=61847335
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711180319.7A Withdrawn CN107907515A (en) | 2017-11-23 | 2017-11-23 | One kind detection glutathione probe reagent box and its detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107907515A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112304930A (en) * | 2020-04-20 | 2021-02-02 | 浙江今复康生物科技有限公司 | Disulfide bond detection method and sputum detection kit containing disulfide bonds |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001072955A2 (en) * | 2000-03-27 | 2001-10-04 | Bayer Aktiengesellschaft | Regulation of human nedd1-related protein |
| CN104677868A (en) * | 2014-11-30 | 2015-06-03 | 陈燕婷 | Homocysteine (HCy) kit based on aptamer fluorescence probe and detection method for concentration of homocysteine (HCy) by using homocysteine (HCy) kit |
-
2017
- 2017-11-23 CN CN201711180319.7A patent/CN107907515A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001072955A2 (en) * | 2000-03-27 | 2001-10-04 | Bayer Aktiengesellschaft | Regulation of human nedd1-related protein |
| CN104677868A (en) * | 2014-11-30 | 2015-06-03 | 陈燕婷 | Homocysteine (HCy) kit based on aptamer fluorescence probe and detection method for concentration of homocysteine (HCy) by using homocysteine (HCy) kit |
Non-Patent Citations (5)
| Title |
|---|
| HUI XU AND MARIA HEPEL: ""Molecular Beacon"-Based Fluorescent Assay for Selective Detection", 《ANALYTICAL CHEMISTRY》 * |
| XIAOFANG JIA, ING LI, ERKANG WANG: "Lighting-Up of the Dye Malachite Green with Mercury(II)–DNA and Its", 《CHEMPUBSOC》 * |
| 刘卫: "核酸探针电化学传感器用于小分子和肌红蛋白检测的研究", 《中国优秀硕士学位论文全文数据库》 * |
| 吴观荣: "应用核酸探针对汞离子的检测", 《中国优秀硕士学位论文全文数据库》 * |
| 靳贵晓 李娟 杨黄浩: "核酸适体的筛选及其在生物医学领域的研究进展", 《福州大学学报(自然科学版)》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112304930A (en) * | 2020-04-20 | 2021-02-02 | 浙江今复康生物科技有限公司 | Disulfide bond detection method and sputum detection kit containing disulfide bonds |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhao et al. | Extracellular vesicles as cancer liquid biopsies: From discovery, validation, to clinical application | |
| CN104597246B (en) | Homocysteine test kit and detection method thereof based on nucleic acid aptamer fluorescence probe HCy2 | |
| Zhou et al. | A portable point-of-care testing system to diagnose lung cancer through the detection of exosomal miRNA in urine and saliva | |
| Sankiewicz et al. | Determination of collagen type IV by Surface Plasmon Resonance Imaging using a specific biosensor | |
| CN103063851B (en) | Free triiodothyronine nanometer magnetic particle chemiluminescence assay kit and preparation method thereof and detection method thereof | |
| CN105548109A (en) | A fluorescence detecting system for heavy metal cadmium and a fluorescence detecting method | |
| Chen et al. | An electricity-and instrument-free infectious disease sensor based on a 3D origami paper-based analytical device | |
| CN102841206B (en) | TnT (Troponin-T, TNT) measures kit | |
| Huang et al. | Sensitive turn-on fluorescent detection of tartrazine based on fluorescence resonance energy transfer | |
| CN105044055B (en) | The detection method and protein detection sensor array of a kind of protein | |
| CN109298177A (en) | Time-resolved fluorescence immunoassay method based on Magneto separate | |
| CN103439491A (en) | Hyaluronic acid chemiluminescence quantitative determination kit and preparation method thereof | |
| Chen et al. | Target-protecting dumbbell molecular probe against exonucleases digestion for sensitive detection of ATP and streptavidin | |
| CN103630517A (en) | Thrombin detection method based on splitter adapter and water-soluble conjugated polymer | |
| Fadhel et al. | Selective nano-sensing approach for the determination of inorganic phosphate in human urine samples | |
| CN103725686A (en) | Aptamer AFB1-20 of aflatoxin B1 and application thereof | |
| CN104597007B (en) | Homocysteine kit based on aptamer fluorescence probe HCy5 and detection method thereof | |
| Hou et al. | A sandwich-type surface-enhanced Raman scattering sensor using dual aptamers and gold nanoparticles for the detection of tumor extracellular vesicles | |
| CN107907515A (en) | One kind detection glutathione probe reagent box and its detection method | |
| CN104597008B (en) | Homocysteine kit based on aptamer fluorescent probe HCy3 and detection method thereof | |
| CN104046682B (en) | The detection method of homocysteine and diagnostic kit thereof | |
| Zhou et al. | Electrochemiluminescence immunoassay based on site-specific labeling using a histidine-binding iridium (III) solvento complex | |
| Huang et al. | Rapid and selective luminescent sensing of allergenic gluten by highly phosphorescent switch-on probe | |
| CN104677868A (en) | Homocysteine (HCy) kit based on aptamer fluorescence probe and detection method for concentration of homocysteine (HCy) by using homocysteine (HCy) kit | |
| Gholami et al. | A paper-based optical sensor for the screening of viruses through the cysteine residues of their surface proteins: A proof of concept on the detection of coronavirus infection |
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 | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20180413 |
|
| WW01 | Invention patent application withdrawn after publication |