CN106525784A - INS5 insulin kit based on aptamer fluorescent probe and detection method of INS5 insulin - Google Patents
INS5 insulin kit based on aptamer fluorescent probe and detection method of INS5 insulin Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
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Abstract
The invention relates to an insulin INS kit based on an aptamer fluorescent probe, further relates to a method for measuring the insulin INS concentration, measuring reagent composition and components, and belongs to the technical field of medical examination and measurement. The kit mainly comprises red cell lysate, a phosphate buffer solution (PBS), an insulin (INS) standard substance and the insulin (INS) aptamer fluorescent probe. The concentration of insulin (INS) is measured through blood sample pyrolysis, ovum cultivation processing and fluorospectro photometer detection. The kit and the method have the advantages that sample treatment is easy, operation is easy, detection time is short, detection specificity is high, sensitivity is high, and detection results are high in repeatability.
Description
Technical field
The invention belongs to medical test determination techniques field, the more particularly to pancreas based on nucleic acid aptamer fluorescence probe INS5
Island element test kit and its detection method.
Background technology
Insulin is by endogenouss or exogenous material such as glucose, Lactose, ribose, essence by the beta Cell of islet in pancreas
The stimulation of propylhomoserin, glucagon etc. and a kind of proteohormone for secreting.Insulin is uniquely to drop hypoglycemic sharp in body
Element, while promote glycogen, fat, protein synthesis.Exogenous insulin is mainly used to treating diabetes.
The biosynthesiss speed of insulin is affected by plasma glucose concentration, when blood sugar concentration is raised, pancreas in β cells
Island element original content increases, and insulin synthesis accelerate.Insulin synthesizes in beta Cell of islet.The molecular weight 5700 of insulin, by two
Bar amino acid peptide chain is constituted.A chains have 21 aminoacid, and B chains have 30 aminoacid.There is disulfide bond at two to be connected between A-B chains.
Insulin is into blood with C peptides with equivalent molecular secretion.Clinically, there is pancreas in the patient of use of exogenous insulin in serum
Island element antibody, affects radioimmunoassay to determine blood insulin levels, can pass through in this case to determine plasma C peptide level,
To understand endogenous insulin secretion state.
The detection method of insulin has three major types at present:Chromatography, Immunological Method, Enzymatic cycling.Chromatography sensitivity height,
Specificity is good, but sample treatment, separation condition, chromatographic column prepare all multi-Varis so as to be difficult to standardization;And hplc device valency
Lattice are expensive, technical conditions have high demands, and need special attendant so as to Difficulty.Immunological Method needs the free INS shapes of reduction
Formula, antibody fluorescence analytic process and turbidimetry are unable to direct detection sequestered Homocysteine, can only detect the total homotype of blood plasma half
Cystine, being educated in 37 DEG C of half an hour ovum with reducing agent carries out reduction treatment to blood sample.Immunological Method needs just go out for more than one hour
As a result, complex operation step, since it is desired that carry out reduction treatment can be affected by some uncertain factors.Enzymatic cycling process is numerous
It is trivial, and test limit is low, it is larger to produce error, it is expensive, therefore cannot promote.
Aptamer is the new identification molecule of a class that developed recently gets up, in recent years by the extensive pass of scientist
Note, it is screened out for the aptamer of important physiologically active molecule in a large number;The various analysis sides based on aptamer
Method and technology are reported;Aptamer medicine " Macugen " was also listed by FDA official approvals in 2005.SELEX skills
The oligonucleotide sequence that art screening is obtained is referred to as aptamer, and domestic its is translated as aptamer, nucleic acid aptamer, nucleic acid to be known
Body or aptamer etc..SELEX technologies refer to that applied chemistry method synthesizes jumbo random oligonucleotide (consolidating by two ends
Sequencing is arranged and middle random sequences composition) library, by applying selection pressure (with reference to target, elutriation and target height
The process of specific bond fragment), and Amplification Technologies are combined, through the circulation selective enrichment of many wheels, obtain and target substance
The oligonucleotide molecules that high special is combined, can be RNA can also be DNA, length is generally 25 ~ 60 nucleotide.
From the foregoing, it will be observed that aptamer is combined presented hypersensitivity and high specific with target substance so as to examine in disease
Have a good application prospect in disconnected, although clinical practice report ripe at present is less, apply fit detection globulin
Research be but on the increase, also continuously emerged based on fit new detecting technique.But it is currently based on being directed to for aptamer
The efficient specific recognition research of insulin also lacks very much, and is directed to the aptamer of insulin and its screens preparation method still
Have no and have been reported that.
The content of the invention
The purpose of the present invention is to be also easy to produce that operating technology error, the not good enough, interference factor of repeatability be a lot, behaviour for existing
Make the deficiencies such as loaded down with trivial details, testing cost is high, there is provided a kind of insulin INS test kits and its detection based on nucleic acid aptamer fluorescence probe
Method.
The solution of the present invention is by being achieved in that:
A kind of Insulin Kit based on nucleic acid aptamer fluorescence probe, it is characterised in that main component includes:Containing NH4Cl、
Tris、EDTA-Na2Erythrocyte cracked liquid;Containing MgCl20.2M phosphate buffers;Insulin standards;Insulin nucleic acid is fitted
Body fluorescent probe;The insulin nucleic acid aptamer fluorescence probe is the nucleoside that mark fluorescent pyrene molecule monomer is distinguished at 5' and 3' two ends
Acid is single-stranded, and the nucleotide single-chain sequence is:taggcctcgg acgttatata cacgatctct acgaccgttc
tggctgcgtt tcggactgt.The sequence designations are INS5.
A kind of above-described Insulin Kit based on nucleic acid aptamer fluorescence probe, it is described containing NH4Cl、Tris、
EDTA-Na2Erythrocyte cracked liquid be containing 1 ~ 280 mmol/L NH4Cl, 1~34 mmol/L Tris,1~2mmol/
LEDTA-Na2, pH 7.0~7.2;It is described containing MgCl20.2M phosphate buffers be containing 1 ~ 10mmol/L MgCl2。
A kind of Insulin Kit based on nucleic acid aptamer fluorescence probe described in any of the above, it is characterised in that described
Containing NH4Cl、Tris、EDTA-Na2Erythrocyte cracked liquid, containing MgCl20.2M phosphate buffers, insulin standards, islets of langerhans
Plain nucleic acid aptamer fluorescence probe is to prepare the liquid reagent that directly uses or use the front dry powder that need to be dissolved in water.
A kind of above-described Insulin Kit based on nucleic acid aptamer fluorescence probe, it is characterised in that the test kit
For detecting anticoagulant heparin whole blood or the insulin concentration in peripheral blood.
It is a kind of with described in any of the above based on the Insulin Kit of nucleic acid aptamer fluorescence probe detecting insulin concentration
Method, it is characterised in that method and step includes:
(1)Blood sample is cracked: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 is stood, supernatant is collected;
(2)Mixing ovum is educated:Mixing ovum is educated:Take 20 ~ 100 μ l steps 1)The supernatant for obtaining and the use of 30 ~ 50ul contain MgCl2's
0.2M phosphate buffers, the insulin nucleic acid aptamer fluorescence probe reagent that dissolving insulin nucleic acid aptamer fluorescence probe is obtained are mixed
Close, under room temperature, ovum educates 5 ~ 15min, insulin nucleic acid aptamer fluorescence probe is fully combined with blood sample, obtains test fluid;
(3)Fluoroscopic examination:Fluorescence detector detecting step 2)Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation;
(4)As a result calculate:Using biomedical mapping software, with reference to the standard curve of insulin standard sample making, with reference to step
Rapid 3)In the fluorescent value that detects calculate the concentration of insulin in blood sample.
Above-described biomedical software is Sigma plot softwares, in being commercially available on the market.
It is above-described it is a kind of with based on the Insulin Kit of nucleic acid aptamer fluorescence probe detecting insulin concentration
Method, it is characterised in that insulin nucleic acid aptamer fluorescence probe concentration is in the insulin nucleic acid aptamer fluorescence probe reagent
200 ~ 400 nmol/L, its characteristic are also resided in, and the insulin nucleic acid aptamer fluorescence probe is that 5' and 3' two ends difference labelling is glimmering
The nucleotide single-chain of light pyrene molecule monomer, the nucleotide single-chain sequence is:taggcctcgg acgttatata cacgatctct
acgaccgttc tggctgcgtt tcggactgt。
When insulin nucleic acid aptamer fluorescence probe is not combined with insulin, aptamer is in more open structure, 5'
Mutually dissociate with the pyrene molecule monomer at 3' two ends, after fluorescence excitation, launch wavelength is between 370 ~ 400nm;Insulin nucleic acid
When fit fluorescent probe is combined with insulin, insulin-induced its recurring structure changes, the pyrene point at aptamer 5' and 3' two ends
Sub- monomer is close to each other, forms excited state dimer, and after fluorescence excitation, excited state dimer launch wavelength arrives 500nm 480
Between.
It is above-described it is a kind of with based on the Insulin Kit of nucleic acid aptamer fluorescence probe detecting insulin concentration
Method, it is characterised in that described blood sample is anticoagulant heparin whole blood or peripheral blood, described containing NH4Cl、Tris、EDTA-Na2It is red
Cell pyrolysis liquid is containing 1 ~ 280 mmol/L NH4Cl, 1~34 mmol/L Tris,1~2mmol/LEDTA-Na2, pH 7.0
~7.2;It is described containing MgCl20.2M phosphate buffers be containing 1 ~ 10mmol/L MgCl2;The fluorescence detector be with when
Between resolved fluorometric determine fluorescence detector.
It is above-described it is a kind of with based on the Insulin Kit of nucleic acid aptamer fluorescence probe detecting insulin concentration
Method, it is characterised in that the detection method is used for detecting anticoagulant heparin whole blood or the insulin concentration in peripheral blood.
The present invention principle be:When not with insulin, aptamer is in more open structure, 5' and 3' two ends
Pyrene molecule monomer mutually dissociate, fluorescence emission wavelengths are 370 between 400nm;When being combined with insulin, insulin
Induction aptamer structure changes, and the pyrene molecule monomer at the 5' and 3' two ends of aptamer is close to each other, forms dimerization
Body, pyrene excited state emission wavelength dimer is 480 between 500nm.Pyrene excited state dimer fluorescence lifetime is up to
100 ns, than the biological sample autofluorescence life-span(About 5 ns)It is long, it is mixed by detecting insulin aptamer probe and sample
Fluorescence intensity and fluorescence lifetime after conjunction, calculates the concentration of insulin in sample.
The present invention substantive distinguishing features and marked improvement be:
(1)Detection is simple to operate quick, processes without the need for complex sample and separates, aptamer probe is directly added into after cracking
Blood sample liquid, with the fluorescent value that can just detect in the spectrofluorophotometer short time at 480 ~ 500nm;
(2)This test kit and its detection method have sensitivity high, and testing result repeatability is high, sample detection error 0.01 ~
Between 0.1%, high specificity, when insulin nucleic acid aptamer fluorescence probe is not combined with insulin, aptamer is in more open
Structure, the pyrene molecule monomer at 5' and 3' two ends mutually dissociates, and after fluorescence excitation, launch wavelength is between 370 ~ 400nm;
When which is combined with insulin, insulin-induced its changes, and the pyrene molecule monomer at aptamer 5' and 3' two ends is close to each other,
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 use using after being front dissolved in water
Dry powder, detectable can be with storage at normal temperature, convenient transportation.
Specific embodiment
Below in conjunction with table 1 and embodiment description insulin INS test kit of the present invention based on nucleic acid aptamer fluorescence probe and
Its detection method.
Kit reagent in 1. embodiment of table is into being grouped into
Embodiment 1
After agent formulations dissolving is prepared needed for embodiment 1 in table 1, bottle is distributed into, lyophilization is carried out, make dry powder examination
Agent;Using front, ultra-pure water is added, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows:
(1)Blood sample is cracked:By anticoagulant heparin whole blood, erythrocyte cracked liquid by volume 1:0.5 mixes, and stands 30min, then middling speed
Centrifugation 7min, collects supernatant;
(2)Mixing ovum is educated:Take 20 μ l steps 1)The use 0.2M phosphate buffer dissolving insulin nucleic acid of the supernatant 45ul for obtaining
The insulin nucleic acid aptamer fluorescence probe reagent mixing that fit fluorescent probe is obtained, under room temperature, ovum educates 5min, obtains test fluid;
(3)Fluoroscopic examination:Fluorescence detector detecting step 2)Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation;
(4)As a result calculate:Using biomedical mapping software, with reference to the standard curve of insulin standard sample making, with reference to step
Rapid 3)In the fluorescent value that detects calculate the concentration of insulin in blood sample.
3 parallel assay errors of sample are 0.05 ± 0.01%.
Embodiment 2
After agent formulations dissolving is prepared needed for embodiment 2 in table 1, bottle is distributed into, lyophilization is carried out, make dry powder examination
Agent;Using front, ultra-pure water is added, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows:
(1)Blood sample is cracked: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)The supernatant 30ul's for obtaining
The insulin nucleic acid aptamer fluorescence probe examination that insulin nucleic acid aptamer fluorescence probe is obtained is dissolved with 0.2M phosphate buffers
Agent mixes, and under room temperature, ovum educates 6min, obtains test fluid;
(3)Fluoroscopic examination:Fluorescence detector detecting step 2)Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation;
(4)As a result calculate:Using biomedical mapping software, with reference to the standard curve of insulin standard sample making, with reference to step
Rapid 3)In the fluorescent value that detects calculate the concentration of insulin in blood sample.3 parallel assay errors of sample be 0.02 ±
0.01%。
Embodiment 3
After agent formulations dissolving is prepared needed for embodiment 3 in table 1, bottle is distributed into, lyophilization is carried out, make dry powder examination
Agent;Using front, ultra-pure water is added, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows:
(1)Blood sample is cracked: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)The supernatant 45ul's for obtaining
The insulin nucleic acid aptamer fluorescence probe examination that insulin nucleic acid aptamer fluorescence probe is obtained is dissolved with 0.2M phosphate buffers
Agent mixes, and under room temperature, ovum educates 7min, obtains test fluid;
(3)Fluoroscopic examination:Fluorescence detector detecting step 2)Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation;
(4)As a result calculate:Using biomedical mapping software, with reference to the standard curve of insulin standard sample making, with reference to step
Rapid 3)In the fluorescent value that detects calculate the concentration of insulin in blood sample.
3 parallel assay errors of sample are 0.04 ± 0.01%.
Embodiment 4
After agent formulations dissolving is prepared needed for embodiment 4 in table 1, bottle is distributed into, lyophilization is carried out, make dry powder examination
Agent;Using front, ultra-pure water is added, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows:
(1)Blood sample is cracked:By anticoagulant heparin whole blood, erythrocyte cracked liquid by volume 1:0.5 mixes, and stands 25min, then middling speed
Centrifugation 8min, collects supernatant;
(2)Mixing ovum is educated:Take 100 μ l steps 1)The use 0.2M phosphate buffer dissolving insulin nucleic acid of the supernatant 50ul for obtaining
The insulin nucleic acid aptamer fluorescence probe reagent mixing that fit fluorescent probe is obtained, under room temperature, ovum educates 8min, obtains test fluid;
(3)Fluoroscopic examination:Fluorescence detector detecting step 2)Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation;
(4)As a result calculate:Using biomedical mapping software, with reference to the standard curve of insulin standard sample making, with reference to step
Rapid 3)In the fluorescent value that detects calculate the concentration of insulin in blood sample.
3 parallel assay errors of sample are 0.06 ± 0.01%.
Embodiment 5
After agent formulations dissolving is prepared needed for embodiment 5 in table 1, bottle is distributed into, lyophilization is carried out, make dry powder examination
Agent;Using front, ultra-pure water is added, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows:
(1)Blood sample is cracked:By anticoagulant heparin whole blood, erythrocyte cracked liquid by volume 1:1.0 mix, and stand 20min, then middling speed
Centrifugation 9min, collects supernatant;
(2)Mixing ovum is educated:Take 80 μ l steps 1)The use 0.2M phosphate buffer dissolving insulin nucleic acid of the supernatant 30ul for obtaining
The insulin nucleic acid aptamer fluorescence probe reagent mixing that fit fluorescent probe is obtained, under room temperature, ovum educates 9min, obtains test fluid;
(3)Fluoroscopic examination:Fluorescence detector detecting step 2)Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation;
(4)As a result calculate:Using biomedical mapping software, with reference to the standard curve of insulin standard sample making, with reference to step
Rapid 3)In the fluorescent value that detects calculate the concentration of insulin in blood sample.
3 parallel assay errors of sample are 0.07 ± 0.01%.
Embodiment 6
After agent formulations dissolving is prepared needed for embodiment 6 in table 1, bottle is distributed into, lyophilization is carried out, make dry powder examination
Agent;Using front, ultra-pure water is added, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows:
(1)Blood sample is cracked:By anticoagulant heparin whole blood, erythrocyte cracked liquid by volume 1:4.5 mix, and stand 15min, then middling speed
Centrifugation 10min, collects supernatant;
(2)Mixing ovum is educated:Take 30 μ l steps 1)The use 0.2M phosphate buffer dissolving insulin nucleic acid of the supernatant 40ul for obtaining
The insulin nucleic acid aptamer fluorescence probe reagent mixing that fit fluorescent probe is obtained, under room temperature, ovum educates 10min, obtains test fluid;
(3)Fluoroscopic examination:Fluorescence detector detecting step 2)Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation;
(4)As a result calculate:Using biomedical mapping software, with reference to the standard curve of insulin standard sample making, with reference to step
Rapid 3)In the fluorescent value that detects calculate the concentration of insulin in blood sample.
3 parallel assay errors of sample are 0.08 ± 0.01%.
Embodiment 7
After agent formulations dissolving is prepared needed for embodiment 7 in table 1, bottle is distributed into, lyophilization is carried out, make dry powder examination
Agent;Using front, ultra-pure water is added, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows:
(1)Blood sample is cracked: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)The use 0.2M phosphate buffer dissolving insulin nucleic acid of the supernatant 20ul for obtaining
The insulin nucleic acid aptamer fluorescence probe reagent mixing that fit fluorescent probe is obtained, under room temperature, ovum educates 12min, obtains test fluid;
(3)Fluoroscopic examination:Fluorescence detector detecting step 2)Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation;
(4)As a result calculate:Using biomedical mapping software, with reference to the standard curve of insulin standard sample making, with reference to step
Rapid 3)In the fluorescent value that detects calculate the concentration of insulin in blood sample.
3 parallel assay errors of sample are 0.1 ± 0.01%.
Embodiment 8
After agent formulations dissolving is prepared needed for embodiment 8 in table 1, bottle is distributed into, lyophilization is carried out, make dry powder examination
Agent;Using front, ultra-pure water is added, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows:
(1)Blood sample is cracked: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)The use 0.2M phosphate buffer dissolving insulin nucleic acid of the supernatant 25ul for obtaining
The insulin nucleic acid aptamer fluorescence probe reagent mixing that fit fluorescent probe is obtained, under room temperature, ovum educates 13min, obtains test fluid;
(3)Fluoroscopic examination:Fluorescence detector detecting step 2)Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation;
(4)As a result calculate:Using biomedical mapping software, with reference to the standard curve of insulin standard sample making, with reference to step
Rapid 3)In the fluorescent value that detects calculate the concentration of insulin in blood sample.
3 parallel assay errors of sample are 0.02 ± 0.01%.
Embodiment 9
After agent formulations dissolving is prepared needed for embodiment 9 in table 1, bottle is distributed into, lyophilization is carried out, make dry powder examination
Agent;Using front, ultra-pure water is added, used after redissolution.Each sample setting 3 is parallel, and detecting step is as follows:
(1)Blood sample is cracked: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)The use 0.2M phosphate buffer dissolving insulin nucleic acid of the supernatant 35ul for obtaining
The insulin nucleic acid aptamer fluorescence probe reagent mixing that fit fluorescent probe is obtained, under room temperature, ovum educates 15min, obtains test fluid;
(3)Fluoroscopic examination:Fluorescence detector detecting step 2)Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation;
(4)As a result calculate:Using biomedical mapping software, with reference to the standard curve of insulin standard sample making, with reference to step
Rapid 3)In the fluorescent value that detects calculate the concentration of insulin in blood sample.
3 parallel assay errors of sample are 0.01 ± 0.01%.
Sequence table
<110>Liuzhou Li Jie Science and Technology Ltd.s
<120>Based on nucleic acid aptamer fluorescence probe INS5 Insulin Kit and its detection method
<130> 2016
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 58
<212> DNA
<213>Artificial sequence
<400> 1
taggcctcgg acgttatata cacgatctct acgaccgttc tggctgcgtt tcggactgt 58
Claims (9)
1. a kind of Insulin Kit based on nucleic acid aptamer fluorescence probe, it is characterised in that main component includes:Containing NH4Cl、
Tris、EDTA-Na2Erythrocyte cracked liquid;Containing MgCl20.2M phosphate buffers;Insulin standards;Insulin nucleic acid is fitted
Body fluorescent probe;The insulin nucleic acid aptamer fluorescence probe is the nucleoside that mark fluorescent pyrene molecule monomer is distinguished at 5' and 3' two ends
Acid is single-stranded, and the nucleotide single-chain sequence is:taggcctcgg acgttatata cacgatctct acgaccgttc
tggctgcgtt tcggactgt。
2. a kind of Insulin Kit based on nucleic acid aptamer fluorescence probe according to claim 1, it is characterised in that institute
State containing NH4Cl、Tris、EDTA-Na2Erythrocyte cracked liquid be containing 1~280 mmol/L NH4Cl, 1~34 mmol/L
Tris,1~2mmol/LEDTA-Na2, pH 7.0~7.2;It is described containing MgCl20.2M phosphate buffers be containing 1 ~ 10mmol/
L MgCl2。
3. a kind of Insulin Kit based on nucleic acid aptamer fluorescence probe according to claim 1 and 2, its feature exist
In described containing NH4Cl、Tris、EDTA-Na2Erythrocyte cracked liquid, containing MgCl20.2M phosphate buffers, insulin standard
Product, insulin nucleic acid aptamer fluorescence probe are to prepare the liquid reagent that directly uses or use the front dry powder that need to be dissolved in water
State.
4. a kind of Insulin Kit based on nucleic acid aptamer fluorescence probe according to claim 1 ~ 3, it is characterised in that
The test kit is used for detecting anticoagulant heparin whole blood or the insulin concentration in peripheral blood.
5. it is a kind of with described in right 1 ~ 3 is arbitrary based on the Insulin Kit of nucleic acid aptamer fluorescence probe detecting insulin concentration
Method, it is characterised in that method and step includes:
(1)Blood sample is cracked: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 is stood, supernatant is collected;
(2)Mixing ovum is educated:Take 20 ~ 100 μ l steps 1)The supernatant for obtaining and the use of 30 ~ 50ul contain MgCl20.2M phosphoric acid delay
Liquid is rushed, the insulin nucleic acid aptamer fluorescence probe reagent mixing that insulin nucleic acid aptamer fluorescence probe is obtained, ovum under room temperature is dissolved
5 ~ 15min is educated, insulin nucleic acid aptamer fluorescence probe is fully combined with blood sample, is obtained test fluid;
(3)Fluoroscopic examination:Fluorescence detector detecting step 2)Test fluid 50ul for obtaining, after fluorescence detector fluorescence excitation,
Fluorescent value of the wavelength in 480 ~ 500nm in 20 to the 100ns time periods after reading fluorescence excitation;
(4)As a result calculate:Using biomedical mapping software, with reference to the standard curve of insulin standard sample making, with reference to step
Rapid 3)In the fluorescent value that detects calculate the concentration of insulin in blood sample.
6. it is according to claim 5 it is a kind of with based on the Insulin Kit of nucleic acid aptamer fluorescence probe detecting insulin
The method of concentration, it is characterised in that insulin nucleic acid aptamer fluorescence probe in the insulin nucleic acid aptamer fluorescence probe reagent
Concentration is 200 ~ 400 nmol/L, and the insulin nucleic acid aptamer fluorescence probe is that mark fluorescent pyrene molecule is distinguished at 5' and 3' two ends
The nucleotide single-chain of monomer, the nucleotide single-chain sequence is:gggctgccta atcgtatata gcatcctact
gacgttcacc tctgcggttg actggtcgt。
7. it is according to claim 6 it is a kind of with based on the Insulin Kit of nucleic acid aptamer fluorescence probe detecting insulin
The method of concentration, it is characterised in that when described insulin nucleic acid aptamer fluorescence probe is not combined with insulin, at aptamer
In more open structure, the pyrene molecule monomer at 5' and 3' two ends mutually dissociates, after fluorescence excitation launch wavelength 370 ~
Between 400nm;When described insulin nucleic acid aptamer fluorescence probe is combined with insulin, insulin-induced its recurring structure changes
Become, the pyrene molecule monomer at aptamer 5' and 3' two ends is close to each other, form excited state dimer, excited state two after fluorescence excitation
Aggressiveness launch wavelength is 480 between 500nm.
8. it is according to claim 5 it is a kind of with based on the Insulin Kit of nucleic acid aptamer fluorescence probe detecting insulin
The method of concentration, it is characterised in that described blood sample is anticoagulant heparin whole blood or peripheral blood, described containing NH4Cl、Tris、EDTA-
Na2Erythrocyte cracked liquid be containing 1 ~ 280 mmol/L NH4Cl, 1~34 mmol/L Tris,1~2mmol/LEDTA-Na2,
pH 7.0~7.2;It is described containing MgCl20.2M phosphate buffers be containing 1 ~ 10mmol/L MgCl2;The fluorescence detector is
Fluorescence detector with time-resolved fluorometry.
9. a kind of according to claim 5 ~ 7 detects islets of langerhans with based on the Insulin Kit of nucleic acid aptamer fluorescence probe
The method of plain concentration, it is characterised in that the detection method is used for detecting anticoagulant heparin whole blood or the insulin concentration in peripheral blood.
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JP2009183192A (en) * | 2008-02-05 | 2009-08-20 | Tokyo Univ Of Agriculture & Technology | Insulin-binding aptamer |
CN104697968A (en) * | 2013-12-06 | 2015-06-10 | 中国科学院深圳先进技术研究院 | Construction method for near infrared fluorescence energy transfer based biosensor |
CN105647932A (en) * | 2016-03-25 | 2016-06-08 | 徐大鹏 | Alpha-fetoprotein nucleic acid aptamer AFP1 and preparation method thereof |
CN105785033A (en) * | 2016-03-25 | 2016-07-20 | 徐大鹏 | Alpha fetal protein kit based on aptamer fluorescent probe AFP5 and detection method thereof |
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2016
- 2016-10-17 CN CN201610901342.XA patent/CN106525784A/en active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2009183192A (en) * | 2008-02-05 | 2009-08-20 | Tokyo Univ Of Agriculture & Technology | Insulin-binding aptamer |
CN104697968A (en) * | 2013-12-06 | 2015-06-10 | 中国科学院深圳先进技术研究院 | Construction method for near infrared fluorescence energy transfer based biosensor |
CN105647932A (en) * | 2016-03-25 | 2016-06-08 | 徐大鹏 | Alpha-fetoprotein nucleic acid aptamer AFP1 and preparation method thereof |
CN105785033A (en) * | 2016-03-25 | 2016-07-20 | 徐大鹏 | Alpha fetal protein kit based on aptamer fluorescent probe AFP5 and detection method thereof |
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