CN106645054A - INS4 insulin kit based on high-affinity aptamer fluorescent probe and detection method of kit - Google Patents
INS4 insulin kit based on high-affinity aptamer fluorescent probe and detection method of kit Download PDFInfo
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- CN106645054A CN106645054A CN201610905827.6A CN201610905827A CN106645054A CN 106645054 A CN106645054 A CN 106645054A CN 201610905827 A CN201610905827 A CN 201610905827A CN 106645054 A CN106645054 A CN 106645054A
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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 insulin INS concentration, a measuring reagent composition and components, and belongs to the technical field of medical examination and measurement. The kit mainly comprises red blood cell lysate, phosphate buffer PBS, an insulin INS standard substance and the insulin INS aptamer fluorescent probe. The concentration of insulin INS is calculated by blood sample pyrolysis, mixed egg culture treatment and detection by a fluorescent spectrophotometer. The kit has the advantages of sample treatment simplicity, simplicity and convenience in operation, short detection time, high detection specificity, sensitivity and detection result repeatability and the like.
Description
Technical field
The invention belongs to medical test determination techniques field, the more particularly to pancreas based on nucleic acid aptamer fluorescence probe INS4
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 promoting 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 to enter blood with equivalent molecular secretion with C peptides.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 to determine plasma C peptide level in this case,
To understand endogenous insulin secretion state.
At present the detection method of insulin has three major types：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, educated in 37 DEG C of half an hour ovum with reducing agent carries out reduction treatment to blood sample.Immunological Method needs just to 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 " has also been listed in 2005 by FDA official approvals.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
High special combine oligonucleotide molecules, 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, using fit detection small peptide egg
White research is but on the increase, and is also continuously emerged based on fit new detecting technique.But it is currently based on being directed to for aptamer
Also lack very much in the efficient specific recognition research of insulin, and be directed to the aptamer and its screening preparation method of insulin
There is not yet report.
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：gtagggatgc tcgaacggta agtctctgat ctccgtgaat
aactcgtccc ccatctgg.The sequence designations are INS4.
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 that insulin concentration is detected based on the Insulin Kit of nucleic acid aptamer fluorescence probe with described in any of the above
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 are stood, collect supernatant；
（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 is mixed
Close, ovum educates 5 ~ 15min under room temperature, 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 a kind of to detect insulin concentration with based on the Insulin Kit of nucleic acid aptamer fluorescence probe
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 is 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：gtagggatgc tcgaacggta agtctctgat
ctccgtgaat aactcgtccc ccatctgg。
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, launch wavelength is between 370 ~ 400nm after fluorescence excitation；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 excited state dimer launch wavelength is 480 to 500nm after fluorescence excitation
Between.
It is above-described a kind of to detect insulin concentration with based on the Insulin Kit of nucleic acid aptamer fluorescence probe
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 a kind of to detect insulin concentration with based on the Insulin Kit of nucleic acid aptamer fluorescence probe
Method, it is characterised in that the detection method is used to detect 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, and aptamer probe is directly added into after cracking
Blood sample liquid, with the fluorescent value that just can be detected 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 launch wavelength is between 370 ~ 400nm after fluorescence excitation；
When it 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 using 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 the present invention based on nucleic acid aptamer fluorescence probe insulin INS test kits and
Its detection method.
Kit reagent in the embodiment of table 1. is into being grouped into
Embodiment 1
After agent formulations dissolving is prepared needed for embodiment 1 in table 1, bottle is distributed into, carries out lyophilization, 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, ovum educates 5min under room temperature, 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 0.15 ± 0.01%.
Embodiment 2
After agent formulations dissolving is prepared needed for embodiment 2 in table 1, bottle is distributed into, carries out lyophilization, 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 ovum educates 6min under room temperature, 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, carries out lyophilization, 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 ovum educates 7min under room temperature, 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, carries out lyophilization, 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, ovum educates 8min under room temperature, 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 5
After agent formulations dissolving is prepared needed for embodiment 5 in table 1, bottle is distributed into, carries out lyophilization, 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, ovum educates 9min under room temperature, 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 0.03 ± 0.01%.
Embodiment 6
After agent formulations dissolving is prepared needed for embodiment 6 in table 1, bottle is distributed into, carries out lyophilization, 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, ovum educates 10min under room temperature, 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, carries out lyophilization, 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, ovum educates 12min under room temperature, 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, carries out lyophilization, 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, ovum educates 13min under room temperature, 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, carries out lyophilization, 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, ovum educates 15min under room temperature, 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>A kind of high-affinity insulin aptamer INS4 and preparation method thereof
<130> 2016
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 58
<212> DNA
<213>Artificial sequence
<400> 1
gtagggatgc tcgaacggta agtctctgat ctccgtgaat aactcgtccc ccatctgg 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：gtagggatgc tcgaacggta agtctctgat ctccgtgaat
aactcgtccc ccatctgg。
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 exists
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 to detect anticoagulant heparin whole blood or the insulin concentration in peripheral blood.
5. a kind of with detecting insulin concentration based on the Insulin Kit of nucleic acid aptamer fluorescence probe described in right 1 ~ 3 is arbitrary
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 are stood, collect supernatant；
（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, obtain 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 a kind of to detect insulin with based on the Insulin Kit of nucleic acid aptamer fluorescence probe
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：ggatgcggta acggtacgta cctgtatagt
gtgacatcct gtctccaacc acctgctgac gtgc。
7. it is according to claim 6 a kind of to detect insulin with based on the Insulin Kit of nucleic acid aptamer fluorescence probe
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 a kind of to detect insulin with based on the Insulin Kit of nucleic acid aptamer fluorescence probe
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 to detect anticoagulant heparin whole blood or the insulin concentration in peripheral blood.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610905827.6A CN106645054A (en) | 2016-10-17 | 2016-10-17 | INS4 insulin kit based on high-affinity aptamer fluorescent probe and detection method of kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610905827.6A CN106645054A (en) | 2016-10-17 | 2016-10-17 | INS4 insulin kit based on high-affinity aptamer fluorescent probe and detection method of kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106645054A true CN106645054A (en) | 2017-05-10 |
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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 |
| CN105606582A (en) * | 2016-03-25 | 2016-05-25 | 徐大鹏 | Alpha fetoprotein kit based on aptamer fluorescent probe AFP4 and detection method thereof |
| CN105647932A (en) * | 2016-03-25 | 2016-06-08 | 徐大鹏 | Alpha-fetoprotein nucleic acid aptamer AFP1 and preparation method thereof |
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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 |
| CN105606582A (en) * | 2016-03-25 | 2016-05-25 | 徐大鹏 | Alpha fetoprotein kit based on aptamer fluorescent probe AFP4 and detection method thereof |
| CN105647932A (en) * | 2016-03-25 | 2016-06-08 | 徐大鹏 | Alpha-fetoprotein nucleic acid aptamer AFP1 and preparation method thereof |
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Application publication date: 20170510 |