CN104597008B - Homocysteine kit based on aptamer fluorescent probe HCy3 and detection method thereof - Google Patents
Homocysteine kit based on aptamer fluorescent probe HCy3 and detection method thereof Download PDFInfo
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- CN104597008B CN104597008B CN201410717094.4A CN201410717094A CN104597008B CN 104597008 B CN104597008 B CN 104597008B CN 201410717094 A CN201410717094 A CN 201410717094A CN 104597008 B CN104597008 B CN 104597008B
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- 108091023037 Aptamer Proteins 0.000 title claims abstract description 27
- 238000001514 detection method Methods 0.000 title claims abstract description 25
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- 238000012360 testing method Methods 0.000 claims description 49
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Abstract
The invention relates to a homocysteine HCy kit based on aptamer fluorescent probe, also relates to a method for determining homocysteine HCy concentration and determining reagent composition and ingredients, and belongs to the technical field of medical science examination determination. The main ingredients of the kit comprise an erythrocyte lysate, a phosphoric acid buffer PBS, a homocysteine HCy standard sample and a homocysteine HCy aptamer fluorescent probe. The concentration of homocysteine HCy is measured and calculated by performing blood sample lysis and mixed ovum culturing treatment, and combining with fluorospectrophotometer detection. The kit and the method have the advantages of simple sample processing, simple operation, short detection time, high detection specificity, high sensitivity, high detection result repeatability and the like.
Description
Technical field
The invention belongs to medical test determination techniques field, the more particularly to homotype based on nucleic acid aptamer fluorescence probe half
Cystine hcy test kit and its detection method.
Background technology
Homocysteine is a kind of sulfur-containing amino acid, is the intermediate product of biological internal Methionine metabolism, with half Guang ammonia
Acid is compared, many methylene in side chain.Homocysteine is the risk factor of multiple diseases, it is now recognized that homotype half Guang ammonia
It is the cardiovascular and cerebrovascular diseases caused the most extensive, paathogenic factor the most independent of atherosclerosiss that acid is increased.Homocysteine is also
The shadow of multiple diseases and some drugses, tumor with neural tube defects, parkinson disease, alzheimer disease, chronic renal failure etc.
Sound is relevant.The main chromatography of method of detection homocysteine, immunological method and Enzymatic cycling.
Chromatography: gas chromatogram-mass spectrometric determination homocysteine.This method can measure cysteine, egg ammonia simultaneously
The many kinds of substances such as acid, cystathionie and methylglycine.Chromatography sensitivity is high, specificity is good, but sample treatment, separation condition,
Chromatographic column prepares all multi-Varis so as to be difficult to standardization.And hplc equipment price is expensive, technical conditions have high demands, need special
Attendant so as to Difficulty.
Immunological Method: the anti-s- adenosyl homocysteine monoclonal technigue of this method application specific, using fluorescence polarization
Method or immunization measure.Homocysteine, in the presence of s SAHH and excessive adenosine, is converted into
S adenosyl homocysteine；The fluorescence s of prediluted mixture, anti-s- adenosyl homocysteine monoclonal antibody and labelling
Adenosyl homocysteine tracer is together incubated, the change of instrument automatic detection polarized light, you can measure the total homotype of specimen half
Cystine level.Antibody fluorescence analytic process and turbidimetry are unable to direct detection Homocysteine, need just can go out for more than one hour
As a result, complex operation step.
Enzymatic cycling；It is the detection method amplifying target substance (measured object) using zymolyte characteristic.Blood plasma oxidized form homotype
Cysteine is reduced into sequestered homocysteine through three second carboxyethyl phosphine (tcep), based on the transfer of homocysteine methyl
Enzyme, circulation enzyme system and the principle of dehydrogenase coenzyme system that s SAHH is constituted, catabolite shows
Color, surveying od with automatic biochemistry analyzer in 600nm wavelength is worth the content of the homocysteine in blood plasma.
Currently without the antibody being capable of Direct Recognition homocysteine and other molecular probe.Therefore design a kind of height
Set the molecular probe of homocysteine, and simple and quick and cheap detection method is set up based on this and will improve to heart and brain blood
Pipe be equal to the related disease of homocysteine monitoring.
Aptamer is that the new identification of a class that developed recently gets up divides, and is to refer to high specific and high-affinity ground
The single stranded nucleotide acid molecule of domain target molecule knot.Aptamer can pass through part index concentration phyletic evolution technology
(systematic evolution of ligands by exponentialenrichment, selex) screening obtains.
Selex technology refers to that applied chemistry method synthesizes jumbo random oligonucleotide (by the fixed sequence program at two ends and middle random
Sequence forms) library, by apply selection pressure (in conjunction with target, the mistake of elutriation and target high special binding fragment
Journey), and combine Amplification Technologies, the circulation selective enrichment through excessive wheel, obtain the few core being combined with target substance high special
Thuja acid molecule, can be rna can also be dna, length be generally 25～60 nucleotide.Aptamer mainly passes through to occur to fit
Answering property folds, and is interacted by hydrogen bond, hydrophobic sedimentation, Van der Waals force and target molecule, with insertion or coated target molecule
Form stable three-D space structure.Not when target molecule is combined, aptamer is in more open structure, 5' to aptamer
Mutually free with 3' two ends.When target molecule is combined, target molecule induction aptamer structure changes, the 5' end of aptamer
All will participate in and the interaction of target molecule specific region (antigen sites similar to antibodies) with 3' end, lead to 5' and
3' two ends are close to each other.This feature makes aptamer be suitable to molecular beacon probe design.One of which molecular beacon design profit
With fluorescent dye pyrene molecule.Can be formed when an excited state pyrene molecule and another ground state pyrene molecule closely meet with
Excited state dimer, can discharge a photon in the position longer than pyrene monomer wavelength.Pyrene excited state dimer emission peak is 480
To 500nm, and the emission peak of pyrene monomer is between 370 to 400nm.In addition, pyrene molecule fluorescence lifetime is than non-in biological sample
Specifically excite the life-span length of fluorescence, during detection can non-specific excite fluorescence to disappear after, more dimeric to collect excited state
Fluorescence signal, greatly improves specificity and the sensitivity of detection.The aptamer of identification homocysteine can apply to same
The Clinical detection of type cysteine, improves detection efficiency, reduces testing cost.
Aptamer is combined presented hypersensitivity and high specific with target substance so as to have good in medical diagnosis on disease
Good application prospect, although clinical practice report ripe at present is less, application is fit to detect that the research of target protein is continuous
Increase, also continuously emerged based on fit new detecting technique.Aptamer becomes new one due to the chemical antibody characteristic of its uniqueness
Generation for the molecular medicine of specific protein or targeting vector, and for detecting the target molecule material of its specific recognition.But
The homocysteine hcy diagnostic reagent that is directed to being currently based on aptamer also lacks very much, and is directed to homocysteine
The exploitation of the nucleic acid aptamer fluorescence probe detection kit of hcy is there is not yet report.
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, provide a kind of homocysteine hcy test kit based on nucleic acid aptamer fluorescence probe and
Its detection method.
The solution of the present invention is by being achieved in that:
A kind of homocysteine test kit based on nucleic acid aptamer fluorescence probe is it is characterised in that main component includes:
Containing nh4cl、tris、edta-na2Erythrocyte cracked liquid；Containing mgcl20.2m phosphate buffer；Homocysteine standard
Product；Homocysteine nucleic acid aptamer fluorescence probe；Described homocysteine nucleic acid aptamer fluorescence probe is 5' and 3' two ends
The nucleotide single-chain of mark fluorescent pyrene molecule monomer respectively, this nucleotide single-chain sequence is: ggcactgtac ggctaagttc
ccggatagtg gattggaact ggcataaccc.This sequence designations is hcy3.
A kind of above-described homocysteine test kit based on nucleic acid aptamer fluorescence probe, described containing nh4cl、
tris、edta-na2Erythrocyte cracked liquid be containing 1～280mmol/l nh4Cl, 1～34mmol/l tris, 1～2mmol/
ledta-na2, ph7.0～7.2；Described containing mgcl20.2m phosphate buffer be containing 1～10mmol/l mgcl2.
A kind of homocysteine test kit based on nucleic acid aptamer fluorescence probe described in any of the above, its feature exists
In, described containing nh4cl、tris、edta-na2Erythrocyte cracked liquid, contain mgcl20.2m phosphate buffer, homotype half Guang ammonia
Sour standard substance, homocysteine nucleic acid aptamer fluorescence probe are to prepare the liquid reagent of direct use or added using front need
Water-soluble dry powder.
A kind of above-described homocysteine test kit based on nucleic acid aptamer fluorescence probe is it is characterised in that be somebody's turn to do
Test kit is used for detecting the homotype semicystinol concentration investigating in anticoagulant heparin whole blood or peripheral blood.
A kind of homocysteine test kit based on nucleic acid aptamer fluorescence probe described in any of the above detects homotype
The method of semicystinol concentration is it is characterised in that method and step includes:
(1) blood sample cracking: by blood sample with containing nh4cl、tris、edta-na2Erythrocyte cracked liquid press 1:0.5～5 volume
Ratio mixing, stands 5～30min, then medium-speed centrifuge 5～10min, collects supernatant；
(2) mixing ovum is educated: mixing ovum is educated: takes 20～100 μ l steps 1) use of the supernatant that obtains and 30～50ul contains
mgcl20.2m phosphate buffer, the dissolving homocysteine nucleic acid that obtains of homocysteine nucleic acid aptamer fluorescence probe
Fit fluorescent probe reagent mixing, under room temperature, ovum educates 5～15min, makes homocysteine nucleic acid aptamer fluorescence probe and blood sample
Fully combine, obtain test fluid；
(3) fluoroscopic examination: fluorescence detector detecting step 2) test fluid 50ul that obtains, in fluorescence detector fluorescence excitation
Afterwards, read after fluorescence excitation in 20 to the 100ns time period wavelength in the fluorescent value of 480～500nm；
(4) result calculates: using biomedical mapping software, the standard making with reference to homocysteine standard sample is bent
Line, in conjunction with step 3) in the fluorescent value that detects calculate the concentration of homocysteine in blood sample.
Above-described biomedicine software is sigma plot software, in being commercially available on the market.
Above-described a kind of with homotype half is detected based on the homocysteine test kit of nucleic acid aptamer fluorescence probe
The method of cystine concentration is it is characterised in that homotype half Guang ammonia in described homocysteine nucleic acid aptamer fluorescence probe reagent
Sour nucleic acid aptamer fluorescence probe concentration is 200～400nmol/l, and its characteristic also resides in, described homocysteine aptamer
Fluorescent probe is the nucleotide single-chain of 5' and 3' two ends difference mark fluorescent pyrene molecule monomer, and this nucleotide single-chain sequence is:
ggcactgtac ggctaagttc ccggatagtg gattggaact ggcataaccc.
Not when homocysteine is combined, it is loose that aptamer is in comparison to homocysteine nucleic acid aptamer fluorescence probe
Scattered structure, the pyrene molecule monomer at 5' and 3' two ends is mutually free, and after fluorescence excitation, launch wavelength is between 370～400nm；With
When homocysteine is combined, homocysteine induces its recurring structure to change to type cysteine nucleic acid aptamer fluorescence probe
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 between 480 to 500nm.
Above-described a kind of with homotype half is detected based on the homocysteine test kit of nucleic acid aptamer fluorescence probe
The method of cystine 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 1～280mmol/l nh4Cl, 1～34mmol/l tris, 1～2mmol/
ledta-na2, ph7.0～7.2；Described containing mgcl20.2m phosphate buffer be containing 1～10mmol/l mgcl2；Described glimmering
Optical detector is the fluorescence detector with time-resolved fluorometry.
Above-described a kind of with homotype half is detected based on the homocysteine test kit of nucleic acid aptamer fluorescence probe
The method of cystine concentration is it is characterised in that this detection method is used for detecting the homotype half in anticoagulant heparin whole blood or peripheral blood
Cystine concentration.
The principle of the present invention is: when not with homocysteine, aptamer is in more open structure, 5' and 3'
The pyrene molecule monomer at two ends is mutually free, and fluorescence emission wavelengths are between 370 to 400nm；When homocysteine is combined, with
Type cysteine induction aptamer structure changes, and the pyrene molecule monomer at the 5' and 3' two ends of aptamer is close to each other,
Form dimer, pyrene excited state emission wavelength dimer is between 480 to 500nm.Pyrene excited state dimer fluorescence lifetime has
Up to 100ns, longer than the biological sample autofluorescence life-span (about 5ns), by detecting homocysteine aptamer probe
With the fluorescence intensity after sample mix and fluorescence lifetime, calculate the concentration of homocysteine in sample.
The substantive distinguishing features of the present invention and marked improvement are:
(1) detection is simple to operate quick, without complex sample processing and separation, aptamer probe is directly added into cracking
Blood sample liquid afterwards, with just detecting the fluorescent value at 480～500nm in the spectrofluorophotometer short time；
(2) this test kit and its detection method have sensitivity height, and testing result repeatability is high, and sample detection error exists
Between 0.01～0.1%, high specificity, homocysteine nucleic acid aptamer fluorescence probe not when homocysteine is combined,
Aptamer is in more open structure, and the pyrene molecule monomer at 5' and 3' two ends is mutually free, launch wavelength after fluorescence excitation
Between 370～400nm；, when homocysteine is combined, homocysteine induces it to change for it, aptamer 5'
Close to each other with the pyrene molecule monomer at 3' two ends, form dimer, after fluorescence excitation, dimer launch wavelength is in 480～500nm
Between.
(3) reagent used by 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
Describe the homocysteine hcy based on nucleic acid aptamer fluorescence probe for the present invention below in conjunction with table 1 and embodiment to try
Agent box and its detection method.
Kit reagent in table 1. embodiment becomes to be grouped into
Embodiment 1
After agent formulations dissolving prepares needed for embodiment 1 in table 1, it is distributed into bottle, carries out lyophilization, make dry powder
Reagent；Using front, add ultra-pure water, use after redissolution.Each sample setting 3 is parallel, and detecting step is as follows:
(1) blood sample cracking: 1:0.5 mixes by volume by anticoagulant heparin whole blood, erythrocyte cracked liquid, stands 30min, then
Medium-speed centrifuge 7min, collects supernatant；
(2) mixing ovum is educated: takes 20 μ l steps 1) the use 0.2m phosphate buffer dissolving homotype half of the supernatant 45ul that obtains
The homocysteine nucleic acid aptamer fluorescence probe reagent mixing that cystine nucleic acid aptamer fluorescence probe obtains, under room temperature, ovum is educated
5min, obtains test fluid；
(3) fluoroscopic examination: fluorescence detector detecting step 2) test fluid 50ul that obtains, in fluorescence detector fluorescence excitation
Afterwards, read after fluorescence excitation in 20 to the 100ns time period wavelength in the fluorescent value of 480～500nm；
(4) result calculates: using biomedical mapping software, the standard making with reference to homocysteine standard sample is bent
Line, in conjunction with step 3) in the fluorescent value that detects calculate the concentration of homocysteine in blood sample.
3 parallel assay errors of sample are 0.05 ± 0.01%.
Embodiment 2
After agent formulations dissolving prepares needed for embodiment 2 in table 1, it is distributed into bottle, carries out lyophilization, make dry powder
Reagent；Using front, add ultra-pure water, use after redissolution.Each sample setting 3 is parallel, and detecting step is as follows:
(1) blood sample cracking: by peripheral blood, erythrocyte cracked liquid by volume 1:2.5 mix, stand 5min, then middling speed from
Heart 6min, collects supernatant；
(2) mixing ovum is educated: takes 50 μ l steps 1) the use 0.2m phosphate buffer dissolving homotype half of the supernatant 30ul that obtains
The homocysteine nucleic acid aptamer fluorescence probe reagent mixing that cystine nucleic acid aptamer fluorescence probe obtains, under room temperature, ovum is educated
6min, obtains test fluid；
(3) fluoroscopic examination: fluorescence detector detecting step 2) test fluid 50ul that obtains, in fluorescence detector fluorescence excitation
Afterwards, read after fluorescence excitation in 20 to the 100ns time period wavelength in the fluorescent value of 480～500nm；
(4) result calculates: using biomedical mapping software, the standard making with reference to homocysteine standard sample is bent
Line, in conjunction with step 3) in the fluorescent value that detects calculate the concentration of homocysteine in blood sample.3 parallel assays of sample
Error is 0.02 ± 0.01%.
Embodiment 3
After agent formulations dissolving prepares needed for embodiment 3 in table 1, it is distributed into bottle, carries out lyophilization, make dry powder
Reagent；Using front, add ultra-pure water, use after redissolution.Each sample setting 3 is parallel, and detecting step is as follows:
(1) blood sample cracking: by peripheral blood, erythrocyte cracked liquid by volume 1:3.5 mix, stand 10min, then middling speed from
Heart 5min, collects supernatant；
(2) mixing ovum is educated: takes 75 μ l steps 1) the use 0.2m phosphate buffer dissolving homotype half of the supernatant 45ul that obtains
The homocysteine nucleic acid aptamer fluorescence probe reagent mixing that cystine nucleic acid aptamer fluorescence probe obtains, under room temperature, ovum is educated
7min, obtains test fluid；
(3) fluoroscopic examination: fluorescence detector detecting step 2) test fluid 50ul that obtains, in fluorescence detector fluorescence excitation
Afterwards, read after fluorescence excitation in 20 to the 100ns time period wavelength in the fluorescent value of 480～500nm；
(4) result calculates: using biomedical mapping software, the standard making with reference to homocysteine standard sample is bent
Line, in conjunction with step 3) in the fluorescent value that detects calculate the concentration of homocysteine in blood sample.
3 parallel assay errors of sample are 0.04 ± 0.01%.
Embodiment 4
After agent formulations dissolving prepares needed for embodiment 4 in table 1, it is distributed into bottle, carries out lyophilization, make dry powder
Reagent；Using front, add ultra-pure water, use after redissolution.Each sample setting 3 is parallel, and detecting step is as follows:
(1) blood sample cracking: 1:0.5 mixes by volume by anticoagulant heparin whole blood, erythrocyte cracked liquid, stands 25min, then
Medium-speed centrifuge 8min, collects supernatant；
(2) mixing ovum is educated: takes 100 μ l steps 1) the use 0.2m phosphate buffer dissolving homotype half of the supernatant 50ul that obtains
The homocysteine nucleic acid aptamer fluorescence probe reagent mixing that cystine nucleic acid aptamer fluorescence probe obtains, under room temperature, ovum is educated
8min, obtains test fluid；
(3) fluoroscopic examination: fluorescence detector detecting step 2) test fluid 50ul that obtains, in fluorescence detector fluorescence excitation
Afterwards, read after fluorescence excitation in 20 to the 100ns time period wavelength in the fluorescent value of 480～500nm；
(4) result calculates: using biomedical mapping software, the standard making with reference to homocysteine standard sample is bent
Line, in conjunction with step 3) in the fluorescent value that detects calculate the concentration of homocysteine in blood sample.
3 parallel assay errors of sample are 0.06 ± 0.01%.
Embodiment 5
After agent formulations dissolving prepares needed for embodiment 5 in table 1, it is distributed into bottle, carries out lyophilization, make dry powder
Reagent；Using front, add ultra-pure water, use after redissolution.Each sample setting 3 is parallel, and detecting step is as follows:
(1) blood sample cracking: 1:1.0 mixes by volume by anticoagulant heparin whole blood, erythrocyte cracked liquid, stands 20min, then
Medium-speed centrifuge 9min, collects supernatant；
(2) mixing ovum is educated: takes 80 μ l steps 1) the use 0.2m phosphate buffer dissolving homotype half of the supernatant 30ul that obtains
The homocysteine nucleic acid aptamer fluorescence probe reagent mixing that cystine nucleic acid aptamer fluorescence probe obtains, under room temperature, ovum is educated
9min, obtains test fluid；
(3) fluoroscopic examination: fluorescence detector detecting step 2) test fluid 50ul that obtains, in fluorescence detector fluorescence excitation
Afterwards, read after fluorescence excitation in 20 to the 100ns time period wavelength in the fluorescent value of 480～500nm；
(4) result calculates: using biomedical mapping software, the standard making with reference to homocysteine standard sample is bent
Line, in conjunction with step 3) in the fluorescent value that detects calculate the concentration of homocysteine in blood sample.
3 parallel assay errors of sample are 0.07 ± 0.01%.
Embodiment 6
After agent formulations dissolving prepares needed for embodiment 6 in table 1, it is distributed into bottle, carries out lyophilization, make dry powder
Reagent；Using front, add ultra-pure water, use after redissolution.Each sample setting 3 is parallel, and detecting step is as follows:
(1) blood sample cracking: 1:4.5 mixes by volume by anticoagulant heparin whole blood, erythrocyte cracked liquid, stands 15min, then
Medium-speed centrifuge 10min, collects supernatant；
(2) mixing ovum is educated: takes 30 μ l steps 1) the use 0.2m phosphate buffer dissolving homotype half of the supernatant 40ul that obtains
The homocysteine nucleic acid aptamer fluorescence probe reagent mixing that cystine nucleic acid aptamer fluorescence probe obtains, under room temperature, ovum is educated
10min, obtains test fluid；
(3) fluoroscopic examination: fluorescence detector detecting step 2) test fluid 50ul that obtains, in fluorescence detector fluorescence excitation
Afterwards, read after fluorescence excitation in 20 to the 100ns time period wavelength in the fluorescent value of 480～500nm；
(4) result calculates: using biomedical mapping software, the standard making with reference to homocysteine standard sample is bent
Line, in conjunction with step 3) in the fluorescent value that detects calculate the concentration of homocysteine in blood sample.
3 parallel assay errors of sample are 0.08 ± 0.01%.
Embodiment 7
After agent formulations dissolving prepares needed for embodiment 7 in table 1, it is distributed into bottle, carries out lyophilization, make dry powder
Reagent；Using front, add ultra-pure water, use after redissolution.Each sample setting 3 is parallel, and detecting step is as follows:
(1) blood sample cracking: by peripheral blood, erythrocyte cracked liquid by volume 1:3.0 mix, stand 18min, then middling speed from
Heart 8min, collects supernatant；
(2) mixing ovum is educated: takes 50 μ l steps 1) the use 0.2m phosphate buffer dissolving homotype half of the supernatant 20ul that obtains
The homocysteine nucleic acid aptamer fluorescence probe reagent mixing that cystine nucleic acid aptamer fluorescence probe obtains, under room temperature, ovum is educated
12min, obtains test fluid；
(3) fluoroscopic examination: fluorescence detector detecting step 2) test fluid 50ul that obtains, in fluorescence detector fluorescence excitation
Afterwards, read after fluorescence excitation in 20 to the 100ns time period wavelength in the fluorescent value of 480～500nm；
(4) result calculates: using biomedical mapping software, the standard making with reference to homocysteine standard sample is bent
Line, in conjunction with step 3) in the fluorescent value that detects calculate the concentration of homocysteine in blood sample.
3 parallel assay errors of sample are 0.1 ± 0.01%.
Embodiment 8
After agent formulations dissolving prepares needed for embodiment 8 in table 1, it is distributed into bottle, carries out lyophilization, make dry powder
Reagent；Using front, add ultra-pure water, use after redissolution.Each sample setting 3 is parallel, and detecting step is as follows:
(1) blood sample cracking: by peripheral blood, erythrocyte cracked liquid by volume 1:2.0 mix, stand 26min, then middling speed from
Heart 6min, collects supernatant；
(2) mixing ovum is educated: takes 60 μ l steps 1) the use 0.2m phosphate buffer dissolving homotype half of the supernatant 25ul that obtains
The homocysteine nucleic acid aptamer fluorescence probe reagent mixing that cystine nucleic acid aptamer fluorescence probe obtains, under room temperature, ovum is educated
13min, obtains test fluid；
(3) fluoroscopic examination: fluorescence detector detecting step 2) test fluid 50ul that obtains, in fluorescence detector fluorescence excitation
Afterwards, read after fluorescence excitation in 20 to the 100ns time period wavelength in the fluorescent value of 480～500nm；
(4) result calculates: using biomedical mapping software, the standard making with reference to homocysteine standard sample is bent
Line, in conjunction with step 3) in the fluorescent value that detects calculate the concentration of homocysteine in blood sample.
3 parallel assay errors of sample are 0.02 ± 0.01%.
Embodiment 9
After agent formulations dissolving prepares needed for embodiment 9 in table 1, it is distributed into bottle, carries out lyophilization, make dry powder
Reagent；Using front, add ultra-pure water, use after redissolution.Each sample setting 3 is parallel, and detecting step is as follows:
(1) blood sample cracking: by peripheral blood, erythrocyte cracked liquid by volume 1:4.0 mix, stand 8min, then middling speed from
Heart 7min, collects supernatant；
(2) mixing ovum is educated: takes 40 μ l steps 1) the use 0.2m phosphate buffer dissolving homotype half of the supernatant 35ul that obtains
The homocysteine nucleic acid aptamer fluorescence probe reagent mixing that cystine nucleic acid aptamer fluorescence probe obtains, under room temperature, ovum is educated
15min, obtains test fluid；
(3) fluoroscopic examination: fluorescence detector detecting step 2) test fluid 50ul that obtains, in fluorescence detector fluorescence excitation
Afterwards, read after fluorescence excitation in 20 to the 100ns time period wavelength in the fluorescent value of 480～500nm；
(4) result calculates: using biomedical mapping software, the standard making with reference to homocysteine standard sample is bent
Line, in conjunction with step 3) in the fluorescent value that detects calculate the concentration of homocysteine in blood sample.
3 parallel assay errors of sample are 0.01 ± 0.01%.
Nucleotides sequence list
<110>the climing Li Hongmei of graceful shen of Chen Yan
<120>the homocysteine test kit based on nucleic acid aptamer fluorescence probe hcy3 and its detection method
<130>2014
<160>1
<170>patentin version 3.3
<210>1
<211>50
<212>dna
<213>artificial sequence
<222>(1) ... (50)
<400>1
ggcactgtac ggctaagttc ccggatagtg gattggaact ggcataaccc
Claims (9)
1. a kind of homocysteine test kit based on nucleic acid aptamer fluorescence probe is it is characterised in that main component includes: contains
nh4cl、tris、edta-na2Erythrocyte cracked liquid；Containing mgcl20.2m phosphate buffer；Homocysteine standard substance；
Homocysteine nucleic acid aptamer fluorescence probe；Described homocysteine nucleic acid aptamer fluorescence probe is 5' and 3' two ends difference
The nucleotide single-chain of mark fluorescent pyrene molecule monomer, this nucleotide single-chain sequence is: ggcactgtac ggctaagttc
ccggatagtg gattggaact ggcataaccc.
2. a kind of homocysteine test kit based on nucleic acid aptamer fluorescence probe according to claim 1, its feature
Be, 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；Described containing mgcl20.2m phosphate buffer be containing 1 ~
10mmol/l mgcl2.
3. a kind of homocysteine test kit based on nucleic acid aptamer fluorescence probe according to claim 1 and 2, it is special
Levy and be, described containing nh4cl、tris、edta-na2Erythrocyte cracked liquid, contain mgcl20.2m phosphate buffer, homotype half
Cystine standard substance, homocysteine nucleic acid aptamer fluorescence probe are to prepare the liquid reagent of direct use or using front
The dry powder that need to be dissolved in water.
4. a kind of homocysteine test kit based on nucleic acid aptamer fluorescence probe according to claim 1, its feature
It is, this test kit is used for detecting the homotype semicystinol concentration investigating in anticoagulant heparin whole blood or peripheral blood.
5. a kind of homocysteine test kit based on nucleic acid aptamer fluorescence probe described in claim 1 detects homotype half
The method of cystine concentration is it is characterised in that method and step includes:
(1) blood sample cracking: by blood sample with containing nh4cl、tris、edta-na2Erythrocyte cracked liquid press 1:0.5 ~ 5 volume ratio mix
Even, stand 5 ~ 30min, then medium-speed centrifuge 5 ~ 10min, collect supernatant；
(2) mixing incubation: take the supernatant that 20 ~ 100 μ l step 1) obtain and 30 ~ 50 μ l containing mgcl20.2m phosphoric acid buffer
Liquid, the homocysteine nucleic acid aptamer fluorescence probe reagent mixing that dissolving homocysteine nucleic acid aptamer fluorescence probe obtains
Liquid, is incubated 5 ~ 15min under room temperature, so that homocysteine nucleic acid aptamer fluorescence probe is fully combined with blood sample, obtain test fluid；
(3) fluoroscopic examination: fluorescence detector detecting step 2) the test fluid 50 μ l that obtains, after fluorescence detector fluorescence excitation,
In 20 to the 100ns time period after reading fluorescence excitation, wavelength is in the fluorescent value of 480 ~ 500nm；
(4) result calculates: using biomedical mapping software, with reference to the standard curve of homocysteine standard sample making,
Calculate the concentration of homocysteine in blood sample in conjunction with the fluorescent value detecting in step 3).
6. a kind of homocysteine test kit based on nucleic acid aptamer fluorescence probe according to claim 5 detects
The method of homotype semicystinol concentration investigating is it is characterised in that homotype in described homocysteine nucleic acid aptamer fluorescence probe reagent
Cysteine nucleic acid aptamer fluorescence probe concentration is 200 ~ 400 nmol/l, described homocysteine nucleic acid aptamer fluorescence probe
For the nucleotide single-chain of 5' and 3' two ends difference mark fluorescent pyrene molecule monomer, this nucleotide single-chain sequence is: ggcactgtac
ggctaagttc ccggatagtg gattggaact ggcataaccc.
7. a kind of homocysteine test kit based on nucleic acid aptamer fluorescence probe according to claim 6 detects
The method of homotype semicystinol concentration investigating it is characterised in that described homocysteine nucleic acid aptamer fluorescence probe not with homotype
When cysteine combines, aptamer is in more open structure, and the pyrene molecule monomer at 5' and 3' two ends is mutually free, glimmering
Light excites rear launch wavelength between 370 ~ 400nm；Described homocysteine nucleic acid aptamer fluorescence probe and homotype half
When cystine combines, homocysteine induces its recurring structure to change, the pyrene molecule single phase at aptamer 5' and 3' two ends
Mutually close, form excited state dimer, after fluorescence excitation, excited state dimer launch wavelength is between 480 to 500nm.
8. a kind of homocysteine test kit based on nucleic acid aptamer fluorescence probe according to claim 5 detects
The method of homotype semicystinol concentration investigating, it is characterised in that described blood sample is anticoagulant heparin whole blood or peripheral blood, described contains
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；Described containing mgcl20.2m phosphate buffer be containing 1 ~ 10mmol/l
mgcl2；Described fluorescence detector is the fluorescence detector with time-resolved fluorometry.
9. a kind of homocysteine test kit based on nucleic acid aptamer fluorescence probe according to claim 5 detects
The method of homotype semicystinol concentration investigating is it is characterised in that this detection method is used for detecting in anticoagulant heparin whole blood or peripheral blood
Homotype semicystinol concentration investigating.
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| CN104597246B (en) * | 2014-11-30 | 2016-06-15 | 李佳华 | Homocysteine test kit and detection method thereof based on nucleic acid aptamer fluorescence probe HCy2 |
| CN108982461A (en) * | 2018-09-04 | 2018-12-11 | 华东师范大学 | A kind of time resolution type fluorescent optical sensor and its preparation method and application |
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| CN102183495A (en) * | 2011-01-26 | 2011-09-14 | 鲁东大学 | Fluorescence detection method of cysteine |
| CN102901720A (en) * | 2012-09-27 | 2013-01-30 | 中国人民解放军军事医学科学院基础医学研究所 | Method for detecting blood homocysteine and kit |
| CN103755672A (en) * | 2014-01-26 | 2014-04-30 | 大连理工常熟研究院有限公司 | Specific fluorescence probe for identifying cysteine and application thereof |
| CN103788076A (en) * | 2014-01-22 | 2014-05-14 | 山西大学 | Reagent and method for detecting cysteine |
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| US8642352B2 (en) * | 2009-02-06 | 2014-02-04 | California Institute Of Technology | Methods and systems for detection of stoichiometry by Förster resonance energy transfer |
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|---|---|---|---|---|
| US5322795A (en) * | 1992-06-23 | 1994-06-21 | Merck & Co., Inc. | HPLC assay for the selective resolution of recombinant proteins |
| CN102183495A (en) * | 2011-01-26 | 2011-09-14 | 鲁东大学 | Fluorescence detection method of cysteine |
| CN102901720A (en) * | 2012-09-27 | 2013-01-30 | 中国人民解放军军事医学科学院基础医学研究所 | Method for detecting blood homocysteine and kit |
| CN103788076A (en) * | 2014-01-22 | 2014-05-14 | 山西大学 | Reagent and method for detecting cysteine |
| CN103755672A (en) * | 2014-01-26 | 2014-04-30 | 大连理工常熟研究院有限公司 | Specific fluorescence probe for identifying cysteine and application thereof |
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