CN102998289B - Glycosylated hemoglobin kit based on nucleic acid aptamer fluorescence probe and detection method thereof - Google Patents
Glycosylated hemoglobin kit based on nucleic acid aptamer fluorescence probe and detection method thereof Download PDFInfo
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- CN102998289B CN102998289B CN201210456410.8A CN201210456410A CN102998289B CN 102998289 B CN102998289 B CN 102998289B CN 201210456410 A CN201210456410 A CN 201210456410A CN 102998289 B CN102998289 B CN 102998289B
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Abstract
The invention relates to a glycosylated hemoglobin (GHb) kit based on a nucleic acid aptamer fluorescence probe, and also relates to a method for testing concentration of glycosylated hemoglobin, and composition and components of the test reagent, belonging to the technical field of medical examination and testing. The main components of the kit include red blood cell lysis solution, phosphate buffer solution (PBS), glycosylated hemoglobin standard sample and glycosylated hemoglobin nucleic acid aptamer fluorescence probe; and the concentration of the glycosylated hemoglobin is calculated by blood sample pyrolysis and mixed egg culturing treatment and combining detection of a fluorospectro photometer. The glycosylated hemoglobin kit and the method has the advantages of being simple in sample treatment, simple and convenient to operate, short in detection time, strong in detection specificity, high in sensitivity, high in detection result repeatability and the like.
Description
Technical field
The invention belongs to medical test determination techniques field, particularly relate to the glycosylated hemoglobin GHb kit based on nucleic acid aptamer fluorescence probe and detection method thereof.
Background technology
Diabetes are worldwide public health problems of human health, and diabetes are a kind of lifelong disease, and its complication is to residual main cause till death, so people wish to find as early as possible and to treat diabetes.Traditional diabetes diagnosis and Treatment monitoring adopt fasting blood-glucose, postprandial blood sugar and oral glucose tolerance test etc., but glycemic parameters only represents moment blood sugar level during blood drawing, and glycosylated hemoglobin (GHb) is as the goldstandard of reflection long-term blood glucose level, it is also the important indicator of monitoring treating diabetes.GHb be in red blood cell haemoglobin and glucose slowly, continue and irreversibly carry out the product of non-enzymatic protein saccharification react.Not easily separate after forming two weeks.When the concentration of glucose in blood is higher, the GHb content that human body is formed also can be relatively high. and under normal physiological conditions, the growing amount of non-enzymatic saccharification react product is directly proportional to the concentration of reactant.Because protein concentration keeps relative stability, saccharification level depends mainly on concentration of glucose, also relevant with the time length of protein and glucose exposure.GHb can reflect the average blood glucose levels of nearest 2-3 months.Periodic detection GHb answers in ADA in 2002 clear stipulaties, and it can be used as the gold mark that monitoring diabetes glucose controls.Universal Diabetes Mellitus Knowledge, upgrades treatment concept, monitors and keep GHb up to standard, more early, more reasonably uses the drug therapies such as insulin, particularly important for the generation development controlling diabetic complication.
The detection method of glycosylated hemoglobin GHb conventional at present has following several types.
High pressure liquid phase method (HPLC): can the automatically variant of separation determination GHb and haemproteins and hypotype, but the operation of instrument maintenance requires higher, within CV value 1%, be difficult to the hospital of relatively basic unit and laboratory universal; Microtrabeculae method manual steps is loaded down with trivial details, and the quality of chromatography time and microtrabeculae is wayward, easily produces operative technique error, and repeatability is not good enough; And disturbing factor is a lot, especially to the sensitive of pH value and temperature, HbF and variation haemoglobin (HbS, HbC, HbE etc.) are to result interference, and degree of disturbing is determined according to the separating power of post, and will examine collection of illustrative plates, so this method can not get generally adopting.
Immunization method: immunization method adopts many detection meanss at present, mainly comprises following several detection mode.
(1) turbidimetry: utilize antigen, the principle of antibody response measures.The B chain N end of GHb provides one easily by the epitope of antibody recognition, with the epitope of last 4 ~ 6 amino acid composition of the B chain N end of monoclonal antibody specific recognition GHb, in conjunction with turbidimetry for Determination.
(2) Ion capture: application antigen-antibody reaction principle, in parallel with fluorescent marker, by connecting electronegative polyanionic compound, be adsorbed onto positively charged fiber surface, after the steps such as a series of thorough cleanings, measure fluorescence intensity change rate, calculate GHb concentration.
(3) Latex Agglutination: utilize antigen-antibody reaction directly to measure the method for the percentage composition of HbAlc in total lib.In sample, total Hb and GHb and latex have identical non-specific adsorption and immobilization, when forming the compound of latex one GHb. mouse-anti people GHb monoclonal antibody after the monoclonal antibody specific adding HbAlc, this compound forms aggegation due to sheep anti-mouse antibody, the difference that aggegation amount is measured because of the immobilised GHb of latex surface and different.By measuring its absorbance and the percentage composition of Hb shared by GHb in sample can being obtained more afterwards with the typical curve of GHb percentage concentration.Immunization measures its accuracy of glycosylated hemoglobin and repeatability is all weak, and measures the interference by materials such as high concentration glucose, high triglyceride, high cholerythrin, and the turbidity due to sample can make photometric determination produce mistake.In addition, antibody is obtained by animal immune, and cost is high and the screening cycle is long, there are differences between batches; Antibody is responsive to temperature humidity, and changeableness is difficult to ensure shortcomings such as depositing.
Affinity chromatography: utilize the affine test of boronation as Cleaning Principle.The reagent energy lysed erythrocyte of this method the specificity precipitate hemoglobin factor, wherein blue boric acid conjugate can and glycosylated hemoglobin on Cis-diols combine.Blood adds in reagent, and red blood cell dissolves immediately, and whole haemoglobins is all precipitated, and the cis-diol structure of boric acid conjugate just and in GHb combines.No matter add filter membrane device, the haemoglobin of all precipitations after blood and reagent mixing, be combine or the unconjugated top being all retained in filter membrane with conjugate.Excessive colour developing conjugate is just washed off by cleansing solution.The intensity of blue (glycosylated hemoglobin) and redness (total hemoglobin) is measured by golden scalar quantity instrument, can the number percent of GHb in calculation sample.
Aptamer is the novel identification molecule of a class that developed recently gets up, and be subject to the extensive concern of scientist in recent years, the aptamer in a large number for important physiologically active molecule is out screened; The various analytical approach based on aptamer and technology are in the news; Aptamer medicine " Macugen " was also gone on the market in 2005 by FDA official approval.The oligonucleotide sequence that SELEX technology screening obtains is called as aptamer, and domestic its is translated as aptamer, nucleic acid aptamer, aptamer or aptamer etc.SELEX technology refers to that applied chemistry method synthesizes jumbo random oligonucleotide (being made up of the fixed sequence program at two ends and the random series of centre) library, by applying selection pressure (in conjunction with target, the process of elutriation and target high special binding fragment), and in conjunction with Amplification Technologies, through the circulation selective enrichment too much taken turns, the oligonucleotide molecules that acquisition is combined with target material high special can be RNA also can be DNA, and length is generally 25 ~ 60 nucleotide.
Pyrene is a kind of flammable faint yellow monoclinic crystal organic compound, and the following Fig. 1 of its molecular structure is water insoluble, is soluble in ethanol, ether.Parental materials can be carried out, as reactions such as halogenation, nitrated, sulfonation.Pyrene is mainly present in the distillation of coal-tar asphalt.Pyrene is organic synthesis raw material, can produce Isosorbide-5-Nitrae, 5,8-naphthalenetetracarbacidic acidic, for fuel, synthetic resin, disperse dyes and engineering plastics through oxidation; Can the gorgeous orange GR of reducing dye processed and other multiple dyestuffs after acidylate; Also can pesticide processed, plastifier etc.; Current pyrene molecule can be used as fluorescence probe for based in the detection method of aptamer.
Fluorescent dye pyrene, can form excited state dimer (* Py+Py) when an excited state molecule (* Py) and another ground state molecule (Py) closely meet with time, can discharge a photon in the position longer than pyrene monomer wavelength.Pyrene excited state dimer has a wide flat emission peak at 480 to 500nm places, this emission peak is easy to differentiate; Even if because the emission peak of pyrene monomer is also very wide, but the emission peak of pyrene monomer is between 370 to 400nm.With fluorescent resonance energy transfer type seemingly, form excited state dimer and there is strict distance dependencies, be conducive to Development of Novel nucleic acid probe.Two pyrene molecules are connected to the two ends of molecular beacon, are designed to " excited state dimer-monomer conversion hysteria " molecular beacon, target dna is detected; Two pyrene molecules are connected to one end of molecular beacon as fluorophor, the other end connects fluorescent quenching group, the combination of aptamer and measured target causes its space structure to change, make the pyrene molecule at two ends close and form excited state dimer, by detecting fluorescence intensity and the fluorescence lifetime of aptamer probe, thus achieve the highly sensitive detection to measured target.
Aptamer is combined presented hypersensitivity and high specific with target material, it is made to have a good application prospect in medical diagnosis on disease, although clinical practice report ripe is at present less, but the research of applying fit detection target protein is on the increase, and also constantly occurs based on fit new detecting technique.Aptamer becomes a new generation for the molecular medicine of specific protein or targeting vector due to the chemical antibody characteristic of its uniqueness, and for detecting the target molecule material of its specific recognition.But the glycosylated hemoglobin GHb diagnostic reagent that is directed at present based on aptamer also lacks very much, and the exploitation being directed to the nucleic acid aptamer fluorescence probe detection kit of glycosylated hemoglobin GHb there is not yet report.
Summary of the invention
The object of the invention is for existing easy generation operative technique error, repeatability is not good enough, disturbing factor a lot, the high deficiency of complex operation, testing cost, provides a kind of glycosylated hemoglobin GHb kit based on nucleic acid aptamer fluorescence probe and detection method thereof.
The solution of the present invention is by realizing like this:
Based on a glycosylated hemoglobin kit for nucleic acid aptamer fluorescence probe, it is characterized in that, principal ingredient comprises: containing NH
4cl, Tris, EDTA-Na
2erythrocyte cracked liquid; Containing MgCl
20.2M phosphate buffer; Glycosylated hemoglobin standard items; Glycosylated hemoglobin nucleic acid aptamer fluorescence probe; Described glycosylated hemoglobin nucleic acid aptamer fluorescence 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 any sequence dna fragment in SEQ ID NO:1 ~ SEQ ID NO:4, and its sequence is:
SEQ ID NO:1：gggaggcatg tgtgctaagg cgtagcaggg ttggaaccc；
SEQ ID NO:2：gggaggcatg tgatttaagg cgcggcagat gttggaaccc；
SEQ ID NO:3：gggaggcatg agacctatgg cgttgcattg gttggaaccc；
SEQ ID NO:4：gggaggcatg ggaaacaatg cgccgcagag ttggaaccc。
Above-described a kind of glycosylated hemoglobin kit based on nucleic acid aptamer fluorescence probe, described containing NH
4cl, Tris, EDTA-Na
2erythrocyte cracked liquid for containing 1 ~ 280mmol/L NH
4cl, 1 ~ 34mmol/LTris, 1 ~ 2mmol/LEDTA-Na
2, pH7.0 ~ 7.2; Described containing MgCl
20.2M phosphate buffer for containing 1 ~ 10mmol/L MgCl
2.
Arbitrary described a kind of glycosylated hemoglobin kit based on nucleic acid aptamer fluorescence probe, is characterized in that above, described containing NH
4cl, Tris, EDTA-Na
2erythrocyte cracked liquid, containing MgCl
20.2M phosphate buffer, glycosylated hemoglobin standard items, glycosylated hemoglobin nucleic acid aptamer fluorescence probe be the dry powder preparing the liquid reagent that directly uses or need before using to be dissolved in water.
Above-described a kind of glycosylated hemoglobin kit based on nucleic acid aptamer fluorescence probe, is characterized in that, this kit is for detecting the glycosylated hemoglobin concentration in anticoagulant heparin whole blood or peripheral blood.
Detect a method for glycosylated hemoglobin concentration in order to upper arbitrary described glycosylated hemoglobin kit based on nucleic acid aptamer fluorescence probe, it is characterized in that, method step comprises:
(1) blood sample cracking: by blood sample with containing NH
4cl, Tris, EDTA-Na
2erythrocyte cracked liquid press the mixing of 1:0.5 ~ 5 volume ratios, leave standstill 5 ~ 30min, then medium-speed centrifuge 5 ~ 10min, collect supernatant;
(2) mix ovum to educate: mixing ovum is educated: get the use of supernatant that 20 ~ 100 μ l step 1) obtain and 30 ~ 50ul containing MgCl
20.2M phosphate buffer, dissolve the glycosylated hemoglobin nucleic acid aptamer fluorescence probe reagent mixing that the fit fluorescence probe of saccharification haemoglobin nucleic acid obtains, under room temperature, ovum educates 5 ~ 15min, glycosylated hemoglobin nucleic acid aptamer fluorescence probe is fully combined with blood sample, obtains test fluid;
(3) fluoroscopic examination: fluorescence detector detecting step 2) the test fluid 50ul that obtains, after fluorescence detector fluorescence excitation, to read after fluorescence excitation in 20 to the 100ns time period wavelength at the fluorescent value of 480 ~ 500nm;
(4) result calculates: use biomedical mapping software, with reference to the typical curve that glycosylated hemoglobin standard model makes, integrating step 3) in the fluorescent value that detects calculate the concentration of glycosylated hemoglobin in blood sample.
Above-described biomedical software is Sigma plot software, in buying on the market.
A kind of above-described method detecting glycosylated hemoglobin concentration with the glycosylated hemoglobin kit based on nucleic acid aptamer fluorescence probe, it is characterized in that, in described glycosylated hemoglobin nucleic acid aptamer fluorescence probe reagent, glycosylated hemoglobin nucleic acid aptamer fluorescence probe concentration is 200 ~ 400nmol/L, its characteristic is also, described glycosylated hemoglobin nucleic acid aptamer fluorescence probe is the nucleotide single-chain of 5' and 3' two ends difference mark fluorescent pyrene molecule monomer, this nucleotide single-chain sequence is any sequence dna fragment in SEQ IDNO:1 ~ SEQ ID NO:4, its sequence is:
SEQ ID NO:1：gggaggcatg tgtgctaagg cgtagcaggg ttggaaccc；
SEQ ID NO:2：gggaggcatg tgatttaagg cgcggcagat gttggaaccc；
SEQ ID NO:3：gggaggcatg agacctatgg cgttgcattg gttggaaccc；
SEQ ID NO:4：gggaggcatg ggaaacaatg cgccgcagag ttggaaccc；
Glycosylated hemoglobin nucleic acid aptamer fluorescence probe not with glycosylated hemoglobin in conjunction with time, aptamer is in more open structure, and the pyrene molecule monomer at 5' and 3' two ends dissociates mutually, and after fluorescence excitation, emission wavelength is between 370 ~ 400nm; Glycosylated hemoglobin nucleic acid aptamer fluorescence probe and glycosylated hemoglobin in conjunction with time, glycosylated hemoglobin induces its recurring structure to change, the pyrene molecule monomer at aptamer 5' and 3' two ends is close to each other, form excited state dimer, after fluorescence excitation, excited state dimer emission wavelength is between 480 to 500nm.
A kind of above-described method detecting glycosylated hemoglobin concentration with the glycosylated hemoglobin kit based on nucleic acid aptamer fluorescence probe, it is characterized in that, described blood sample is anticoagulant heparin whole blood or peripheral blood, described containing NH
4cl, Tris, EDTA-Na
2erythrocyte cracked liquid for containing 1 ~ 280mmol/L NH
4cl, 1 ~ 34mmol/L Tris, 1 ~ 2mmol/LEDTA-Na
2, pH7.0 ~ 7.2; Described containing MgCl
20.2M phosphate buffer for containing 1 ~ 10mmol/L MgCl
2; Described fluorescence detector is the fluorescence detector with time-resolved fluorometry.
A kind of above-described method detecting glycosylated hemoglobin concentration with the glycosylated hemoglobin kit based on nucleic acid aptamer fluorescence probe, is characterized in that, this detection method is for detecting the glycosylated hemoglobin concentration in anticoagulant heparin whole blood or peripheral blood.
Substantive distinguishing features of the present invention and marked improvement are:
(1) detect fast simple to operate, without the need to complex sample processing and be separated, aptamer probe is directly added the blood sample liquid after cracking, with the fluorescent value that just can detect 480 ~ 500nm place in the fluorospectrophotometer short time.
(2) this kit and detection method thereof have highly sensitive, testing result repeatability is high, sample detection error is between 0.01 ~ 0.1%, high specificity, glycosylated hemoglobin nucleic acid aptamer fluorescence probe not with glycosylated hemoglobin in conjunction with time, aptamer is in more open structure, and the pyrene molecule monomer at 5' and 3' two ends dissociates mutually, and after fluorescence excitation, emission wavelength is between 370 ~ 400nm; Its with glycosylated hemoglobin in conjunction with time, glycosylated hemoglobin induces it to change, and the pyrene molecule monomer at aptamer 5' and 3' two ends is close to each other, and form dimer, after fluorescence excitation, dimer emission wavelength is between 480 ~ 500nm.
(3) reagent used can make to prepare the liquid reagent that can directly use or the dry powder used after being dissolved in water before using, and detecting reagent can storage at normal temperature, convenient transportation.
Accompanying drawing explanation
Fig. 1. pyrene molecular structure.
Embodiment
Below in conjunction with table 1 and embodiment, the glycosylated hemoglobin GHb kit and the detection method thereof that the present invention is based on nucleic acid aptamer fluorescence probe are described.
Kit reagent in table 1. embodiment becomes to be grouped into
Embodiment 1
After agent formulations dissolving prepares needed for embodiment in table 11, be distributed into bottle, carry out freeze drying, make powdered reagent; Before using, add ultrapure water, use after redissolving.Each sample set 3 parallel, detecting step is as follows:
(1) blood sample cracking: by the 1:0.5 mixing by volume of anticoagulant heparin whole blood, erythrocyte cracked liquid, leave standstill 30min, then medium-speed centrifuge 7min, collect supernatant.
(2) mix ovum to educate: the glycosylated hemoglobin nucleic acid aptamer fluorescence probe reagent mixing obtained with the fit fluorescence probe of 0.2M phosphate buffer dissolving saccharification haemoglobin nucleic acid of getting the upper cleer and peaceful 45ul that 20 μ l step 1) obtain, under room temperature, ovum educates 5min, obtains test fluid.
(3) fluoroscopic examination: fluorescence detector detecting step 2) the test fluid 50ul that obtains, after fluorescence detector fluorescence excitation, to read after fluorescence excitation in 20 to the 100ns time period wavelength at the fluorescent value of 480 ~ 500nm.
(4) result calculates: use biomedical mapping software, with reference to the typical curve that glycosylated hemoglobin standard model makes, integrating step 3) in the fluorescent value that detects calculate the concentration of glycosylated hemoglobin in blood sample.
3 replicate determination errors of sample are 0.05 ± 0.01%.
Embodiment 2
After agent formulations dissolving prepares needed for embodiment in table 12, be distributed into bottle, carry out freeze drying, make powdered reagent; Before using, add ultrapure water, use after redissolving.Each sample set 3 parallel, detecting step is as follows:
(1) blood sample cracking: by the 1:2.5 mixing by volume of peripheral blood, erythrocyte cracked liquid, leave standstill 5min, then medium-speed centrifuge 6min, collect supernatant.
(2) mix ovum to educate: the glycosylated hemoglobin nucleic acid aptamer fluorescence probe reagent mixing obtained with the fit fluorescence probe of 0.2M phosphate buffer dissolving saccharification haemoglobin nucleic acid of getting the upper cleer and peaceful 30ul that 50 μ l step 1) obtain, under room temperature, ovum educates 6min, obtains test fluid.
(3) fluoroscopic examination: fluorescence detector detecting step 2) the test fluid 50ul that obtains, after fluorescence detector fluorescence excitation, to read after fluorescence excitation in 20 to the 100ns time period wavelength at the fluorescent value of 480 ~ 500nm.
(4) result calculates: use biomedical mapping software, with reference to the typical curve that glycosylated hemoglobin standard model makes, integrating step 3) in the fluorescent value that detects calculate the concentration of glycosylated hemoglobin in blood sample.
3 replicate determination errors of sample are 0.02 ± 0.01%.
Embodiment 3
After agent formulations dissolving prepares needed for embodiment in table 13, be distributed into bottle, carry out freeze drying, make powdered reagent; Before using, add ultrapure water, use after redissolving.Each sample set 3 parallel, detecting step is as follows:
(1) blood sample cracking: by the 1:3.5 mixing by volume of peripheral blood, erythrocyte cracked liquid, leave standstill 10min, then medium-speed centrifuge 5min, collect supernatant.
(2) mix ovum to educate: the glycosylated hemoglobin nucleic acid aptamer fluorescence probe reagent mixing obtained with the fit fluorescence probe of 0.2M phosphate buffer dissolving saccharification haemoglobin nucleic acid of getting the upper cleer and peaceful 45ul that 75 μ l step 1) obtain, under room temperature, ovum educates 7min, obtains test fluid.
(3) fluoroscopic examination: fluorescence detector detecting step 2) the test fluid 50ul that obtains, after fluorescence detector fluorescence excitation, to read after fluorescence excitation in 20 to the 100ns time period wavelength at the fluorescent value of 480 ~ 500nm.
(4) result calculates: use biomedical mapping software, with reference to the typical curve that glycosylated hemoglobin standard model makes, integrating step 3) in the fluorescent value that detects calculate the concentration of glycosylated hemoglobin in blood sample.
3 replicate determination errors of sample are 0.04 ± 0.01%.
Embodiment 4
After agent formulations dissolving prepares needed for embodiment in table 14, be distributed into bottle, carry out freeze drying, make powdered reagent; Before using, add ultrapure water, use after redissolving.Each sample set 3 parallel, detecting step is as follows:
(1) blood sample cracking: by the 1:0.5 mixing by volume of anticoagulant heparin whole blood, erythrocyte cracked liquid, leave standstill 25min, then medium-speed centrifuge 8min, collect supernatant.
(2) mix ovum to educate: the glycosylated hemoglobin nucleic acid aptamer fluorescence probe reagent mixing obtained with the fit fluorescence probe of 0.2M phosphate buffer dissolving saccharification haemoglobin nucleic acid of getting the upper cleer and peaceful 50ul that 100 μ l step 1) obtain, under room temperature, ovum educates 8min, obtains test fluid.
(3) fluoroscopic examination: fluorescence detector detecting step 2) the test fluid 50ul that obtains, after fluorescence detector fluorescence excitation, to read after fluorescence excitation in 20 to the 100ns time period wavelength at the fluorescent value of 480 ~ 500nm.
(4) result calculates: use biomedical mapping software, with reference to the typical curve that glycosylated hemoglobin standard model makes, integrating step 3) in the fluorescent value that detects calculate the concentration of glycosylated hemoglobin in blood sample.
3 replicate determination errors of sample are 0.06 ± 0.01%.
Embodiment 5
After agent formulations dissolving prepares needed for embodiment in table 15, be distributed into bottle, carry out freeze drying, make powdered reagent; Before using, add ultrapure water, use after redissolving.Each sample set 3 parallel, detecting step is as follows:
(1) blood sample cracking: by the 1:1.0 mixing by volume of anticoagulant heparin whole blood, erythrocyte cracked liquid, leave standstill 20min, then medium-speed centrifuge 9min, collect supernatant.
(2) mix ovum to educate: the glycosylated hemoglobin nucleic acid aptamer fluorescence probe reagent mixing obtained with the fit fluorescence probe of 0.2M phosphate buffer dissolving saccharification haemoglobin nucleic acid of getting the upper cleer and peaceful 30ul that 80 μ l step 1) obtain, under room temperature, ovum educates 9min, obtains test fluid.
(3) fluoroscopic examination: fluorescence detector detecting step 2) the test fluid 50ul that obtains, after fluorescence detector fluorescence excitation, to read after fluorescence excitation in 20 to the 100ns time period wavelength at the fluorescent value of 480 ~ 500nm.
(4) result calculates: use biomedical mapping software, with reference to the typical curve that glycosylated hemoglobin standard model makes, integrating step 3) in the fluorescent value that detects calculate the concentration of glycosylated hemoglobin in blood sample.
3 replicate determination errors of sample are 0.07 ± 0.01%.
Embodiment 6
After agent formulations dissolving prepares needed for embodiment in table 16, be distributed into bottle, carry out freeze drying, make powdered reagent; Before using, add ultrapure water, use after redissolving.Each sample set 3 parallel, detecting step is as follows:
(1) blood sample cracking: by the 1:4.5 mixing by volume of anticoagulant heparin whole blood, erythrocyte cracked liquid, leave standstill 15min, then medium-speed centrifuge 10min, collect supernatant.
(2) mix ovum to educate: the glycosylated hemoglobin nucleic acid aptamer fluorescence probe reagent mixing obtained with the fit fluorescence probe of 0.2M phosphate buffer dissolving saccharification haemoglobin nucleic acid of getting the upper cleer and peaceful 40ul that 30 μ l step 1) obtain, under room temperature, ovum educates 10min, obtains test fluid.
(3) fluoroscopic examination: fluorescence detector detecting step 2) the test fluid 50ul that obtains, after fluorescence detector fluorescence excitation, to read after fluorescence excitation in 20 to the 100ns time period wavelength at the fluorescent value of 480 ~ 500nm.
(4) result calculates: use biomedical mapping software, with reference to the typical curve that glycosylated hemoglobin standard model makes, integrating step 3) in the fluorescent value that detects calculate the concentration of glycosylated hemoglobin in blood sample.
3 replicate determination errors of sample are 0.08 ± 0.01%.
Embodiment 7
After agent formulations dissolving prepares needed for embodiment in table 17, be distributed into bottle, carry out freeze drying, make powdered reagent; Before using, add ultrapure water, use after redissolving.Each sample set 3 parallel, detecting step is as follows:
(1) blood sample cracking: by the 1:3.0 mixing by volume of peripheral blood, erythrocyte cracked liquid, leave standstill 18min, then medium-speed centrifuge 8min, collect supernatant.
(2) mix ovum to educate: the glycosylated hemoglobin nucleic acid aptamer fluorescence probe reagent mixing obtained with the fit fluorescence probe of 0.2M phosphate buffer dissolving saccharification haemoglobin nucleic acid of getting the upper cleer and peaceful 20ul that 50 μ l step 1) obtain, under room temperature, ovum educates 12min, obtains test fluid.
(3) fluoroscopic examination: fluorescence detector detecting step 2) the test fluid 50ul that obtains, after fluorescence detector fluorescence excitation, to read after fluorescence excitation in 20 to the 100ns time period wavelength at the fluorescent value of 480 ~ 500nm.
(4) result calculates: use biomedical mapping software, with reference to the typical curve that glycosylated hemoglobin standard model makes, integrating step 3) in the fluorescent value that detects calculate the concentration of glycosylated hemoglobin in blood sample.
3 replicate determination errors of sample are 0.1 ± 0.01%.
Embodiment 8
After agent formulations dissolving prepares needed for embodiment in table 18, be distributed into bottle, carry out freeze drying, make powdered reagent; Before using, add ultrapure water, use after redissolving.Each sample set 3 parallel, detecting step is as follows:
(1) blood sample cracking: by the 1:2.0 mixing by volume of peripheral blood, erythrocyte cracked liquid, leave standstill 26min, then medium-speed centrifuge 6min, collect supernatant.
(2) mix ovum to educate: the glycosylated hemoglobin nucleic acid aptamer fluorescence probe reagent mixing obtained with the fit fluorescence probe of 0.2M phosphate buffer dissolving saccharification haemoglobin nucleic acid of getting the upper cleer and peaceful 25ul that 60 μ l step 1) obtain, under room temperature, ovum educates 13min, obtains test fluid.
(3) fluoroscopic examination: fluorescence detector detecting step 2) the test fluid 50ul that obtains, after fluorescence detector fluorescence excitation, to read after fluorescence excitation in 20 to the 100ns time period wavelength at the fluorescent value of 480 ~ 500nm.
(4) result calculates: use biomedical mapping software, with reference to the typical curve that glycosylated hemoglobin standard model makes, integrating step 3) in the fluorescent value that detects calculate the concentration of glycosylated hemoglobin in blood sample.
3 replicate determination errors of sample are 0.02 ± 0.01%.
Embodiment 9
After agent formulations dissolving prepares needed for embodiment in table 19, be distributed into bottle, carry out freeze drying, make powdered reagent; Before using, add ultrapure water, use after redissolving.Each sample set 3 parallel, detecting step is as follows:
(1) blood sample cracking: by the 1:4.0 mixing by volume of peripheral blood, erythrocyte cracked liquid, leave standstill 8min, then medium-speed centrifuge 7min, collect supernatant.
(2) mix ovum to educate: the glycosylated hemoglobin nucleic acid aptamer fluorescence probe reagent mixing obtained with the fit fluorescence probe of 0.2M phosphate buffer dissolving saccharification haemoglobin nucleic acid of getting the upper cleer and peaceful 35ul that 40 μ l step 1) obtain, under room temperature, ovum educates 15min, obtains test fluid.
(3) fluoroscopic examination: fluorescence detector detecting step 2) the test fluid 50ul that obtains, after fluorescence detector fluorescence excitation, to read after fluorescence excitation in 20 to the 100ns time period wavelength at the fluorescent value of 480 ~ 500nm.
(4) result calculates: use biomedical mapping software, with reference to the typical curve that glycosylated hemoglobin standard model makes, integrating step 3) in the fluorescent value that detects calculate the concentration of glycosylated hemoglobin in blood sample.
3 replicate determination errors of sample are 0.01 ± 0.01%.
Sequence table
Glad bio tech ltd, Anren, <110> Guangxi
<120> is based on the glycosylated hemoglobin kit of nucleic acid aptamer fluorescence probe and detection method thereof
<130>2012
<160>4
<170>PatentIn version 3.3
<210>1
<211>39
<212>DNA
<213> artificial sequence
<400>1
gggaggcatg tgtgctaagg cgtagcaggg ttggaaccc 39
<210>2
<211>40
<212>DNA
<213> artificial sequence
<400>2
gggaggcatg tgatttaagg cgcggcagat gttggaaccc 40
<210>3
<211>40
<212>DNA
<213> artificial sequence
<400>3
gggaggcatg agacctatgg cgttgcattg gttggaaccc 40
<210>4
<211>39
<212>DNA
<213> artificial sequence
<400>4
gggaggcatg ggaaacaatg cgccgcagag ttggaaccc 39
Claims (9)
1., based on a glycosylated hemoglobin kit for nucleic acid aptamer fluorescence probe, it is characterized in that, principal ingredient comprises: containing NH
4cl, Tris, EDTA-Na
2erythrocyte cracked liquid; Containing MgCl
20.2M phosphate buffer; Glycosylated hemoglobin standard items; Glycosylated hemoglobin nucleic acid aptamer fluorescence probe; Described glycosylated hemoglobin nucleic acid aptamer fluorescence probe is the nucleotide single-chain of 5' and 3' two ends difference mark fluorescent pyrene molecule monomer, this nucleotide single-chain sequence is any sequence dna fragment in SEQ ID NO:1 ~ SEQ ID NO:4, and its sequence is SEQ ID NO:1:gggaggcatg tgtgctaagg cgtagcaggg ttggaaccc.
2. a kind of glycosylated hemoglobin kit based on nucleic acid aptamer fluorescence probe according to claim 1, is characterized in that, described containing NH
4cl, Tris, EDTA-Na
2erythrocyte cracked liquid for containing 1 ~ 280mmol/L NH
4cl, 1 ~ 34mmol/L Tris, 1 ~ 2mmol/LEDTA-Na
2, pH 7.0 ~ 7.2; Described containing MgCl
20.2M phosphate buffer for containing 1 ~ 10mmol/L MgCl
2.
3. a kind of glycosylated hemoglobin kit based on nucleic acid aptamer fluorescence probe according to claim 1, is characterized in that, described containing NH
4cl, Tris, EDTA-Na
2erythrocyte cracked liquid, containing MgCl
20.2M phosphate buffer, glycosylated hemoglobin standard items, glycosylated hemoglobin nucleic acid aptamer fluorescence probe be the dry powder preparing the liquid reagent that directly uses or need before using to be dissolved in water.
4. a kind of glycosylated hemoglobin kit based on nucleic acid aptamer fluorescence probe according to any one of claims 1 to 3, is characterized in that, this kit is for detecting the glycosylated hemoglobin concentration in anticoagulant heparin whole blood or peripheral blood.
5. detect a method for glycosylated hemoglobin concentration with the glycosylated hemoglobin kit based on nucleic acid aptamer fluorescence probe described in any one of claims 1 to 3, it is characterized in that, method step comprises:
(1) blood sample cracking: by blood sample with containing NH
4cl, Tris, EDTA-Na
2erythrocyte cracked liquid press the mixing of 1:0.5 ~ 5 volume ratios, leave standstill 5 ~ 30min, then medium-speed centrifuge 5 ~ 10min, collect supernatant;
(2) mixing ovum to educate: get 20 ~ 100 μ l steps 1) use of the supernatant that obtains and 30 ~ 50ul is containing MgCl
20.2M phosphate buffer, dissolve the glycosylated hemoglobin nucleic acid aptamer fluorescence probe reagent mixing that the fit fluorescence probe of saccharification haemoglobin nucleic acid obtains, under room temperature, ovum educates 5 ~ 15min, glycosylated hemoglobin nucleic acid aptamer fluorescence probe is fully combined with blood sample, obtains test fluid;
(3) fluoroscopic examination: fluorescence detector detecting step 2) the test fluid 50ul that obtains, after fluorescence detector fluorescence excitation, to read after fluorescence excitation in 20 to the 100ns time period wavelength at the fluorescent value of 480 ~ 500nm;
(4) result calculates: use biomedical mapping software, with reference to the typical curve that glycosylated hemoglobin standard model makes, integrating step 3) in the fluorescent value that detects calculate the concentration of glycosylated hemoglobin in blood sample.
6. a kind of method detecting glycosylated hemoglobin concentration with the glycosylated hemoglobin kit based on nucleic acid aptamer fluorescence probe according to claim 5, it is characterized in that, in described glycosylated hemoglobin nucleic acid aptamer fluorescence probe reagent, glycosylated hemoglobin nucleic acid aptamer fluorescence probe concentration is 200 ~ 400nmol/L, described glycosylated hemoglobin nucleic acid aptamer fluorescence probe is the nucleotide single-chain of 5' and 3' two ends difference mark fluorescent pyrene molecule monomer, this nucleotide single-chain sequence is any sequence dna fragment in SEQ ID NO:1 ~ SEQ ID NO:4, its sequence is: SEQ ID NO:1:gggaggcatg tgtgctaagg cgtagcaggg ttggaaccc.
7. a kind of method detecting glycosylated hemoglobin concentration with the glycosylated hemoglobin kit based on nucleic acid aptamer fluorescence probe according to claim 6, it is characterized in that, described glycosylated hemoglobin nucleic acid aptamer fluorescence probe not with glycosylated hemoglobin in conjunction with time, aptamer is in more open structure, the pyrene molecule monomer at 5' and 3' two ends dissociates mutually, and after fluorescence excitation, emission wavelength is between 370 ~ 400nm; Described glycosylated hemoglobin nucleic acid aptamer fluorescence probe and glycosylated hemoglobin in conjunction with time, glycosylated hemoglobin induces its recurring structure to change, the pyrene molecule monomer at aptamer 5' and 3' two ends is close to each other, form excited state dimer, after fluorescence excitation, excited state dimer emission wavelength is between 480 to 500nm.
8. a kind of method detecting glycosylated hemoglobin concentration with the glycosylated hemoglobin kit based on nucleic acid aptamer fluorescence probe according to claim 5, it is characterized in that, described blood sample is anticoagulant heparin whole blood or peripheral blood, described containing NH
4cl, Tris, EDTA-Na
2erythrocyte cracked liquid for containing 1 ~ 280mmol/L NH
4cl, 1 ~ 34mmol/L Tris, 1 ~ 2mmol/LEDTA-Na
2, pH 7.0 ~ 7.2; Described containing MgCl
20.2M phosphate buffer for containing 1 ~ 10mmol/L MgCl
2; Described fluorescence detector is the fluorescence detector with time-resolved fluorometry.
9. a kind of method detecting glycosylated hemoglobin concentration with the glycosylated hemoglobin kit based on nucleic acid aptamer fluorescence probe according to any one of claim 5 ~ 7, it is characterized in that, this detection method is for detecting the glycosylated hemoglobin concentration in anticoagulant heparin whole blood or peripheral blood.
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