CN102998289A - 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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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 glycosylated hemoglobin GHb kit and detection method thereof based on the aptamer fluorescence probe.
Background technology
Diabetes are worldwide public health problems of human health, and diabetes are a kind of lifelong property diseases, and its complication is to residual main cause till death, so people wish to find as early as possible and to treat diabetes.Fasting blood-glucose, postprandial blood sugar and oral glucose tolerance test etc. are adopted in traditional diabetes diagnosis and treatment monitoring, the moment blood sugar level when but the blood sugar parameter only represents blood drawing, and glycosylated hemoglobin (GHb) also is the important indicator of monitoring treating diabetes as the goldstandard of the long-term blood sugar level of reflection.GHb be in the red blood cell haemoglobin and glucose slowly, continue and irreversibly carry out the product of non-enzymatic albumen saccharification react.Be difficult for separately after forming for two weeks.When the concentration of glucose in blood was higher, the formed GHb content of human body also can be relatively high. and under the normal physiological conditions, the growing amount of non-enzymatic saccharification react product is directly proportional with the concentration of reactant.Because protein concentration keeps relative stability, the saccharification level depends mainly on concentration of glucose, and the time length that also contacts with glucose with protein is relevant.GHb can reflect nearest 2-3 months average blood sugar level.GHb should regularly detect in ADA in 2002 clear, and with its gold mark as monitoring diabetes glycemic control.Popularize Diabetes Mellitus Knowledge, upgrade the treatment theory, monitoring also keeps GHb up to standard, more early, more reasonably uses the drug therapies such as insulin, and is particularly important for the genesis of control diabetic complication.
The detection method of glycosylated hemoglobin GHb commonly used has following several types at present:
High pressure liquid phase method (HPLC): the automatically variant of separation determination GHb and haemproteins and hypotype, but the operation of instrument maintenance has relatively high expectations in the CV value 1%, are difficult in hospital and the laboratory of basic unit relatively universal; Microtrabeculae method manual steps is loaded down with trivial details, and the quality of chromatography time and microtrabeculae is wayward, easily produces the 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.) disturb the result, and the degree of disturbing is decided according to the separating power of post, and examine collection of illustrative plates, so this method can not get generally adopting.
Immunization method: immunization method is to adopt at present many detection meanss, mainly comprises following several detection mode.
(1) turbidimetry: utilize the principle of antigen, antibody response to measure.The B chain N end of GHb provides one easily by the epitope of antibody recognition, with the epitope of terminal last 4～6 amino acid composition of the B chain N of monoclonal antibody specific recognition GHb, in conjunction with turbidimetry for Determination.
(2) Ion capture: use the 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 the fluorescence intensity rate of change, calculate GHb concentration.
(3) latex agglutination method: utilize antigen-antibody reaction directly to measure the method for the percentage composition of HbAlc among total lib.Total Hb has identical non-specific adsorption and immobilization with GHb with latex in the sample, behind the monoclonal antibody specific that adds HbAlc, form the compound of latex one GHb. mouse-anti people GHb monoclonal antibody, this compound is owing to sheep anti-mouse antibody forms aggegation, and the aggegation amount is different because of the difference of the immobilised GHb amount in latex surface.By measuring its absorbance and can obtaining more afterwards the percentage composition of the shared Hb of GHb in the sample with the typical curve of GHb percentage concentration.Immunization is measured its accuracy of glycosylated hemoglobin and repeatability all is weak, and measures the interference that is subjected to the materials such as high concentration glucose, high triglyceride, high cholerythrin, because the turbidity of 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 the shortcoming such as deposits.
Affinity chromatography: utilize the affine test of boronation as detecting principle.The reagent energy lysed erythrocyte of this method and the specificity precipitation haemoglobin factor, wherein blue boric acid conjugate energy and the Cis-diols on the glycosylated hemoglobin combine.Blood adds in the reagent, and red blood cell dissolves immediately, and whole haemoglobins is all precipitated, the boric acid conjugate just and the cis-diol structure among the GHb combine.Add filter membrane device after blood and the reagent mix, the haemoglobin of all precipitations is no matter be and the top that all the is retained in filter membrane conjugate combination or unconjugated.Excessive colour developing conjugate just is washed liquid and washes off.Measure the intensity of blue (glycosylated hemoglobin) and redness (total hemoglobin) by golden scalar quantity instrument, the number percent of the GHb in can calculation sample.
Aptamer is the novel identification molecule of a class that developed recently gets up, and is subject in recent years scientist's extensive concern, and is a large amount of out screened for the aptamer of important physiologically active molecule; Various analytical approach and technology based on aptamer are in the news; Aptamer medicine " Macugen " was also gone on the market by the FDA official approval in 2005.The oligonucleotide sequence that the SELEX technology screening obtains is called as aptamer, and domestic its is translated as aptamer, nucleic acid aptamer, aptamer or aptamer etc.The SELEX technology refers to that the applied chemistry method synthesizes jumbo random oligonucleotide (being comprised of the fixed sequence program at two ends and middle random series) 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 too much circulation selective enrichment of wheel, the oligonucleotide molecules that acquisition is combined with target material high special, can be that RNA also can be DNA, length be generally 25 ~ 60 nucleotide.
Pyrene is a kind of flammable faint yellow
Monoclinic crystalOrganic compound, the following Fig. 1 of its molecular structure, water insoluble, be soluble in ethanol, ether.Can carry out
Parent's electricity replaces, such as reactions such as halogenation, nitrated, sulfonation.Pyrene mainly is present in
Coal tarIn the distillation of pitch.Pyrene is the organic synthesis raw material, can produce Isosorbide-5-Nitrae through oxidation, and 5,8-naphthalenetetracarbacidic acidic is used for
Dyestuff, synthetic resin, disperse dyes and engineering plastics; Can the gorgeous orange GR of reducing dye processed and other multiple dyestuffs after the acidylate; Also can make
Pesticide, plastifier etc.; The pyrene molecule can be used as fluorescence probe for the detection method based on aptamer at present.
The fluorescent dye pyrene can form excited state dimer (* Py+Py) in the time of an excited state molecule (* Py) and another ground state molecule (Py) close encounters, 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 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 the excited state dimer and have strict Range-dependent, be conducive to the 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 an end of molecular beacon as fluorophor, the other end connects the fluorescent quenching group, the combination of aptamer and measured target causes that its space structure changes, the pyrene molecule that makes two ends near and form the excited state dimer, by detecting fluorescence intensity and the fluorescence lifetime of aptamer probe, thereby realized the highly sensitive detection to measured target.
Aptamer is combined hypersensitivity and the high specific that presents with the target material, it is had a good application prospect in medical diagnosis on disease, although ripe clinical practice report is less at present, but the research of using fit detection target protein is on the increase, and also constantly occurs based on fit detection new technology.Aptamer is because the chemical antibody characteristic of its uniqueness becomes a new generation for molecular medicine or the targeting vector of specific protein, and for detection of the target molecule material of its specific recognition.But at present the glycosylated hemoglobin GHb diagnostic reagent that is directed to based on aptamer also lacks very much, and the exploitation that is directed to the aptamer fluorescence probe detection kit of glycosylated hemoglobin GHb there is not yet report.
Summary of the invention
The objective of the invention is, complex operation a lot of for existing easy generation operative technique error, not good enough, the disturbing factor of repeatability, the high deficiency of testing cost, a kind of glycosylated hemoglobin GHb kit and detection method thereof based on the aptamer fluorescence probe is provided.
The solution of the present invention is by such realization:
A kind of glycosylated hemoglobin kit based on the aptamer fluorescence probe is characterized in that principal ingredient comprises: contain NH
4Cl, Tris, EDTA-Na
2Erythrocyte cracked liquid; Contain MgCl
2The 0.2M phosphate buffer; The glycosylated hemoglobin standard items; Glycosylated hemoglobin aptamer fluorescence probe; Described glycosylated hemoglobin 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 among 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 the aptamer fluorescence probe, the described NH that contains
4Cl, Tris, EDTA-Na
2Erythrocyte cracked liquid for containing 1 ~ 280 mmol/L NH
4Cl, 1 ~ 34 mmol/L Tris, 1 ~ 2mmol/LEDTA-Na
2, pH 7.0 ~ 7.2; The described MgCl that contains
2The 0.2M phosphate buffer for containing 1 ~ 10mmol/L MgCl
2
Above arbitrary described a kind of glycosylated hemoglobin kit based on the aptamer fluorescence probe is characterized in that the described NH that contains
4Cl, Tris, EDTA-Na
2Erythrocyte cracked liquid, contain MgCl
20.2M phosphate buffer, glycosylated hemoglobin standard items, glycosylated hemoglobin aptamer fluorescence probe be prepare the liquid reagent of direct use or use before the dry powder that need be dissolved in water.
Above-described a kind of glycosylated hemoglobin kit based on the aptamer fluorescence probe is characterized in that this kit is for detection of the glycosylated hemoglobin concentration in anticoagulant heparin whole blood or the peripheral blood.
A kind ofly it is characterized in that in order to upper arbitrary described method that detects glycosylated hemoglobin concentration based on the glycosylated hemoglobin kit of aptamer fluorescence probe, method step comprises:
(1) blood sample cracking: with blood sample with contain NH
4Cl, Tris, EDTA-Na
2Erythrocyte cracked liquid press 1:0.5 ~ 5 volume ratio mixings, leave standstill 5 ~ 30min, the centrifugal 5 ~ 10min of middling speed again collects supernatant;
(2) mixing ovum educates: mix ovum and educate: get supernatant that 20 ~ 100 μ l step 1) obtain and the usefulness of 30 ~ 50ul and contain MgCl
2The 0.2M phosphate buffer, the glycosylated hemoglobin aptamer fluorescence probe reagent mix that dissolving glycosylated hemoglobin aptamer fluorescence probe obtains, ovum is educated 5 ~ 15min under the room temperature, makes the abundant combination of glycosylated hemoglobin aptamer fluorescence probe and blood sample, obtains test fluid;
(3) fluoroscopic examination: the test fluid 50ul that fluorescence detector detecting step 2) obtains, behind the fluorescence detector fluorescence excitation, read behind the fluorescence excitation in 20 to 100ns time periods wavelength at the fluorescent value of 480 ~ 500nm;
(4) fluorescent value that detects result's calculating: use biomedical mapping software, with reference to the typical curve that the glycosylated hemoglobin standard model is made, integrating step 3) calculates the concentration of glycosylated hemoglobin in the blood sample.
Above-described biomedical software is Sigma plot software, in buying on the market.
Above-described a kind of method that detects glycosylated hemoglobin concentration based on the glycosylated hemoglobin kit of aptamer fluorescence probe of using, it is characterized in that, glycosylated hemoglobin aptamer fluorescence probe concentration is 200 ~ 400 nmol/L in the described glycosylated hemoglobin aptamer fluorescence probe reagent, its characteristic also is, described glycosylated hemoglobin 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 among 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；
Glycosylated hemoglobin aptamer fluorescence probe is not when glycosylated hemoglobin is combined, and aptamer is in more open structure, and the pyrene molecule monomer at 5' and 3' two ends is mutually free, and emission wavelength is between 370 ~ 400nm behind the fluorescence excitation; Glycosylated hemoglobin aptamer fluorescence probe is when glycosylated hemoglobin is combined, glycosylated hemoglobin induces its recurring structure to change, the pyrene molecule monomer at aptamer 5' and 3' two ends is mutually close, form the excited state dimer, excited state dimer emission wavelength is between 480 to 500nm behind the fluorescence excitation.
Above-described a kind of method that detects glycosylated hemoglobin concentration based on the glycosylated hemoglobin kit of aptamer fluorescence probe of using is characterized in that described blood sample is anticoagulant heparin whole blood or peripheral blood, the described NH that contains
4Cl, Tris, EDTA-Na
2Erythrocyte cracked liquid for containing 1 ~ 280 mmol/L NH
4Cl, 1 ~ 34 mmol/L Tris, 1 ~ 2mmol/LEDTA-Na
2, pH 7.0 ~ 7.2; The described MgCl that contains
2The 0.2M phosphate buffer for containing 1 ~ 10mmol/L MgCl
2Described fluorescence detector is the fluorescence detector with time-resolved fluorometry.
Above-described a kind of method that detects glycosylated hemoglobin concentration based on the glycosylated hemoglobin kit of aptamer fluorescence probe of using is characterized in that this detection method is for detection of the glycosylated hemoglobin concentration in anticoagulant heparin whole blood or the peripheral blood.
Substantive distinguishing features of the present invention and marked improvement are:
(1) detects and simple to operately need not complex sample processing and separates fast, aptamer probe is directly added blood sample liquid after the cracking, with the interior fluorescent value that just can detect 480 ~ 500nm place of fluorospectrophotometer short time;
(2) this kit and detection method thereof have highly sensitive, testing result repeatability is high, the sample detection error is between 0.01 ~ 0.1%, high specificity, glycosylated hemoglobin aptamer fluorescence probe is not when glycosylated hemoglobin is combined, aptamer is in more open structure, and the pyrene molecule monomer at 5' and 3' two ends is mutually free, and emission wavelength is between 370 ~ 400nm behind the fluorescence excitation; When it was combined with glycosylated hemoglobin, glycosylated hemoglobin induced it to change, and the pyrene molecule monomer at aptamer 5' and 3' two ends is mutually close, formed dimer, and the dimer emission wavelength is between 480 ~ 500nm behind the fluorescence excitation.
(3) used reagent can make prepare the liquid reagent that can directly use or use before the be dissolved in water dry powder of rear use, detecting reagent can storage at normal temperature, convenient transportation.
Description of drawings
Fig. 1. the pyrene molecular structure.
Embodiment
Below in conjunction with table 1 and embodiment glycosylated hemoglobin GHb kit and the detection method thereof that the present invention is based on the aptamer fluorescence probe described.
Kit reagent among table 1. embodiment becomes to be grouped into
Embodiment 1
After preparing according to the required reagent composition dissolving of embodiment in the table 11, be distributed into bottle, carry out freeze drying, make powdered reagent; Before the use, add ultrapure water, use after redissolving.Each 3 of sample setting is parallel, and detecting step is as follows:
(1) blood sample cracking: with anticoagulant heparin whole blood, erythrocyte cracked liquid 1:0.5 mixing by volume, leave standstill 30min, the centrifugal 7min of middling speed collects supernatant again;
(2) mixing ovum educates: that gets upper cleer and peaceful 45ul that 20 μ l step 1) obtain dissolves the glycosylated hemoglobin aptamer fluorescence probe reagent mix that glycosylated hemoglobin aptamer fluorescence probe obtains with the 0.2M phosphate buffer, ovum is educated 5min under the room temperature, obtains test fluid;
(3) fluoroscopic examination: the test fluid 50ul that fluorescence detector detecting step 2) obtains, behind the fluorescence detector fluorescence excitation, read behind the fluorescence excitation in 20 to 100ns time periods wavelength at the fluorescent value of 480 ~ 500nm;
(4) fluorescent value that detects result's calculating: use biomedical mapping software, with reference to the typical curve that the glycosylated hemoglobin standard model is made, integrating step 3) calculates the concentration of glycosylated hemoglobin in the blood sample.
3 replicate determination errors of sample are 0.05 ± 0.01%.
Embodiment 2
After preparing according to the required reagent compositions dissolving of embodiment in the table 12, be distributed into bottle, carry out freeze drying, make powdered reagent; Before the use, add ultrapure water, use after redissolving.Each 3 of sample setting is parallel, and detecting step is as follows:
(1) blood sample cracking: with peripheral blood, erythrocyte cracked liquid 1:2.5 mixing by volume, leave standstill 5min, the centrifugal 6min of middling speed collects supernatant again;
(2) mixing ovum educates: get upper cleer and peaceful 30ul's that 50 μ l step 1) obtain
With the glycosylated hemoglobin aptamer fluorescence probe reagent mix that 0.2M phosphate buffer dissolving glycosylated hemoglobin aptamer fluorescence probe obtains, ovum is educated 6min under the room temperature, obtains test fluid;
(3) fluoroscopic examination: the test fluid 50ul that fluorescence detector detecting step 2) obtains, behind the fluorescence detector fluorescence excitation, read behind the fluorescence excitation in 20 to 100ns time periods wavelength at the fluorescent value of 480 ~ 500nm;
(4) fluorescent value that detects result's calculating: use biomedical mapping software, with reference to the typical curve that the glycosylated hemoglobin standard model is made, integrating step 3) calculates the concentration of glycosylated hemoglobin in the blood sample.3 replicate determination errors of sample are 0.02 ± 0.01%.
Embodiment 3
After preparing according to the required reagent compositions dissolving of embodiment in the table 13, be distributed into bottle, carry out freeze drying, make powdered reagent; Before the use, add ultrapure water, use after redissolving.Each 3 of sample setting is parallel, and detecting step is as follows:
(1) blood sample cracking: with peripheral blood, erythrocyte cracked liquid 1:3.5 mixing by volume, leave standstill 10min, the centrifugal 5min of middling speed collects supernatant again;
(2) mixing ovum educates: get upper cleer and peaceful 45ul's that 75 μ l step 1) obtain
With the glycosylated hemoglobin aptamer fluorescence probe reagent mix that 0.2M phosphate buffer dissolving glycosylated hemoglobin aptamer fluorescence probe obtains, ovum is educated 7min under the room temperature, obtains test fluid;
(3) fluoroscopic examination: the test fluid 50ul that fluorescence detector detecting step 2) obtains, behind the fluorescence detector fluorescence excitation, read behind the fluorescence excitation in 20 to 100ns time periods wavelength at the fluorescent value of 480 ~ 500nm;
(4) fluorescent value that detects result's calculating: use biomedical mapping software, with reference to the typical curve that the glycosylated hemoglobin standard model is made, integrating step 3) calculates the concentration of glycosylated hemoglobin in the blood sample.
3 replicate determination errors of sample are 0.04 ± 0.01%.
Embodiment 4
After preparing according to the required reagent compositions dissolving of embodiment in the table 14, be distributed into bottle, carry out freeze drying, make powdered reagent; Before the use, add ultrapure water, use after redissolving.Each 3 of sample setting is parallel, and detecting step is as follows:
(1) blood sample cracking: with anticoagulant heparin whole blood, erythrocyte cracked liquid 1:0.5 mixing by volume, leave standstill 25min, the centrifugal 8min of middling speed collects supernatant again;
(2) mixing ovum educates: that gets upper cleer and peaceful 50ul that 100 μ l step 1) obtain dissolves the glycosylated hemoglobin aptamer fluorescence probe reagent mix that glycosylated hemoglobin aptamer fluorescence probe obtains with the 0.2M phosphate buffer, ovum is educated 8min under the room temperature, obtains test fluid;
(3) fluoroscopic examination: the test fluid 50ul that fluorescence detector detecting step 2) obtains, behind the fluorescence detector fluorescence excitation, read behind the fluorescence excitation in 20 to 100ns time periods wavelength at the fluorescent value of 480 ~ 500nm;
(4) fluorescent value that detects result's calculating: use biomedical mapping software, with reference to the typical curve that the glycosylated hemoglobin standard model is made, integrating step 3) calculates the concentration of glycosylated hemoglobin in the blood sample.
3 replicate determination errors of sample are 0.06 ± 0.01%.
Embodiment 5
After preparing according to the required reagent compositions dissolving of embodiment in the table 15, be distributed into bottle, carry out freeze drying, make powdered reagent; Before the use, add ultrapure water, use after redissolving.Each 3 of sample setting is parallel, and detecting step is as follows:
(1) blood sample cracking: with anticoagulant heparin whole blood, erythrocyte cracked liquid 1:1.0 mixing by volume, leave standstill 20min, the centrifugal 9min of middling speed collects supernatant again;
(2) mixing ovum educates: that gets upper cleer and peaceful 30ul that 80 μ l step 1) obtain dissolves the glycosylated hemoglobin aptamer fluorescence probe reagent mix that glycosylated hemoglobin aptamer fluorescence probe obtains with the 0.2M phosphate buffer, ovum is educated 9min under the room temperature, obtains test fluid;
(3) fluoroscopic examination: the test fluid 50ul that fluorescence detector detecting step 2) obtains, behind the fluorescence detector fluorescence excitation, read behind the fluorescence excitation in 20 to 100ns time periods wavelength at the fluorescent value of 480 ~ 500nm;
(4) fluorescent value that detects result's calculating: use biomedical mapping software, with reference to the typical curve that the glycosylated hemoglobin standard model is made, integrating step 3) calculates the concentration of glycosylated hemoglobin in the blood sample.
3 replicate determination errors of sample are 0.07 ± 0.01%.
Embodiment 6
After preparing according to the required reagent compositions dissolving of embodiment in the table 16, be distributed into bottle, carry out freeze drying, make powdered reagent; Before the use, add ultrapure water, use after redissolving.Each 3 of sample setting is parallel, and detecting step is as follows:
(1) blood sample cracking: with anticoagulant heparin whole blood, erythrocyte cracked liquid 1:4.5 mixing by volume, leave standstill 15min, the centrifugal 10min of middling speed collects supernatant again;
(2) mixing ovum educates: that gets upper cleer and peaceful 40ul that 30 μ l step 1) obtain dissolves the glycosylated hemoglobin aptamer fluorescence probe reagent mix that glycosylated hemoglobin aptamer fluorescence probe obtains with the 0.2M phosphate buffer, ovum is educated 10min under the room temperature, obtains test fluid;
(3) fluoroscopic examination: the test fluid 50ul that fluorescence detector detecting step 2) obtains, behind the fluorescence detector fluorescence excitation, read behind the fluorescence excitation in 20 to 100ns time periods wavelength at the fluorescent value of 480 ~ 500nm;
(4) fluorescent value that detects result's calculating: use biomedical mapping software, with reference to the typical curve that the glycosylated hemoglobin standard model is made, integrating step 3) calculates the concentration of glycosylated hemoglobin in the blood sample.
3 replicate determination errors of sample are 0.08 ± 0.01%.
Embodiment 7
After preparing according to the required reagent compositions dissolving of embodiment in the table 17, be distributed into bottle, carry out freeze drying, make powdered reagent; Before the use, add ultrapure water, use after redissolving.Each 3 of sample setting is parallel, and detecting step is as follows:
(1) blood sample cracking: with peripheral blood, erythrocyte cracked liquid 1:3.0 mixing by volume, leave standstill 18min, the centrifugal 8min of middling speed collects supernatant again;
(2) mixing ovum educates: that gets upper cleer and peaceful 20ul that 50 μ l step 1) obtain dissolves the glycosylated hemoglobin aptamer fluorescence probe reagent mix that glycosylated hemoglobin aptamer fluorescence probe obtains with the 0.2M phosphate buffer, ovum is educated 12min under the room temperature, obtains test fluid;
(3) fluoroscopic examination: the test fluid 50ul that fluorescence detector detecting step 2) obtains, behind the fluorescence detector fluorescence excitation, read behind the fluorescence excitation in 20 to 100ns time periods wavelength at the fluorescent value of 480 ~ 500nm;
(4) fluorescent value that detects result's calculating: use biomedical mapping software, with reference to the typical curve that the glycosylated hemoglobin standard model is made, integrating step 3) calculates the concentration of glycosylated hemoglobin in the blood sample.
3 replicate determination errors of sample are 0.1 ± 0.01%.
Embodiment 8
After preparing according to the required reagent compositions dissolving of embodiment in the table 18, be distributed into bottle, carry out freeze drying, make powdered reagent; Before the use, add ultrapure water, use after redissolving.Each 3 of sample setting is parallel, and detecting step is as follows:
(1) blood sample cracking: with peripheral blood, erythrocyte cracked liquid 1:2.0 mixing by volume, leave standstill 26min, the centrifugal 6min of middling speed collects supernatant again;
(2) mixing ovum educates: that gets upper cleer and peaceful 25ul that 60 μ l step 1) obtain dissolves the glycosylated hemoglobin aptamer fluorescence probe reagent mix that glycosylated hemoglobin aptamer fluorescence probe obtains with the 0.2M phosphate buffer, ovum is educated 13min under the room temperature, obtains test fluid;
(3) fluoroscopic examination: the test fluid 50ul that fluorescence detector detecting step 2) obtains, behind the fluorescence detector fluorescence excitation, read behind the fluorescence excitation in 20 to 100ns time periods wavelength at the fluorescent value of 480 ~ 500nm;
(4) fluorescent value that detects result's calculating: use biomedical mapping software, with reference to the typical curve that the glycosylated hemoglobin standard model is made, integrating step 3) calculates the concentration of glycosylated hemoglobin in the blood sample.
3 replicate determination errors of sample are 0.02 ± 0.01%.
Embodiment 9
After preparing according to the required reagent compositions dissolving of embodiment in the table 19, be distributed into bottle, carry out freeze drying, make powdered reagent; Before the use, add ultrapure water, use after redissolving.Each 3 of sample setting is parallel, and detecting step is as follows:
(1) blood sample cracking: with peripheral blood, erythrocyte cracked liquid 1:4.0 mixing by volume, leave standstill 8min, the centrifugal 7min of middling speed collects supernatant again;
(2) mixing ovum educates: that gets upper cleer and peaceful 35ul that 40 μ l step 1) obtain dissolves the glycosylated hemoglobin aptamer fluorescence probe reagent mix that glycosylated hemoglobin aptamer fluorescence probe obtains with the 0.2M phosphate buffer, ovum is educated 15min under the room temperature, obtains test fluid;
(3) fluoroscopic examination: the test fluid 50ul that fluorescence detector detecting step 2) obtains, behind the fluorescence detector fluorescence excitation, read behind the fluorescence excitation in 20 to 100ns time periods wavelength at the fluorescent value of 480 ~ 500nm;
(4) fluorescent value that detects result's calculating: use biomedical mapping software, with reference to the typical curve that the glycosylated hemoglobin standard model is made, integrating step 3) calculates the concentration of glycosylated hemoglobin in the blood sample.
3 replicate determination errors of sample are 0.01 ± 0.01%.
Sequence table
＜110〉the glad bio tech ltd in Anren, Guangxi
＜120〉based on glycosylated hemoglobin kit and the detection method thereof of aptamer fluorescence probe
<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. the glycosylated hemoglobin kit based on the aptamer fluorescence probe is characterized in that, principal ingredient comprises: contain NH
4Cl, Tris, EDTA-Na
2Erythrocyte cracked liquid; Contain MgCl
2The 0.2M phosphate buffer; The glycosylated hemoglobin standard items; Glycosylated hemoglobin aptamer fluorescence probe; Described glycosylated hemoglobin 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 among 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。
2. a kind of glycosylated hemoglobin kit based on the aptamer fluorescence probe according to claim 1 is characterized in that the described NH that contains
4Cl, Tris, EDTA-Na
2Erythrocyte cracked liquid for containing 1～280 mmol/L NH
4Cl, 1 ~ 34 mmol/L Tris, 1 ~ 2mmol/LEDTA-Na
2, pH 7.0 ~ 7.2; The described MgCl that contains
2The 0.2M phosphate buffer for containing 1 ~ 10mmol/L MgCl
2
3. a kind of glycosylated hemoglobin kit based on the aptamer fluorescence probe according to claim 1 and 2 is characterized in that the described NH that contains
4Cl, Tris, EDTA-Na
2Erythrocyte cracked liquid, contain MgCl
20.2M phosphate buffer, glycosylated hemoglobin standard items, glycosylated hemoglobin aptamer fluorescence probe be prepare the liquid reagent of direct use or use before the dry powder that need be dissolved in water.
4. described a kind of glycosylated hemoglobin kit based on the aptamer fluorescence probe is characterized in that according to claim 1 ~ 3, and this kit is for detection of the glycosylated hemoglobin concentration in anticoagulant heparin whole blood or the peripheral blood.
5. one kind with right 1 ~ 3 arbitrary described method that detects glycosylated hemoglobin concentration based on the glycosylated hemoglobin kit of aptamer fluorescence probe, it is characterized in that, method step comprises:
(1) blood sample cracking: with blood sample with contain NH
4Cl, Tris, EDTA-Na
2Erythrocyte cracked liquid press 1:0.5 ~ 5 volume ratio mixings, leave standstill 5 ~ 30min, the centrifugal 5 ~ 10min of middling speed again collects supernatant;
(2) mixing ovum educates: get supernatant that 20 ~ 100 μ l step 1) obtain and the usefulness of 30 ~ 50ul and contain MgCl
2The 0.2M phosphate buffer, the glycosylated hemoglobin aptamer fluorescence probe reagent mix that dissolving glycosylated hemoglobin aptamer fluorescence probe obtains, ovum is educated 5 ~ 15min under the room temperature, makes the abundant combination of glycosylated hemoglobin aptamer fluorescence probe and blood sample, obtains test fluid;
(3) fluoroscopic examination: the test fluid 50ul that fluorescence detector detecting step 2) obtains, behind the fluorescence detector fluorescence excitation, read behind the fluorescence excitation in 20 to 100ns time periods wavelength at the fluorescent value of 480 ~ 500nm;
(4) fluorescent value that detects result's calculating: use biomedical mapping software, with reference to the typical curve that the glycosylated hemoglobin standard model is made, integrating step 3) calculates the concentration of glycosylated hemoglobin in the blood sample.
6. a kind of method that detects glycosylated hemoglobin concentration based on the glycosylated hemoglobin kit of aptamer fluorescence probe of using according to claim 5, it is characterized in that, glycosylated hemoglobin aptamer fluorescence probe concentration is 200 ~ 400 nmol/L in the described glycosylated hemoglobin aptamer fluorescence probe reagent, described glycosylated hemoglobin 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 among 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。
7. a kind of method that detects glycosylated hemoglobin concentration based on the glycosylated hemoglobin kit of aptamer fluorescence probe of using according to claim 6, it is characterized in that, described glycosylated hemoglobin aptamer fluorescence probe is not when glycosylated hemoglobin is combined, aptamer is in more open structure, the pyrene molecule monomer at 5' and 3' two ends is mutually free, and emission wavelength is between 370 ~ 400nm behind the fluorescence excitation; Described glycosylated hemoglobin aptamer fluorescence probe is when glycosylated hemoglobin is combined, glycosylated hemoglobin induces its recurring structure to change, the pyrene molecule monomer at aptamer 5' and 3' two ends is mutually close, form the excited state dimer, excited state dimer emission wavelength is between 480 to 500nm behind the fluorescence excitation.
8. a kind of method that detects glycosylated hemoglobin concentration based on the glycosylated hemoglobin kit of aptamer fluorescence probe of using according to claim 5 is characterized in that, described blood sample is anticoagulant heparin whole blood or peripheral blood, the described NH that contains
4Cl, Tris, EDTA-Na
2Erythrocyte cracked liquid for containing 1 ~ 280 mmol/L NH
4Cl, 1 ~ 34 mmol/L Tris, 1 ~ 2mmol/LEDTA-Na
2, pH 7.0 ~ 7.2; The described MgCl that contains
2The 0.2M phosphate buffer for containing 1 ~ 10mmol/L MgCl
2Described fluorescence detector is the fluorescence detector with time-resolved fluorometry.
9. described a kind of method that detects glycosylated hemoglobin concentration based on the glycosylated hemoglobin kit of aptamer fluorescence probe of using according to claim 5 ~ 7, it is characterized in that, this detection method is for detection of the glycosylated hemoglobin concentration in anticoagulant heparin whole blood or the peripheral blood.
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