CN109900910A - A nucleic acid aptamer fluorescent sensor for detecting myoglobin and its construction method - Google Patents
A nucleic acid aptamer fluorescent sensor for detecting myoglobin and its construction method Download PDFInfo
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Abstract
本发明公开了一种检测肌红蛋白的核酸适配体荧光传感器及其构建方法，属于分析检测技术领域。所述构建方法，包括：将修饰有淬灭基团的肌红蛋白核酸适配体和修饰有荧光基团的核酸适配体互补链加入缓冲体系中杂交，制得所述的核酸适配体荧光传感器。当加入肌红蛋白，核酸适配体与互补短链分离，与肌红蛋白结合，连有荧光基团的互补短链脱离连有淬灭基团的核酸适配体后，荧光基团的荧光得到恢复，其荧光恢复的强度与肌红蛋白的浓度成正比，由此可测定肌红蛋白的浓度。本发明采用价廉易制备的核酸适配体荧光传感器对肌红蛋白进行检测，可实现肌红蛋白的精准检测。该方法设计简单、成本低、检测步骤少，操作简便，灵敏度高，选择性好。
The invention discloses a nucleic acid aptamer fluorescence sensor for detecting myoglobin and a construction method thereof, belonging to the technical field of analysis and detection. The construction method includes: adding the myoglobin nucleic acid aptamer modified with a quenching group and the complementary chain of the nucleic acid aptamer modified with a fluorescent group into a buffer system for hybridization to prepare the nucleic acid aptamer Fluorescence sensor. When myoglobin is added, the nucleic acid aptamer is separated from the complementary short chain, binds to myoglobin, and the complementary short chain with the fluorescent group is separated from the nucleic acid aptamer with the quenching group, the fluorescence of the fluorescent group It is recovered, and the intensity of fluorescence recovery is proportional to the concentration of myoglobin, from which the concentration of myoglobin can be determined. The invention adopts the nucleic acid aptamer fluorescence sensor which is cheap and easy to prepare to detect the myoglobin, and can realize the accurate detection of the myoglobin. The method has the advantages of simple design, low cost, few detection steps, simple operation, high sensitivity and good selectivity.
Description
Technical field
The present invention relates to technical field of analysis and detection, and in particular to a kind of aptamer fluorescence biography for detecting myoglobins
Sensor and its construction method.
Background technique
Myoglobins is a kind of small molecular protein, and transhipment is played in myocyte and stores the function of oxygen.In normal human
In serum, the content of myoglobins is seldom, about 30-90ng/mL, and when myocardial damage, myoglobins can be rapidly from impaired thin
It is discharged into blood in born of the same parents, causes the content of myoglobins in blood significantly raised, 200-900ng/mL can be risen to, therefore can make
For the marker of early diagnosis of acute myocardial infarction.
Acute myocardial infarction AMI is clinically common cardiovascular disease.The disease be by myocardial ischemia, anoxic caused by, have
The features such as onset urgency, death rate height, seriously threaten the life security of patient.The key of the clinical treatment disease be as early as possible discovery,
It makes a definite diagnosis as early as possible, treated as soon as possible, the successful treatment rate of patient could be promoted in this way, improve prognosis.
Modern medicine study shows that serologic test has great significance in acute myocardial infarction AMI clinical diagnosis, to the heart
Flesh marker (such as myoglobins) is precisely detected, and can reflect conditions of patients, is early diagnosis, the painstaking effort of acute myocardial infarction AMI
The clinical Accurate Diagnosis of pipe disease, antidiastole and judge that therapeutic effect provides important evidence.Therefore clinically establish quickly,
Accurately and efficiently the detection method of Applications of Cardiac Markers is particularly important.
Currently, the method for detection myoglobins mainly has liquid chromatography, mass spectrography, Surface enhanced Raman scattering method, huge
Magnetoimpedance immunization, electro-chemistry immunity method, fluorescence method, surface plasmon resonance, enzyme-linked immunosorbent assay, colorimetric method
With chemoluminescence method etc., enzyme-linked immunosorbent assay be it is a kind of it is classical, be widely used in clinical method of immunity,
It is suitable for batch Samples detection, but this method influence factor is more, there are separation and washing step, and cumbersome, the measurement period is grown partially
The problems such as.Chemoluminescence method is although safe, sensitive, versatile, but this method instrument cost is relatively high.Although mass spectrography is clever
Sensitivity, specificity are higher, but instrument is more expensive, and at present since its database is still unsound, so also failing to widely be answered
With.In addition, the method for above-mentioned detection myoglobins is the detection architecture for being mutually distinguishable building based on antibody antigen mostly, it is excellent
Gesture is immunoassay selectivity with higher and sensitivity, but Antibody preparation is needed by immune zoopery or cell reality
It tests, it is cumbersome time-consuming, it is at high cost.And influence of the antibody vulnerable to external condition especially temperature, harshness is required to storage conditions, very
The flexible Application of these methods is limited in big degree.
Aptamer be obtained through external SELEX technology (the phyletic evolution technology of index concentration ligand) screening it is one small
Section single strand oligonucleotide acid sequence.It is a kind of microRNA or single stranded DNA with specific three dimensional conformation, and length is generally
20~80 bases, relative molecular mass are 6000~30000.Due to the diversity of aptamer structure and space conformation,
Some stable three can be formed by the pairing and electrostatic interaction of complementary bases certain in chain, hydrogen bond action and target molecule
Dimension space structure (such as hair fastener, false knot, bulge loop, G- tetrad), and it is in combination with high-affinity, thus can specific recognition
Small molecule, protein, bacterium, virus and cell.Compared with antibody, aptamer has high specific, high-affinity, nothing
(or low) immunogenicity, low cost, be easily-synthesized, easily modify, being fixed easily, target range is wide, can Reusability and long-term preservation
The advantages that.
Fluorescence analysis have many advantages, such as low detection limit, high sensitivity, quickly, instrument it is simple, by aptamer and glimmering
Both optical analysis combine, and can develop reliable, quick, highly selective, highly sensitive aptamer fluorescence analysis side
Method realizes the accurate, quick of myoglobins, efficient detection in buffer solution or even human serum.
Summary of the invention
Using aptamer fluorescent optical sensor myoglobins is detected the purpose of the present invention is to provide a kind of
Kit realizes the accurate detection to myoglobins, solves the problems, such as the early diagnosis of acute myocardial infarction AMI.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of construction method for the aptamer fluorescent optical sensor detecting myoglobins, comprising: base is quenched by being modified with
The myoglobins aptamer of group and the aptamer complementary strand for being modified with fluorophor, which are added in buffer system, to be hybridized, and is made
Obtain the aptamer fluorescent optical sensor.
The working principle for the fluorescent optical sensor detection myoglobins that the present invention constructs:
In buffer solution, the aptamer for being connected with quenching group and the complementary short-chain for being connected with fluorophor are carried out miscellaneous
It hands over, wherein fluorophor and quenching group contact with each other, and the fluorescence of fluorophor is quenched group and is quenched, which is
For the fluorescent optical sensor based on aptamer.
Certain density myoglobins is added in the above system, after a period of time, aptamer and complementary short-chain
Separation, with myoglobins ining conjunction with, be connected with fluorophor complementary short-chain be detached from be connected with the aptamer of quenching group after, fluorescence
The fluorescence of group is restored, as shown in Figure 1.The intensity that its fluorescence restores is directly proportional to the concentration of myoglobins, thus can survey
Determine the concentration of myoglobins.
The present invention research shows that: compared to the prior art amplifying nucleic acid aptamers connection fluorophor be quenched with complementary chain link
The fluorescent optical sensor of group building, using connecting quenching group in aptamer long-chain, connect on complementary short-chain in the present invention
It connects the fluorescent optical sensor fluorescent quenching significant effect that fluorophor mode constructs to rise, and subsequent fluorescence rear in conjunction with myoglobins
Recovery time significantly shortens, and is obviously improved the accuracy and sensitivity of testing result.
Preferably, the quenching group is 4- [4- (dimethylamino) phenylazo] benzoic acid (DABCYL), it is described
Fluorophor is 6- Fluoresceincarboxylic acid (FAM).
Preferably, the base sequence of the myoglobins aptamer is 5 '-CCCTCCTTTCCTTCGACGTAGA
TCTGCTGCGTTGTTCCGA-3 ', the base sequence of the aptamer complementary strand are 5 '-GGAACAACGC-3 ', 5 '-
TCGGAACAAC-3 ', 5 '-CGGAACAACG-3 ', any one in 5 '-GAACAACGCA-3 '.
The bases longs of amplifying nucleic acid aptamers complementary strand of the present invention are 10, if complementary short-chain bases longs are too long, such as
15-30, complementary short-chain is difficult to separate with aptamer, causes fluorescence recovery strength not high, and fluorescence recovery time is long, complementary
Short chain it is too short such as 5 hereinafter, if complementary short-chain be not sufficiently stable in conjunction with aptamer, can be easily separated.
Preferably, the buffer system is phosphate buffer solution.Specifically, using the PBS of the 0.01M of pH=7.4
Buffer solution.
Preferably, the concentration for being modified with the myoglobins aptamer of quenching group in hybridization system is 1-
100nmol/L, the aptamer complementary strand concentration for being modified with fluorophor is 1-100nmol/L.More preferably, nucleic acid is adapted to
The concentration of body and complementary strand is 25nmol/L.
Preferably, the condition of hybridization is to be incubated for 1-100min at 20-25 DEG C.More preferably, hybridization time is 20 minutes.
The present invention also provides the aptamer fluorescent optical sensors as made from the construction method.
The present invention also provides a kind of methods based on above-mentioned aptamer fluorescent optical sensor detection myoglobins, including
Following steps:
(1) standard curve is drawn:
In the aptamer fluorescent optical sensor, the myoglobins standard solution of concentration in gradient distribution is added
It is incubated for myoglobins final concentration in system in 0.1-5ng/mL, then measures respective fluorescence intensity level F, while measuring blank
The fluorescent value F of sample0, according to fluorescence intensity change value F-F0Standard curve is drawn with myoglobin concentration；
(2) unknown sample detects:
Sample to be tested solution is added in the aptamer fluorescent optical sensor, is incubated for, measures fluorescence intensity change
Value calculates myoglobin concentration in sample to be tested according to standard curve.
When drawing standard curve, the certain density myoglobins of addition in the above system, wherein in 0.1-
In the concentration range of 5ng/mL, the intensity that system fluorescence restores is directly proportional to the myoglobin concentration of addition.Equation of linear regression
Are as follows: y=79.4c+22.59, R2=0.991.Wherein y is fluorescence recovery value, and c is that the concentration ng/mL, R of myoglobins are correlation
Coefficient.
Preferably, the condition of the incubation is to be incubated for 1-100min at 20-25 DEG C.More preferably, incubation time is 50
Minute.
Fluorescence detection condition is fluorescence intensity of the measurement system at 517nm, excitation wavelength 495nm.
It is that the present invention has the utility model has the advantages that
The present invention detects myoglobins using standby aptamer fluorescent optical sensor cheap and easy to manufacture, it can be achieved that flesh
The accurate detection of Lactoferrin.Compared with existing method, this method design is simple, at low cost, detecting step is few, easy to operate, spirit
Sensitivity is high, and selectivity is good.
Detailed description of the invention
Fig. 1 is the working principle diagram that aptamer fluorescent optical sensor of the present invention detects myoglobins.
Fig. 2 be four kinds of short chains of DNA for being connected with fluorophor respectively be connected with the aptamer of quenching group buffer it is molten
Hybridized in liquid, the comparison result of the fluorescence decreasing value (Δ I) of fluorescent quenching has occurred.
The hybridization time of the short chain of DNA1 and the aptamer for being connected with quenching group that Fig. 3 is connected with fluorophor optimizes.
Fig. 4 is hybridize 20 minutes with the aptamer for being connected with quenching group by the short chain of the DNA1 for being connected with fluorophor
Afterwards, the incubation time optimization of the myoglobins sample of final concentration 5ng/mL is added.
Fig. 5 is hybridize 20 minutes with the aptamer for being connected with quenching group by the short chain of the DNA1 for being connected with fluorophor
Afterwards, then it is separately added into the myoglobins sample of various concentration (0.1-100ng/mL), and is incubated for 50min, obtained various samples
The fluorescence intensity figure of product.
Fig. 6 be by the short chain of the DNA1 for being connected with fluorophor (25nM) be connected with the aptamer (25nM) of quenching group into
Row hybridization after twenty minutes, then is separately added into the myoglobins sample of various concentration (0.1-5ng/mL), and be incubated for 50min, gained
The fluorescence intensity figure of the various samples arrived.
Fig. 7 is the myoglobins sample that various concentration (0.1-5ng/mL) is added, and the fluorescence recovery value and flesh detected is red
Protein concentration relationship.
Fig. 8 is the fluorescence intensity recovery figure that aptamer fluorescent optical sensor of the present invention detects various protein examples.
Specific embodiment
The present invention is further illustrated below by embodiment, but cannot limit the contents of the present invention.
Embodiment 1:
1, it is connected with the Base series structure of the aptamer of quenching group are as follows:
5’-CCCTCCTTTCCTTCGACGTAGATCTGCTGCGTTGTTCCGA-
DABCYL-3 ', wherein DABCYL is quenching group, molecular structure are as follows: [4- (dimethylamino) phenyl is even by 4-
Nitrogen] benzoic acid.
It is connected with the Base series structure of the complementary short-chain of fluorophor are as follows: DNA1:5 '-FAM-GGAACAACGC-3 '；
DNA2:5 '-FAM-TCGGAACAAC-3 '；DNA3:5 '-FAM-CGGAACAACG-3 '；DNA4:5 '-FAM-GAACAACGCA-
3 ', wherein FAM is fluorophor, molecular structure are as follows: 6- Fluoresceincarboxylic acid.
2, by 1760 μ L 0.01M PBS buffer solutions (pH=7.4), the complementation that 100 μ L 500nM are connected with fluorophor is short
Chain and 100 μ L 500nM are connected with the aptamer of quenching group, sequentially add in 5mL plastic tube.It is connected with the mutual of fluorophor
The ultimate density for the aptamer mended short chain and be connected with quenching group is 25nM.Mixed solution is incubated for 20 points at 25 DEG C
Clock.
Four kinds of short chains of DNA (25nM) for being connected with fluorophor exist with the aptamer (25nM) for being connected with quenching group respectively
Hybridization 20 minutes is carried out in buffer solution, fluorescent quenching has occurred, and Fig. 2 is the comparison result of fluorescence decreasing value (Δ I), wherein Δ
I is bigger, and quenching effect is better, therefore the quenching effects of DNA1 are optimal.
3, optimize hybridization time
By 1760 μ L 0.01M PBS buffer solutions (pH=7.4), 100 μ L 500nM are connected with the complementary short-chain of fluorophor
It is connected with the aptamer of quenching group with 100 μ L 500nM, sequentially adds in 5mL plastic tube.It is connected with the complementation of fluorophor
The ultimate density of short chain and the aptamer for being connected with quenching group is 25nM.Mixed solution is incubated for 5 respectively at 25 DEG C,
10,15,20,25 minutes.
Be connected with the short chain of DNA1 (25nM) of fluorophor be connected with when the hybridizing of aptamer (25nM) of quenching group
Between optimize, as shown in figure 3, reach 20 minutes when, fluorescence intensity minimizes, quenching effect it is best.
4, optimize incubation time
By 1760 μ L 0.01M PBS buffer solutions (pH=7.4), 100 μ L 500nM are connected with the complementary short-chain of fluorophor
It is connected with the aptamer of quenching group with 100 μ L 500nM, sequentially adds in 5mL plastic tube.It is connected with the complementation of fluorophor
The ultimate density of short chain and the aptamer for being connected with quenching group is 25nM.Mixed solution is incubated for 20 points at 25 DEG C
Clock.
Then, 1960 μ L mixed solutions are divided into two equal portions, every part of solution is 980 μ L, wherein being added in a solution
20 μ L 0.01M PBS buffer solutions (pH=7.4) afterwards and are uniformly mixed (final volume be 1000 μ L), are labeled as blank sample；
The myoglobins standard solution of 20 μ L 250ng/mL is added in another solution, and be uniformly mixed (final volume be 1000 μ
L), two parts of solution are incubated for 10,20,30,40,50,60 minutes respectively at 25 DEG C.Finally, solution is transferred in cuvette,
It is strong in the fluorescence that launch wavelength is two samples of (excitation wavelength 495nm) acquisition at 517nm using F-7000 fluophotometer
Degree.
As shown in figure 4, fluorescence intensity increases to maximum, and fluorescence recovery effects are best when incubation time is 50min.
5, the detection of myoglobins standard sample
By 1760 μ L 0.01M PBS buffer solutions (pH=7.4), 100 μ L 500nM are connected with the complementary short-chain of fluorophor
It is connected with the aptamer of quenching group with 100 μ L 500nM, sequentially adds in 5mL plastic tube.It is connected with the complementation of fluorophor
The ultimate density of short chain and the aptamer for being connected with quenching group is 25nM.Mixed solution is incubated for 20 points at 25 DEG C
Clock.
Then, 1960 μ L mixed solutions are divided into two equal portions, every part of solution is 980 μ L, wherein being added in a solution
20 μ L 0.01M PBS buffer solutions (pH=7.4) afterwards and are uniformly mixed (final volume be 1000 μ L), are labeled as blank sample；
20 μ L concentration ranges are added in another solution and are the myoglobins standard solution of 5-5000ng/mL, and are uniformly mixed (final
Volume is 1000 μ L), then two parts of solution are incubated for 50 minutes at 25 DEG C.Finally, solution is transferred in cuvette, use
F-7000 fluophotometer, will in the fluorescence intensity that launch wavelength is that (excitation wavelength 495nm) obtains two samples at 517nm
The fluorescence intensity of myoglobins sample subtracts the fluorescence intensity of blank sample, the as recovery value of fluorescence intensity,
As illustrated in figs. 5-7, wherein in the concentration range of 0.1-5ng/mL, the flesh of intensity and addition that system fluorescence restores
Hemoglobin concentration is directly proportional.Equation of linear regression are as follows: y=79.4c+22.59, R2=0.991.Wherein y is fluorescence recovery value, c
For the concentration of myoglobins.R is related coefficient.
6, as shown in figure 8, by the short chain of the DNA1 for being connected with fluorophor (25nM) and the aptamer for being connected with quenching group
(25nM) is hybridized after twenty minutes, then is separately added into the various protein examples that ultimate density is 5ng/mL (wherein Mb is flesh
Lactoferrin, BSA are bovine serum albumin(BSA), and AFP is alpha-fetoprotein, and lgA is immunoglobulin lgA, and lgG is immunoglobulin G,
HSA is human serum albumins, and cTnI is cardiac muscle troponin I), and be incubated for 50min, obtained various protein examples it is glimmering
Luminous intensity restores figure.Wherein △ I is bigger, and fluorescence intensity recovery effects are better, shows aptamer to the affinity of the protein
Power is bigger, and selectivity is better.
Embodiment 2: the detection of human serum myoglobins sample and mark-on sample
1. firstly, human serum sample (33.4ng/mL, the testing result of hospital) is used 0.01M PBS buffer solution (pH
=7.4) 6.68 times are diluted, then the macro-molecular protein in serum is filtered off with ultra-filtration centrifuge tube (30kD), can be prepared by human body
Serum myoglobin sample.
Secondly, 100 μ L 500nM are connected with the mutual of fluorophor by 1760 μ L 0.01M PBS buffer solutions (pH=7.4)
It mends short chain and 100 μ L 500nM is connected with the aptamer of quenching group, sequentially add in 5mL plastic tube.It is connected with fluorophor
Complementary short-chain and the ultimate density of the aptamer that is connected with quenching group be 25nM.Mixed solution is incubated at 25 DEG C
20 minutes.
Then, 1960 μ L mixed solutions are divided into two equal portions, every part of solution is 980 μ L, wherein being added in a solution
20 μ L 0.01M PBS buffer solutions (pH=7.4) afterwards and are uniformly mixed (final volume be 1000 μ L), are labeled as blank sample；
The above-mentioned human serum myoglobins sample solution prepared of 20 μ L is added in another solution, and is uniformly mixed (final
Volume is 1000 μ L), then two parts of solution are incubated for 50 minutes at 25 DEG C.
It the use of F-7000 fluophotometer in launch wavelength is that the place 517nm is (sharp finally, solution is transferred in cuvette
Hair wavelength obtains the fluorescence intensity of two samples for 495nm), and the fluorescence intensity of human serum myoglobins sample is subtracted blank
The fluorescence intensity of sample, the as recovery value of fluorescence intensity, further according to equation of linear regression (y=79.4c+22.59) and dilute
Multiple is released, the concentration of human serum myoglobins sample can be calculated.By the data of the blood serum sample 1 of table 1 it is found that sample
The rate of recovery is 113.5%.
2. firstly, by 50 μ L human serum samples (33.4ng/mL, the testing result of hospital) and 50 μ L 133.4ng/mL
Myoglobins standard solution mixed, then by mixed liquor with 0.01M PBS buffer solution (pH=7.4) dilute 10 times,
Then the macro-molecular protein in serum is filtered off with ultra-filtration centrifuge tube (30kD), can be prepared by human serum myoglobins sample.
Secondly, 100 μ L 500nM are connected with the mutual of fluorophor by 1760 μ L 0.01M PBS buffer solutions (pH=7.4)
It mends short chain and 100 μ L 500nM is connected with the aptamer of quenching group, sequentially add in 5mL plastic tube.It is connected with fluorophor
Complementary short-chain and the ultimate density of the aptamer that is connected with quenching group be 25nM.Mixed solution is incubated at 25 DEG C
20 minutes.
Then, 1960 μ L mixed solutions are divided into two equal portions, every part of solution is 980 μ L, wherein being added in a solution
20 μ L 0.01M PBS buffer solutions (pH=7.4) afterwards and are uniformly mixed (final volume be 1000 μ L), are labeled as blank sample；
The above-mentioned human serum myoglobins sample solution prepared of 20 μ L is added in another solution, and is uniformly mixed (final
Volume is 1000 μ L), then two parts of solution are incubated for 50 minutes at 25 DEG C.
It the use of F-7000 fluophotometer in launch wavelength is that the place 517nm is (sharp finally, solution is transferred in cuvette
Hair wavelength obtains the fluorescence intensity of two samples for 495nm), and the fluorescence intensity of human serum myoglobins sample is subtracted blank
The fluorescence intensity of sample, the as recovery value of fluorescence intensity, further according to equation of linear regression (y=79.4c+22.59) and dilute
Multiple is released, the concentration of human serum myoglobins sample can be calculated.By the data of the blood serum sample 2 of table 1 it is found that sample
The rate of recovery is 99.1%.
3. firstly, by 50 μ L human serum samples (33.4ng/mL, the testing result of hospital) with 50 μ L433.4ng/mL's
Myoglobins standard solution is mixed, and mixed liquor 0.01M PBS buffer solution (pH=7.4) is then diluted 10 times, so
The macro-molecular protein in serum is filtered off with ultra-filtration centrifuge tube (30kD) afterwards, can be prepared by human serum myoglobins sample.
Secondly, 100 μ L 500nM are connected with the mutual of fluorophor by 1760 μ L 0.01M PBS buffer solutions (pH=7.4)
It mends short chain and 100 μ L 500nM is connected with the aptamer of quenching group, sequentially add in 5mL plastic tube.It is connected with fluorophor
Complementary short-chain and the ultimate density of the aptamer that is connected with quenching group be 25nM.Mixed solution is incubated at 25 DEG C
20 minutes.
Then, 1960 μ L mixed solutions are divided into two equal portions, every part of solution is 980 μ L, wherein being added in a solution
20 μ L 0.01M PBS buffer solutions (pH=7.4) afterwards and are uniformly mixed (final volume be 1000 μ L), are labeled as blank sample；
The above-mentioned human serum myoglobins sample solution prepared of 20 μ L is added in another solution, and is uniformly mixed (final
Volume is 1000 μ L), then two parts of solution are incubated for 50 minutes at 25 DEG C.
It the use of F-7000 fluophotometer in launch wavelength is that the place 517nm is (sharp finally, solution is transferred in cuvette
Hair wavelength obtains the fluorescence intensity of two samples for 495nm), and the fluorescence intensity of human serum myoglobins sample is subtracted blank
The fluorescence intensity of sample, the as recovery value of fluorescence intensity, further according to equation of linear regression (y=79.4c+22.59) and dilute
Multiple is released, the concentration of human serum myoglobins sample can be calculated.By the data of the blood serum sample 3 of table 1 it is found that sample
The rate of recovery is 104.8%.
4. firstly, by 50 μ L human serum samples (33.4ng/mL, the testing result of hospital) and 50 μ L 933.4ng/mL
Myoglobins standard solution mixed, then by mixed liquor with 0.01M PBS buffer solution (pH=7.4) dilute 10 times,
Then the macro-molecular protein in serum is filtered off with ultra-filtration centrifuge tube (30kD), can be prepared by human serum myoglobins sample.
Secondly, 100 μ L 500nM are connected with the mutual of fluorophor by 1760 μ L 0.01M PBS buffer solutions (pH=7.4)
It mends short chain and 100 μ L 500nM is connected with the aptamer of quenching group, sequentially add in 5mL plastic tube.It is connected with fluorophor
Complementary short-chain and the ultimate density of the aptamer that is connected with quenching group be 25nM.Mixed solution is incubated at 25 DEG C
20 minutes.
Then, 1960 μ L mixed solutions are divided into two equal portions, every part of solution is 980 μ L, wherein being added in a solution
20 μ L 0.01M PBS buffer solutions (pH=7.4) afterwards and are uniformly mixed (final volume be 1000 μ L), are labeled as blank sample；
The above-mentioned human serum myoglobins sample solution prepared of 20 μ L is added in another solution, and is uniformly mixed (final
Volume is 1000 μ L), then two parts of solution are incubated for 50 minutes at 25 DEG C.
It the use of F-7000 fluophotometer in launch wavelength is that the place 517nm is (sharp finally, solution is transferred in cuvette
Hair wavelength obtains the fluorescence intensity of two samples for 495nm), and the fluorescence intensity of human serum myoglobins sample is subtracted blank
The fluorescence intensity of sample, the as recovery value of fluorescence intensity, further according to equation of linear regression (y=79.4c+22.59) and dilute
Multiple is released, the concentration of human serum myoglobins sample can be calculated.By the data of the blood serum sample 4 of table 1 it is found that sample
The rate of recovery is 107.2%.
The result that table 1. detects human serum myoglobins sample and mark-on sample
From the data in table 1, it can be seen that either myoglobins actual sample still detects mark-on sample, sample
The rate of recovery shows measuring method accuracy with higher between 99-114%.Comparative example
1, it is connected with the Base series structure of the aptamer of fluorophor are as follows:
5 '-FAM-CCCTCCTTTCCTTCGACGTAGATCTGCTGCGTTGTTCCGA--3 ', wherein FAM is fluorescent base
Group, molecular structure are as follows: 6- Fluoresceincarboxylic acid.
It is connected with the Base series structure of the complementary short-chain of quenching group are as follows: DNA1:5 '-GGAACAACGC-DABCYL-3 '；
Wherein DABCYL is quenching group, molecular structure are as follows: 4- [4- (dimethylamino) phenylazo] benzoic acid.
2, firstly, by 1760 μ L 0.01M PBS buffer solutions (pH=7.4), 100 μ L 500nM are connected with quenching group
Complementary short-chain and 100 μ L 500nM are connected with the aptamer of fluorophor, sequentially add in 5mL plastic tube.It is connected with and base is quenched
The ultimate density of the complementary short-chain of group and the aptamer for being connected with fluorophor is 25nM.Mixed solution is incubated at 25 DEG C
It educates 30 minutes, it is 652 that fluorescence, which declines intensity, and fluorescence rate of descent is 20%, and system fluorescence background is stronger.
Then, 1960 μ L mixed solutions are divided into two equal portions, every part of solution is 980 μ L, wherein being added in a solution
20 μ L 0.01M PBS buffer solutions (pH=7.4) afterwards and are uniformly mixed (final volume be 1000 μ L), are labeled as blank sample；
20 μ L 100ng/mL human serum myoglobins sample solution well prepared in advance is added in another solution, and is uniformly mixed
Two parts of solution, are then incubated for 120 minutes, the fluorescence intensity of authentic sample rises to most by (final volume is 1000 μ L) at 25 DEG C
It is high.
It the use of F-7000 fluophotometer in launch wavelength is that the place 517nm is (sharp finally, solution is transferred in cuvette
Hair wavelength obtains the fluorescence intensity of two samples for 495nm), and the fluorescence intensity of human serum myoglobins sample is subtracted blank
The fluorescence intensity of sample, the as recovery value (192) of fluorescence intensity.
Conclusion: fluorophor is connected on aptamer, and quenching group is connected on complementary short-chain, leads to first step fluorescence
Quenching time lengthens, and the decline of fluorescent quenching rate, system fluorescence background is excessively high, to the inspection of the range of linearity of myoglobin concentration below
It is unfavorable that measuring tape comes, its range of linearity can be made to become smaller；Second step fluorescence recovery time is too long, and fluorescence recovery strength is not high, and fluorescence is extensive
It is multiple unobvious, it makes troubles to the detection of authentic sample, this is totally unfavorable to the rescue for the dangerous patient for suffering from myocardial infarction.
Sequence table
<110>Institutes Of Jiaxing
<120>a kind of aptamer fluorescent optical sensor and its construction method for detecting myoglobins
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| US20050089864A1 (en) * | 2002-01-22 | 2005-04-28 | Yingfu Li | Signalling aptamer complexes |
| JP5223237B2 (en) * | 2007-05-14 | 2013-06-26 | ソニー株式会社 | Method for detecting binding or interaction between nucleic acid strand for detection and substance |
| CN101812528B (en) * | 2010-04-26 | 2012-07-04 | 湖南大学 | Switch mode aptamer probe and application thereof in tumor living cell and vital detection |
| CN102621120B (en) * | 2012-03-31 | 2014-04-23 | 江苏省农业科学院 | A Method for Detecting Mercury Ion Residue Based on Nucleic Aptamer Structure-Based Fluorescent Signal Transformation Mechanism |
| CN102703454B (en) * | 2012-06-27 | 2014-02-26 | 湖南大学 | Nucleic acid aptamer that can be used to detect myoglobin, microfluidic chip for screening, screening method and application thereof |
| CN104807865B (en) * | 2015-05-08 | 2017-06-30 | 江苏省农业科学院 | It is applied to the preparation method of the electrochemical aptamer sensor of myoglobins detection |
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