CN103630517A - A thrombin detection method based on split aptamer and water-soluble conjugated polymer - Google Patents
A thrombin detection method based on split aptamer and water-soluble conjugated polymer Download PDFInfo
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Abstract
本发明是一种基于拆分适配体和水溶性共轭聚合物的凝血酶检测方法，该方法利用了水溶性聚合物对DNA链构象变化敏感，以及核酸适配体具有特异性结合能力等特点，设计了一种基于水溶性共轭聚合物/核酸适配体的超分子检测平台。当溶液中不存在凝血酶或存在其他非特异性蛋白时，这两段序列不能形成G-四链体结构，复合物与聚合物的之间的距离较近，从而能与聚合物发生有效的能量转移；当溶液中存在凝血酶时，凝血酶将诱导这两段序列形成G-四链体结构，增加了复合物与聚合物的之间的距离，从而不能发生有效的能量转移。本发明方法简便、快速、具有较高的灵敏度和选择性，在凝血酶检测方面具有重要的意义。
The invention is a thrombin detection method based on splitting aptamers and water-soluble conjugated polymers, which utilizes the sensitivity of water-soluble polymers to DNA chain conformation changes, and the specific binding ability of nucleic acid aptamers, etc. characteristics, a supramolecular detection platform based on water-soluble conjugated polymer/nucleic acid aptamer was designed. When there is no thrombin or other non-specific proteins in the solution, the two sequences cannot form a G-quadruplex structure, and the distance between the complex and the polymer is relatively close, so that effective energy can be generated with the polymer. Transfer; when there is thrombin in the solution, thrombin will induce the two sequences to form a G-quadruplex structure, increasing the distance between the complex and the polymer, so that effective energy transfer cannot occur. The method of the invention is simple and fast, has high sensitivity and selectivity, and has important significance in the aspect of thrombin detection.
Description
Technical field
The invention belongs to bio-sensing and analysis field, particularly a kind of fluorescence detection method that splits aptamers and soluble conjugated polymer detection fibrin ferment that utilizes.
Background technology
Fibrin ferment is a kind of proteolytic enzyme being formed by fibrin ferment precursor (neccessary composition in blood plasma), can become fibrin and impel blood clotting by catalysis fibre proteinogen.The detection of fibrin ferment all has more important meaning to the monitoring of the diagnosis of clinical disease, disease, prognosis and curative effect and assessment etc.
Due to the anticoagulant substances neutralization in the early stage very fast body of fibrin ferment producing of blood coagulation, therefore it is very difficult directly to measure fibrin ferment.Fibrin ferment detects the method for multiplex indirect detection clinically, by detecting thrombin activation, reflects the generation of fibrin ferment to the specific Blood Coagulation Markers in thrombosis.Traditional Indirect Detecting Method has fibrinopeptide A (FPA) to measure, and SFMC (SFMC) is measured, thrombin-antithrombin complex (TAT) mensuration etc.These detection method application are comparatively extensive, but its specificity and sensitivity are relatively low.
The sensitivity of fibrin ferment direct Detection Method and specificity based on aptamers and polymkeric substance effect are higher, and clinical more, the laboratory data more intuitively that provides is provided.Yet complete thrombin aptamer base number is more, self easily form secondary structure, before detecting, need to add heat abstraction secondary structure, make complex operation; In addition, also have the problems such as blank sample background is higher, these factors all can affect accuracy and the sensitivity of detection.Comparatively speaking, the thrombin aptamer of fractionation, base negligible amounts, self is difficult for forming secondary structure, thereby can reduces operation steps, reduces the background of blank sample.Research of the present invention, take polyparaphenylene's acetylene as signal magnify tool, take the thrombin aptamer that splits as identification probe, take fluoroscopic examination as Main Analysis means, has realized the high-sensitivity detection to fibrin ferment.This highly sensitive, specificity good, easy and simple to handle, detect the bio-sensing strategy fast, cost is low, will provide valuable theory and experimental basis for the development from now on of this field.
Summary of the invention
The technical problem to be solved in the present invention is the deficiency existing for existing fibrin ferment detection method, and a kind of fluorescent method that splits aptamers and soluble conjugated polymer detection fibrin ferment that utilizes is provided, and it has higher specificity and sensitivity.
The present invention is based on the method that splits aptamers and soluble conjugated polymer detection fibrin ferment, comprise the following steps:
A. aptamers fragment 1, aptamers fragment 2 and soluble conjugated polymer are mixed 1:1:5 ~ 10 in molar ratio, form the compound of aptamers/polymkeric substance, then join in buffer solution, take 404 nm as excitation wavelength, carry out fluoroscopic examination, record the fluorescent emission bands of a spectrum of this compound, and calculate the ratio I of its 525 nm and 440 nm place fluorescence intensities
525/ I
440; Wherein, the mark material of having illicit sexual relations in described aptamers fragment 1 or aptamers fragment 2;
B. by aptamers fragment 1 and aptamers fragment 2 and containing the testing sample of fibrin ferment, join in conjunction with reacting in liquid, form fibrin ferment/aptamers compound; The mol ratio 1:1 of aptamers fragment 1 and aptamers fragment 2, the amount of fibrin ferment is variable, minimum is zero, identical with the amount of aptamers when maximum.
C. in above-mentioned compound, add soluble conjugated polymer, the mol ratio of fibrin ferment/aptamers compound and soluble conjugated polymer be 0～1:5(herein the amount of aptamers fix, amount containing fibrin ferment in the testing sample of fibrin ferment is variable, minimum is zero, identical with the amount of aptamers when maximum, so the amount of fibrin ferment/aptamers compound is according to the amount meter of fibrin ferment, so the mol ratio of fibrin ferment/aptamers compound and soluble conjugated polymer is 0～1:5), thrombin induction aptamers fragment 1 and aptamers fragment 2 form G-tetra-chain body structures, thereby can not form the compound of aptamers/polymkeric substance, then join in buffer solution, take 404 nm as excitation wavelength, carry out fluoroscopic examination, calculate the ratio I of its 525 nm and 440 nm place fluorescence intensities
525/ I
440, its I
525/ I
440ratio declines, according to I
525/ I
440the degree that ratio declines realizes the qualitative of fibrin ferment and quantitatively detects.
Wherein: described aptamers is fibrin ferment specific single-chain oligonucleotides, and the base composition of aptamers fragment 1 is: 5 '-FITC-GGTTGGTG-3 '; The base composition of aptamers fragment 2 is: 5 '-TGGTTGG-3 '.
The mark material of having illicit sexual relations in aptamers fragment 1 or aptamers fragment 2 described in step a), described dyestuff is fluorescein or fluorescein isothiocynate; Dye marker is at 5 ' end of aptamers fragment 1 or 3 ' end of aptamers fragment 2.
Buffer solution used is 0.1M NaHCO
3, 0.1M Na
2cO
3, pH=10.6; Fluorescence detection method used is: the solution of 2.5 μ L soluble conjugated polymers, 2.5 μ L aptamers fragments 1 and 2.5 μ L aptamers fragments 2 is joined to the scanning of carrying out fluorescence emission spectrum in 500 μ L buffer solution, excitation wavelength is 404 nm, and emission wavelength sweep limit is 420-650 nm.The concentration of aptamers fragment 1 is that the concentration of 10 μ M, aptamers fragment 2 is that the concentration of 10 μ M, soluble conjugated polymer is 50 μ M.
The formation of the fibrin ferment/aptamers compound described in step b), is under the induction of fibrin ferment, by non-covalent interaction, makes two sections of aptamers fragments form G-tetra-chain body structures; Fibrin ferment is joined in the combination liquid that contains aptamers, place 40 minutes for 37 ℃; Combination liquid used is 140 mM NaCl, 10 mM Tris-HCl, pH=8.0.
Described fluorescence signal detects with fluorospectrophotometer, and excitation wavelength is 404 nm, and emission wavelength sweep limit is 420-650 nm.
beneficial effect:according to fluorescence detection method of the present invention, can realize the detection to fibrin ferment.While containing other enzyme or protein in system, still can realize its high-sensitivity detection.Therefore, the inventive method is expected to detection for fibrin ferment in actual clinical sample provides a kind of easy, method fast.
Accompanying drawing explanation
Fig. 1 is the fundamental diagram that the present invention utilizes the fluorescence detection method that splits aptamers and soluble conjugated polymer detection fibrin ferment.
Fig. 2 is that the present invention utilizes the feasibility analysis figure that splits aptamers and soluble conjugated polymer detection fibrin ferment.A is polymkeric substance+total length thrombin aptamer; B is polymkeric substance+total length thrombin aptamer+fibrin ferment; C is that polymkeric substance+fractionation aptamers 1+ splits aptamers 2; D is polymkeric substance+aptamers fragment 1+ aptamers fragment 2+ fibrin ferment.As can be seen from the figure, total length thrombin aptamer, after being combined with fibrin ferment, can cause the decline of FRET efficiency, i.e. I
525/ I
440ratio declines; The aptamers fragment splitting and fibrin ferment still can, in conjunction with forming G-tetra-chain body structures, cause the decline of FRET efficiency.Different, while using the aptamers splitting, the effect of FRET decrease in efficiency is better than total length aptamers.
Fig. 3 is that the present invention utilizes the specificity analyses figure that splits aptamers and soluble conjugated polymer detection fibrin ferment.
Fig. 4 is that the present invention utilizes the sensitivity analysis figure that splits aptamers and soluble conjugated polymer detection fibrin ferment.This chart understands FRET efficiency change rate 1-F/F
0with the relation of concentration of thrombin c, wherein, F is the FRET efficiency containing fibrin ferment sample, F
0for not containing the blank FRET efficiency of fibrin ferment.
Embodiment
Below in conjunction with accompanying drawing, with the detection embodiment of fibrin ferment, further illustrate the present invention.But being not limited in detection fibrin ferment, the present invention can also detect K
+and Pb
2+deng, be not subject to the restriction of other conditions in embodiment yet.
Fluoroscopic examination instrument used is fluorescent sub-photometer (RF-5301, Japan).Fluorescence spectral measuring condition: xenon lamp excites, exciting and launching slit width is 5.0 nm and 5.0 nm, voltage is 950 V, response time Auto, excitation wavelength is 404 nm, emission wavelength sweep limit 420～650 nm; With 600 μ L quartz colorimetric utensils, measure sample volume 500 μ L; Room temperature.
In the embodiment of the present invention, aptamers fragment 1 used and aptamers fragment 2 are all purchased from Shanghai biotechnology company limited, and sequence is respectively 5 '-FITC-GGTTGGTG-3 ' and 5 '-TGGTTGG-3 '; Fibrin ferment is purchased from Sigma-Aldrich company; In conjunction with liquid composition, be: 140 mM NaCl, 10 mM Tris-HCl, pH=8.0; Buffer solution composition used is: 0.1M NaHCO
3, 0.1M Na
2cO
3, pH=10.6.
Embodiment 1:
1) the aptamers fragment 1 of dye marker, aptamers fragment 2 and soluble conjugated polymer are mixed for 1: 1: 5 in molar ratio, form the compound of aptamers/polymkeric substance, then join in buffer solution, take 404 nm as excitation wavelength, carry out fluoroscopic examination, record the fluorescent emission bands of a spectrum of this compound, and calculate the ratio I of its 525 nm and 440 nm place fluorescence intensities
525/ I
440; Aptamers fragment 1 and aptamers fragment 2 are special single nucleotide sequence, and 5 ' end of aptamers fragment 1 is marked with fluorescein isothiocynate, and labeling method is: 5 '-6-FITC.
2), by aptamers fragment 1 and aptamers fragment 2 and join in binding soln 37 ℃ containing the testing sample of fibrin ferment and place 40 minutes, form fibrin ferment/aptamers compound.
3) in above-mentioned compound, add soluble conjugated polymer, thrombin induction also forms G-tetra-chain body structures with aptamers fragment 1 and 2 combinations of aptamers fragment, now aptamers and polymkeric substance is distant, effective energy can not occur to be shifted, the fluorescence signal that is marked at the dyestuff of aptamers end declines, under the environment of buffer solution, carry out fluoroscopic examination, calculate the ratio I of its 525 nm and 440 nm place fluorescence intensities
525/ I
440, its I
525/ I
440ratio declines, according to I
525/ I
440the degree that ratio declines realizes the qualitative of fibrin ferment and quantitatively detects.
Embodiment 2:
1) the aptamers fragment 2 of aptamers fragment 1, dye marker and soluble conjugated polymer are mixed for 1: 1: 10 in molar ratio, form the compound of aptamers/polymkeric substance, then join in buffer solution, take 404 nm as excitation wavelength, carry out fluoroscopic examination, record the fluorescent emission bands of a spectrum of this compound, and calculate the ratio I of its 525 nm and 440 nm place fluorescence intensities
525/ I
440; Aptamers fragment 1 and aptamers fragment 2 are special single nucleotide sequence, and 3 ' end of aptamers fragment 2 is marked with fluorescein, and labeling method is: 3 '-6-FAM mark.
2), by aptamers fragment 1 and aptamers fragment 2 and join in binding soln 37 ℃ containing the testing sample of fibrin ferment and place 40 minutes, form fibrin ferment/aptamers compound.
3) in above-mentioned compound, add soluble conjugated polymer, thrombin induction also forms G-tetra-chain body structures with aptamers fragment 1 and 2 combinations of aptamers fragment, now aptamers and polymkeric substance is distant, effective energy can not occur to be shifted, the fluorescence signal that is marked at the dyestuff of aptamers end declines, under the environment of buffer solution, carry out fluoroscopic examination, calculate the ratio I of its 525 nm and 440 nm place fluorescence intensities
525/ I
440, its I
525/ I
440ratio declines, according to I
525/ I
440the degree that ratio declines realizes the qualitative of fibrin ferment and quantitatively detects.
The specificity analyses that embodiment 3 fibrin ferments detect
To adding 2.5 μ L concentration in each centrifuge tube, be that aptamers fragment 1 and the 2.5 μ L concentration of 10 μ M are the aptamers fragment 2 of 10 μ M, then adding respectively 1 μ L concentration is that the fibrin ferment (Thro) of 10 μ M, the bovine serum albumin (BSA) that 6.8 μ L concentration are 0.1 mg/ml, lysozyme (Lys) and the 1.6 μ L concentration that 2 μ L concentration are 5 μ M are 1mg/ml immunoglobulin (Ig) (lgG), making each final concentration of protein is 20 nM, and the final concentration of aptamers fragment 1 and aptamers fragment 2 is 50 nM; Finally adding 2.5 μ L concentration is the soluble conjugated polymer of 50 μ M, at pH=10.6, under the environment of 0.1 M CBS buffer solution, carry out fluoroscopic examination, the results are shown in Figure 3, as can be seen from the figure, blank and add the FRET of nonspecific proteins close and all higher, add the FRET of fibrin ferment obviously to reduce.Because when not having Thro or having other nonspecific proteinses in solution, two sections of sequences that split can not form G-tetra-chain body structures, and the close together between compound and polymkeric substance shifts thereby can there is effective energy with polymkeric substance; While there is fibrin ferment in solution, fibrin ferment will induce this two sections of sequences to form G-tetra-chain body structures, increased compound and polymkeric substance between distance, thereby can not there is effective energy transfer.Show that the method has good specificity.
The sensitivity analysis that embodiment 4 fibrin ferments detect
1) to each centrifuge tube, adding 2.5 μ L concentration is the aptamers fragment 1 of the marked by fluorescein isothiocyanate of 10 μ M, 2.5 μ L concentration are that 10 μ M aptamers fragments 2 and 2.5 μ L concentration are the soluble conjugated polymer of 50 μ M, form the compound of aptamers/polymkeric substance, then join in the buffer solution of 492.5 μ L, take 404 nm as excitation wavelength, carry out fluoroscopic examination, record the fluorescent emission bands of a spectrum of this compound, the ratio I of its 525 nm and 440 nm place fluorescence intensities
525/ I
440;
2) to each centrifuge tube, adding 2.5 μ L concentration is the aptamers fragment 1 of 10 μ M and the fibrin ferment of aptamers fragment 2 and variable concentrations, make fibrin ferment final concentration be respectively 2 nM, 5 nM, 8 nM, 10 nM, 20 nM, 30 nM, 50 nM, in the combination liquid of 37 ℃, hatch 40 minutes; With the detection liquid that does not contain fibrin ferment, make blank, then adding 2.5 μ L concentration is the soluble conjugated polymer of 50 μ M, carries out fluoroscopic examination under the environment of buffer solution, calculates the ratio I of its 525 nm and 440 nm place fluorescence intensities simultaneously
525/ I
440, along with the increase of concentration of thrombin, energy transfer efficiency reduces gradually, i.e. I
525/ I
440ratio reduces gradually.The results are shown in accompanying drawing 4, as can be seen from the figure, along with the increase of concentration of thrombin, 1-F/F
0raise gradually, this is due to the aptamers fragment 1 in fibrin ferment and solution and aptamers fragment 2 generation specific bindings, causes increasing aptamers fragment 1 and aptamers fragment 2 to be combined into G-tetra-chain body structures.Can find, in the scope of 2.0nM ~ 20 nM, concentration of thrombin and 1-F/F
0close to linear relationship.Linear relationship quantitatively detects fibrin ferment accordingly.
SEQUENCE LISTING
<110> Nanjing Univ. of Posts and Telecommunications
Mono-kind of the <120> fibrin ferment detection method based on splitting aptamers and soluble conjugated polymer
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 8
<212> DNA
<213> artificial sequence
<400> 1
ggttggtg 8
<210> 2
<211> 7
<212> DNA
<213> artificial sequence
<400> 2
tggttgg 7
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