CN108956991B - Fluorescence resonance energy transfer biosensor and application thereof - Google Patents
Fluorescence resonance energy transfer biosensor and application thereof Download PDFInfo
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- CN108956991B CN108956991B CN201810829706.7A CN201810829706A CN108956991B CN 108956991 B CN108956991 B CN 108956991B CN 201810829706 A CN201810829706 A CN 201810829706A CN 108956991 B CN108956991 B CN 108956991B
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Abstract
The invention discloses a fluorescence resonance energy transfer biosensor and application thereof. The sensor comprises a first binding probe and a second binding probe; the first binding probe is hybridized by aptamer1 and block1 to form a double-helix structure, and both aptamer1 and block1 are DNA sequences; the 3 'end of the aptamer1 sequence is modified with a fluorescent molecule A, and the 5' end is assembled with a first prostate cancer antigen (PSA) aptamer at a site for binding with a target substance; the second binding probe is aptamer2, the aptamer2 is a DNA sequence; assembling a second PSA aptamer for binding to a second site of target substance PSA on the 3 'end of the aptamer2 sequence, and modifying a fluorescent molecule B on the 5' end; in the presence of a target substance PSA, aptamer2 replaces block1 and hybridizes with aptamer1 to form a double-helix structure, and a fluorescent molecule A and a fluorescent molecule B are close to each other and form a strong fluorescence resonance energy transfer effect. The sensor is applied to the detection of serum prostate specific antigen PSA and has the advantages of high sensitivity, simple detection process, low analysis cost, good marker diagnosis accuracy and the like.
Description
Technical Field
The invention belongs to the technical field of biosensors, and particularly relates to a fluorescence resonance energy transfer biosensor and application thereof.
Background
Prostate cancer is one of the most common malignancies of urology. There are data statistics that the incidence and mortality of prostate cancer rank first and second in male malignancies, respectively, in the united states and europe. Serum Prostate Specific Antigen (PSA) is produced by prostate epithelial cells and has strong organ specificity. PSA has been widely used clinically as a tumor marker, and the detection rate of prostate cancer is obviously improved.
Clinical biochemical tests generally require small sample volumes, rapid, accurate, and preferably high throughput analysis. At present, conventional clinical biochemical analysis is generally by means of immunoassay. Enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), fluoroimmunoassay and time-resolved fluoroimmunoassay, chemiluminescence immunoassay and other techniques are commonly used for detecting clinical serum markers. Although the methods have certain value for disease diagnosis, the methods have the defects of long analysis and detection period, complicated operation steps, high cost, low analysis flux and the like, and limit the further clinical application of the methods. There is still a great development space and application demand for developing new methods for PSA detection by using a more convenient and sensitive labeling method.
Disclosure of Invention
The invention aims to provide a fluorescence resonance energy transfer biosensor and application thereof, wherein the biosensor can be applied to serum prostate specific antigen PSA detection and has the characteristics of simple operation, high detection speed and accurate detection result.
The invention is thus achieved, a fluorescence resonance energy transfer biosensor comprising a first binding probe and a second binding probe; wherein,
the first binding probe is in a double-helix structure formed by hybridization of aptamer1 and block1, and both aptamer1 and block1 are DNA sequences; modifying fluorescent molecule A at the 3 'end of aptamer1 sequence, assembling a first PSA aptamer at a site for binding target substance PSA at the 5' end;
the second binding probe is aptamer2, and the aptamer2 is a DNA sequence; assembling a second PSA aptamer for binding to a second site of target substance PSA on the 3 'end of the aptamer2 sequence, and modifying a fluorescent molecule B on the 5' end;
in the presence of a target substance PSA, aptamer2 replaces block1 and hybridizes with aptamer1 to form a double-helix structure, and a fluorescent molecule A and a fluorescent molecule B are close to each other and form a strong fluorescence resonance energy transfer effect.
Preferably, the DNA sequence of aptamer1 is:
5’-TTTTTAATTAAAGCTCGCCATCAAATAGCTGGGGGTTTTTTTTTTTTTTTTTTTTCCTCAAGATGGTT-3’；
the DNA sequence of the Aptamer2 is as follows:
5’-TTCCATCTTGAGTTTTTTTTTTTTTTTTTTTTGCAATGGTACGGTACTTCCTATGGCGATGTGTTGGCTGTGTGTGGGGTGCAAAAGTGCACGCTACTTTGCTAA-3’；
the DNA sequence of block1 is as follows: 5'-CCATCTTGAGG-3' are provided.
Preferably, the fluorescent molecule A is a fluorescent molecule Cy3, and the fluorescent molecule B is a fluorescent molecule Cy 5.
The invention further discloses application of the fluorescence resonance energy transfer biosensor in detection of serum prostate specific antigen PSA.
Preferably, the application comprises the steps of:
(1) adding the aptamer1 stock solution and the block1 stock solution into a buffer solution, and annealing at 95-4 ℃ to obtain a first binding probe solution; adding the aptamer2 stock solution into a buffer solution, and annealing at 95-4 ℃ to obtain a second binding probe solution;
(2) and mixing the first binding probe solution and the second binding probe solution, adding the mixed solution into a PSA solution, uniformly mixing, carrying out a reaction at room temperature in a dark place for 1 hour, and then measuring fluorescence.
The invention overcomes the defects of the prior art and provides a fluorescence resonance energy transfer biosensor and a preparation method and application thereof. The basic principle of the present invention is shown in FIG. 1, wherein two different PSA aptamers are assembled on two binding probes, and can simultaneously bind to different sites on PSA, and the binding probe 1 is obtained by hybridizing aptamer1 and block 1. Fluorescent molecules Cy3 and Cy5 were modified in aptamer1 and aptamer2, respectively. In the absence of the target substance PSA, the aptamer1 and the block1 form a double-helix hybridization structure, and at the moment, the hybridization of the aptamer2 and the aptamer1 is blocked, so that the distance between Cy3 and Cy5 is far, and effective fluorescence resonance energy transfer cannot be formed. When two binding probes are hybridized with PSA at the same time, due to the ortho-mediated hybridization principle, the aptamer2 and the aptamer1 form a double-helix structure, and block1 is replaced. Therefore, the close distance between Cy3 and Cy5 can form a strong fluorescence resonance energy transfer effect, and the effect is proportional to the concentration of PSA.
The invention is based on the ortho-position immunoassay, utilizes the fluorescence resonance energy transfer technology to realize the high-sensitivity detection of PSA, and solves the defects of low sensitivity, fussy detection process, high analysis cost, poor marker diagnosis accuracy and difficulty in meeting the field detection in the conventional tumor marker diagnosis.
Compared with the defects and shortcomings of the prior art, the invention has the following beneficial effects: the fluorescence resonance energy transfer biosensor is simple and rapid to prepare, is used for specific detection of clinical cancer markers, is beneficial to early screening and diagnosis of prostate cancer, and has the advantages of high sensitivity, simple detection process, low analysis cost, good marker diagnosis accuracy and the like.
Drawings
FIG. 1 is a schematic diagram of a fluorescence resonance energy transfer biosensor according to the present invention;
FIG. 2 is a graph of fluorescence resonance energy transfer signal as a function of time;
FIG. 3 shows the fluorescence detection results of standard solutions of PSA series concentrations;
FIG. 4 shows the signal response results of different tumor markers in this detection system;
FIG. 5 is a graph showing the results of PSA signals for reactions carried out in different substrates.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
1. Preparation of first binding Probe solution
The stock solutions of aptamer1 and block1 were quantified using an ultraviolet fluorescence spectrophotometer to a concentration of 100. mu.M. Then, 2. mu.L each of the stock solutions was dissolved in 100. mu.L of a buffer (20mM HEPES, 500mM NaCl, pH7.4), annealed at 95 ℃ to 4 ℃ for ten minutes at 95 ℃ and then cooled to 4 ℃ and held at 4 ℃ for at least 10 minutes.
2. Preparation of second binding Probe solution
The aptamer2 stock solution was quantified using an ultraviolet fluorescence spectrophotometer to a concentration of 100. mu.M. Then 2. mu.L of the stock solution was dissolved in 100. mu.L of a buffer (20mM HEPES, 500mM NaCl, pH7.4), annealed at 95 ℃ to 4 ℃ for ten minutes at 95 ℃ and then cooled to 4 ℃ and held at 4 ℃ for at least 10 minutes.
3. PSA detection process
And (3) taking 49 microliter of the prepared first binding probe solution and second binding probe solution respectively, mixing in a centrifuge tube, then adding 2 microliter of PSA solution with different concentrations, uniformly mixing the solutions, and reacting at room temperature in a dark place for 1 hour to obtain the solution to be detected.
4. Fluorescence detection process
Placing the solution to be detected after the reaction is finished in a 100-mu-L fluorescence cuvette, and detecting by a fluorescence spectrophotometer under the following detection conditions: the excitation wavelength is 488nm, and the detection wavelength range is 540-750 nm.
5. Effect of different reaction times on PSA assay results
The prepared first binding probe solution and second binding probe solution are mixed in a centrifuge tube in 49 μ L, then 2uL of PSA solution (0.5 μ M) is added, the solutions are mixed uniformly, and then the mixture is placed in a 100 μ L fluorescence cuvette for reaction at room temperature and in a dark place. Fluorescence was measured every 20 minutes and was terminated at 100 minutes. Detecting by a fluorescence spectrophotometer under the following detection conditions: the excitation wavelength is 488nm, and the detection wavelength range is 540-750 nm.
The fluorescence intensity of the reaction system at different reaction time points is shown in FIG. 2. As can be seen from FIG. 2, the intensity of the absorption peak at 565nm gradually decreased and the intensity of the absorption peak at 670nm gradually increased with the increase of the reaction time. After 60 minutes, the change of the peak intensity tends to be stable, so that the reaction time reaches a maximum value within 60 minutes.
6. Fluorescence detection of PSA series concentration standard solution
Taking 49 mu L of the prepared first binding probe and the second binding probe, mixing the first binding probe and the second binding probe in a centrifuge tube, then adding 2uL of PSA series concentration standard solution (0.5 mu M), uniformly mixing the solutions, placing the solutions in a 100 mu L fluorescence cuvette, and detecting by a fluorescence spectrophotometer under the following detection conditions: the excitation wavelength is 488nm, and the detection wavelength range is 540-750 nm.
The increase in fluorescence absorption at 670nm was linearly regressed with the PSA concentration, and the results are shown in FIG. 3 below. The regression equation is that y is 1.5+123.8x, and the regression coefficient is as follows: 0.991. in the equation, y represents the change in fluorescence intensity obtained at 670nm, and x represents the concentration of the target PSA.
7. Selectivity of the reaction system to PSA
Taking 49 microliter of the prepared first binding probe solution and the prepared second binding probe solution, mixing in a centrifuge tube, then adding 2 microliter of PSA solution (0.5 microliter), mixing the solutions uniformly, placing in a 100 microliter fluorescence cuvette, reacting for 1h at room temperature in a dark place, and detecting by a fluorescence spectrophotometer under the following detection conditions: the excitation wavelength is 488nm, and the detection wavelength range is 540-750 nm. In the same manner, the change in fluorescence was measured after adding 2. mu.L of carcinoembryonic antigen (2. mu.M), cancer antigen 125 (2. mu.M) and tumor necrosis factor (2. mu.M) to the probe, respectively.
The fluorescence intensity at 670nm after addition of the different substances is shown in FIG. 4. As can be seen from FIG. 4, only after the target substance PSA is added, the fluorescence intensity at this wavelength is greatly increased, while other tumor markers cannot cause the increase of the signal.
8. Effect of different Complex matrices on PSA detection
Taking 49 microliter of the prepared first binding probe solution and second binding probe solution, mixing in a centrifuge tube, then adding 2 microliter of PSA solution (0.5 microliter), adding 10 microliter of serum or 10 microliter of cell lysate, uniformly mixing the solutions, placing in a 100 microliter fluorescence cuvette, reacting for 1 hour at room temperature in a dark place, and detecting by a fluorescence spectrophotometer under the following detection conditions: the excitation wavelength is 488nm, and the detection wavelength range is 540-750 nm.
The fluorescence intensity at 670nm is shown in FIG. 5. As can be seen from FIG. 5, the results obtained by the reactions in the buffer solution, the serum and the cell lysate are not very different, and the constructed biosensing system can effectively eliminate the interference of the complex matrix.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
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Claims (4)
1. A fluorescence resonance energy transfer biosensor, comprising a first binding probe and a second binding probe; wherein,
the first binding probe is in a double-helix structure formed by hybridization of aptamer1 and block1, and both aptamer1 and block1 are DNA sequences; modifying fluorescent molecule A at the 3 'end of aptamer1 sequence, assembling a first PSA aptamer at a site for binding target substance PSA at the 5' end;
the second binding probe is aptamer2, and the aptamer2 is a DNA sequence; assembling a second PSA aptamer for binding to a second site of target substance PSA on the 3 'end of the aptamer2 sequence, and modifying a fluorescent molecule B on the 5' end;
in the presence of a target substance PSA, aptamer2 replaces block1 and hybridizes with aptamer1 to form a double-helix structure, and a fluorescent molecule A and a fluorescent molecule B are close to each other and form a strong fluorescence resonance energy transfer effect;
the DNA sequence of aptamer1 is:
5’-TTTTTAATTAAAGCTCGCCATCAAATAGCTGGGGGTTTTTTTTTTTTTTTTTTTTCCTCAAGATGGTT-3’；
the DNA sequence of the Aptamer2 is as follows:
5’-TTCCATCTTGAGTTTTTTTTTTTTTTTTTTTTGCAATGGTACGGTACTTCCTATGGCGATGTGTTGGCTGTGTGTGGGGTGCAAAAGTGCACGCTACTTTGCTAA-3’；
the DNA sequence of block1 is as follows: 5'-CCATCTTGAGG-3' are provided.
2. The fluorescence resonance energy transfer biosensor according to claim 1, wherein the fluorescent molecule a is a fluorescent molecule Cy3, and the fluorescent molecule B is a fluorescent molecule Cy 5.
3. Use of the fluorescence resonance energy transfer biosensor according to any one of claims 1-2 for the detection of serum Prostate Specific Antigen (PSA).
4. The use according to claim 3, characterized in that the use comprises the steps of:
(1) adding the aptamer1 stock solution and the block1 stock solution into a buffer solution, and annealing at 95-4 ℃ to obtain a first binding probe solution; adding the aptamer2 stock solution into a buffer solution, and annealing at 95-4 ℃ to obtain a second binding probe solution;
(2) and mixing the first binding probe solution and the second binding probe solution, adding the mixed solution into a PSA solution, uniformly mixing, carrying out a reaction at room temperature in a dark place for 1 hour, and then measuring fluorescence.
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