CN108956991A - A kind of fluorescence resonance energy transfer biosensor and its application - Google Patents
A kind of fluorescence resonance energy transfer biosensor and its application Download PDFInfo
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- CN108956991A CN108956991A CN201810829706.7A CN201810829706A CN108956991A CN 108956991 A CN108956991 A CN 108956991A CN 201810829706 A CN201810829706 A CN 201810829706A CN 108956991 A CN108956991 A CN 108956991A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6417—Spectrofluorimetric devices
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The invention discloses a kind of fluorescence resonance energy transfer biosensor and its applications.The sensor includes the first bonding probes and the second bonding probes；Wherein, the first bonding probes are hybridized by aptamer1 and block1 forms double-spiral structure, and aptamer1 and block1 are DNA sequence dna；Fluorescent molecule A is modified on 3 ' end of aptamer1 sequence, 5 ' end over-assembles one are used to combine the first PSA aptamers on the site target substance prostate cancer antigen (PSA)；Second bonding probes are aptamer2, which is DNA sequence dna；3 ' end over-assemble of aptamer2 sequence one, for combining the 2nd PSA aptamers on the another site target substance PSA, is modified fluorescent molecule B on 5 ' ends；In the presence of target substance PSA, aptamer2 replaces block1 and hybridizes to form double-spiral structure with aptamer1, and fluorescent molecule A, fluorescent molecule B are close to each other and form hyperfluorescence Resonance energy transfer effect.The sensor application has many advantages, such as that high sensitivity, detection process are simple, analysis cost is low, marker diagnostic accuracy is good in the context of detection to serum prostate specific-antigen PSA.
Description
Technical field
The invention belongs to biosensor technology field more particularly to a kind of fluorescence resonance energy transfer biosensor and
It is applied.
Background technique
Prostate cancer is one of most common malignant tumour of Urology Surgery.There is data statistics, in US and European, prostate
The morbidity and mortality of cancer come first and second of male malignancy respectively.Serum prostate specific antigen
(PSA) it is to be generated by prostate epithelial cell, there is stronger organ specificity.PSA has existed as a kind of tumor markers
It is clinically widely applied, the recall rate of prostate cancer is made to be significantly improved.
Biochemistry test generally requires amount of samples few, quick, accurate, it is desirable to carry out high-throughput analysis.Mesh
Before, conventional clinical biochemical is analyzed generally by immunoassay.Enzyme-linked immunosorbent assay (ELISA), radioimmunoassay
The technologies such as method (RIA), fluoroimmunoassay and time-resolved fluoroimmunoassay, chemiluminescence immune assay are usually used in clinical blood
The detection of clear marker.Although these methods have certain value to medical diagnosis on disease, there are analysis detection period length, operation step
Rapid cumbersome, the at high cost, disadvantages such as analysis throughput is low limit it and clinical further apply.Using more easy sensitive mark
Note mode develops PSA new detecting method and still grows a lot space and application demand.
Summary of the invention
The purpose of the present invention is to provide a kind of fluorescence resonance energy transfer biosensor and its application, the bio-sensings
Device can be applied to the detection of serum prostate specific-antigen PSA, with easy to operate, detection speed is fast, testing result is accurately special
Point.
The invention is realized in this way a kind of fluorescence resonance energy transfer biosensor, which includes the first knot
Close probe and the second bonding probes；Wherein,
First bonding probes are hybridized by aptamer1 and block1 forms double-spiral structure, aptamer1 and block1
It is DNA sequence dna；Fluorescent molecule A is modified on 3 ' end of aptamer1 sequence, 5 ' end over-assembles one are used to combine target substance
The first PSA aptamers on mono- site PSA；
Second bonding probes are aptamer2, which is DNA sequence dna；Group on 3 ' end of aptamer2 sequence
Fill one for combine the another site target substance PSA on the 2nd PSA aptamers, modification fluorescent molecule B on 5 ' ends；
In the presence of target substance PSA, aptamer2 replaces block1 and hybridizes to form double helix with aptamer1
Structure, fluorescent molecule A, fluorescent molecule B are close to each other and form hyperfluorescence Resonance energy transfer effect.
Preferably, the DNA sequence dna of the aptamer1 are as follows:
5’-TTTTTAATTAAAGCTCGCCATCAAATAGCTGGGGGTTTTTTTTTTTTTTTTTTTTCCTCAAGATGG
TT-3'；
The DNA sequence dna of the Aptamer2 are as follows:
5’-TTCCATCTTGAGTTTTTTTTTTTTTTTTTTTTGCAATGGTACGGTACTTCCTATGGCGATGTGTTG
GCTGTGTGTGGGGTGCAAAAGTGCACGCTACTTTGCTAA-3'；
The DNA sequence dna of the block1 are as follows: 5 '-CCATCTTGAGG-3 '.
Preferably, the fluorescent molecule A is fluorescent molecule Cy3, and fluorescent molecule B is fluorescent molecule Cy5.
The present invention further discloses above-mentioned fluorescence resonance energy transfer biosensors specifically to resist to serum prostate
The application of the context of detection of former PSA.
Preferably, the application the following steps are included:
(1) by 95 DEG C to 4 DEG C annealing after aptamer1 stock solution and block1 stock solution addition buffer, the first knot is obtained
Close probe solution；By 95 DEG C to 4 DEG C annealing after aptamer2 stock solution addition buffer, the second bonding probes solution is obtained；
(2) PSA solution is added after mixing the first bonding probes solution, the second bonding probes solution, is uniformly mixed, room temperature
Fluorescence is surveyed after being protected from light 1 hour.
The present invention overcomes the deficiencies of the prior art and provide a kind of fluorescence resonance energy transfer biosensor and its preparation side
Method and application.Basic principle of the invention is as shown in Figure 1, wherein two basic change probe over-assemble two different PSA adaptations
Body, can be simultaneously in conjunction with the site different from PSA on, and bonding probes 1 are obtained by aptamer1 and block1 hybridization.
Fluorescent molecule Cy3 and and Cy5 have been modified on aptamer1 and aptamer2 respectively.In the case where no target substance PSA,
Aptamer1 and block1 form double helix hybrid structure, at this point, hybridizing for aptamer2 and aptamer1 is blocked, to make
It obtains Cy3 and Cy5 distance farther out, effective fluorescence resonance energy transfer can not be formed.And when two basic change probe is miscellaneous with PSA simultaneously
After friendship, since ortho position mediates Hybridization principle, aptamer2 and aptamer1 will form double-spiral structure, and block1 is replaced and is replaced
It changes.Cy3 and Cy5 is closer as a result, is capable of forming very strong fluorescence resonance energy transfer effect, also, this effect with
The concentration of PSA is directly proportional.
The present invention, using fluorescence resonance energy transfer technology, realizes the highly sensitive of PSA based on the immunoassay of ortho position
Detection, the present invention solve that the sensitivity in the presence of current markers in diagnosis is low, detection process is cumbersome, analysis cost
Height, marker diagnostic accuracy are poor and are difficult to meet on-site test deficiency.
Compared with the prior art the shortcomings that and deficiency, the invention has the following advantages: fluorescence resonance energy of the present invention
The preparation for shifting biosensor is simple, quick, and obtained fluorescence resonance energy transfer biosensor is used for clinical cancer
The specific detection of marker is conducive to early screening and the diagnosis of prostate cancer, have high sensitivity, detection process it is simple,
The advantages that analysis cost is low, marker diagnostic accuracy is good.
Detailed description of the invention
Fig. 1 is the schematic illustration of fluorescence resonance energy transfer biosensor of the present invention；
Fig. 2 is that fluorescence resonance energy transfer signal changes with time；
Fig. 3 is the fluorescence detection result of PSA series of concentrations standard solution；
Fig. 4 is signal response results of the different tumor markers in this detection architecture；
Fig. 5 is the PSA signal results figure reacted in different substrates.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
1, the preparation of the first bonding probes solution
Aptamer 1 and block1 stock solution are quantified with Ultraluminescence spectrophotometer, make 100 μ of its concentration
M.Then 2 μ L stock solutions is respectively taken to be dissolved in 100 μ L buffers (20mM HEPES, 500mM NaCl, pH 7.4), 95 DEG C to 4
It DEG C anneals, annealing process is 95 DEG C and cools to 4 DEG C ten minutes later, and is at least kept for 10 minutes at 4 DEG C.
2, the preparation of the second bonding probes solution
2 stock solution of aptamer is quantified with Ultraluminescence spectrophotometer, makes 100 μM of its concentration.Then 2 are taken
μ L stock solution is dissolved in 100 μ L buffers (20mM HEPES, 500mM NaCl, pH7.4), and 95 DEG C to 4 DEG C are annealed, and are moved back
Fiery process is 95 DEG C and cools to 4 DEG C ten minutes later, and is at least kept for 10 minutes at 4 DEG C.
3, PSA detection process
Take above-mentioned the first bonding probes solution prepared and each 49 μ L of the second bonding probes solution 2, be mixed in from
In heart pipe, then be added 2uL various concentration PSA solution, after mixing by solution, room temperature is protected from light 1 hour, obtain to
Detect solution.
4, fluorescence detection
The solution to be detected of above-mentioned reacted end is placed in 100 μ L fluorescence cuvettes, fluorescence spectrophotometry is passed through
Meter is detected, testing conditions are as follows: excitation wavelength 488nm, Detection wavelength range are 540~750nm.
5, influence of the differential responses time to PSA testing result
Above-mentioned the first bonding probes solution prepared and each 49 μ L of the second bonding probes solution are taken, centrifugation is mixed in
Then Guan Zhong is added 2uL PSA solution (0.5 μM) and after mixing by solution is placed in room temperature in 100 μ L fluorescence cuvettes and keeps away
Light reaction.Every 20 minutes survey first order fluorescences, measuring 100 minutes terminated.It is detected by sepectrophotofluorometer, detector bar
Part are as follows: excitation wavelength 488nm, Detection wavelength range are 540~750nm.
The fluorescence intensity of differential responses time point reaction system is as shown in Figure 2.When as can be drawn from Figure 2, with reaction
Between increase, absorption peak strength gradually decreases at 565nm, and the intensity of absorption peak gradually increases at 670nm.And more than 60 minutes
Afterwards, the variation of this peak intensity tends towards stability, therefore reacts 60 minutes and can reach maximum value.
6, the fluorescence detection of PSA series of concentrations standard solution
Above-mentioned the first bonding probes prepared and each 49 μ L of the second bonding probes are taken, are mixed in centrifuge tube, so
2uL PSA series of concentrations standard solution solution (0.5 μM) is added afterwards and after mixing by solution is placed in 100 μ L fluorescence cuvettes
In detected by sepectrophotofluorometer, testing conditions are as follows: excitation wavelength 488nm, Detection wavelength range be 540~
750nm。
The value added of fluorescent absorption and PSA concentration at 670nm is taken to carry out linear regression, obtained result is illustrated in fig. 3 shown below.
Regression equation is y=1.5+123.8x, regression coefficient are as follows: 0.991.Y indicates the fluorescence intensity obtained at 670nm in equation
Changing value, x indicate the concentration of object PSA.
7, selectivity of this reaction system to PSA
Above-mentioned the first bonding probes solution prepared and each 49 μ L of the second bonding probes solution are taken, centrifugation is mixed in
Then Guan Zhong is added 2uL PSA solution (0.5 μM) and after mixing by solution is placed in room temperature in 100 μ L fluorescence cuvettes and keeps away
Light reaction 1h, is detected by sepectrophotofluorometer, testing conditions are as follows: excitation wavelength 488nm, Detection wavelength range are
540~750nm.With same method, measures 2 μ L carcinomebryonic antigens (2 μM) are added within the probe respectively, cancer antigen 125 (2 μM) and swollen
After tumor necrosis factor (2 μM), the variation of fluorescence.
After different material is added, the fluorescence intensity at 670nm is as shown in Figure 4.As can be drawn from Figure 4, target is only added
After Substance P SA, the fluorescence intensity at this wavelength has very big promotion, and other tumor markers can not cause the increasing of signal
Add.
8, the influence that different complex matrices detect PSA
Above-mentioned the first bonding probes solution prepared and each 49 μ L of the second bonding probes solution are taken, centrifugation is mixed in
Then 2uL PSA solution (0.5 μM) is added in Guan Zhong, 10 μ L serum or 10 μ L cell pyrolysis liquids are added, solution is uniformly mixed
Afterwards, it is placed in room temperature in 100 μ L fluorescence cuvettes and is protected from light 1h, detected by sepectrophotofluorometer, testing conditions are as follows:
Excitation wavelength is 488nm, and Detection wavelength range is 540~750nm.
Fluorescence intensity at 670nm is as shown in Figure 5.From figure 5 it can be seen that either in buffer or in serum
And reacted in cell pyrolysis liquid, acquired results are not much different, and constructed bio-sensing system can effectively exclude multiple
The interference of miscellaneous matrix.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
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<120>a kind of fluorescence resonance energy transfer biosensor and its application
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Claims (5)
1. a kind of fluorescence resonance energy transfer biosensor, which is characterized in that the sensor includes the first bonding probes and the
Two bonding probes；Wherein,
First bonding probes are hybridized by aptamer1 and block1 forms double-spiral structure, and aptamer1 and block1 are
DNA sequence dna；Fluorescent molecule A is modified on 3 ' end of aptamer1 sequence, 5 ' end over-assembles one are used to combine target substance PSA mono-
The first PSA aptamers on site；
Second bonding probes are aptamer2, which is DNA sequence dna；3 ' end over-assemble one of aptamer2 sequence
A the 2nd PSA aptamers for combining on the another site target substance PSA, fluorescent molecule B is modified on 5 ' ends；
In the presence of target substance PSA, aptamer2 replaces block1 and hybridizes to form double-spiral structure with aptamer1,
Fluorescent molecule A, fluorescent molecule B are close to each other and form hyperfluorescence Resonance energy transfer effect.
2. fluorescence resonance energy transfer biosensor as described in claim 1, which is characterized in that the aptamer1's
DNA sequence dna are as follows:
5’-TTTTTAATTAAAGCTCGCCATCAAATAGCTGGGGGTTTTTTTTTTTTTTTTTTTTCCTCAAGATGGTT-
3'；
The DNA sequence dna of the Aptamer2 are as follows:
5’-TTCCATCTTGAGTTTTTTTTTTTTTTTTTTTTGCAATGGTACGGTACTTCCTATGGCGATGTGTTGGCTG
TGTGTGGGGTGCAAAAGTGCACGCTACTTTGCTAA-3'；
The DNA sequence dna of the block1 are as follows: 5 '-CCATCTTGAGG-3 '.
3. fluorescence resonance energy transfer biosensor as described in claim 1, which is characterized in that the fluorescent molecule A is
Fluorescent molecule Cy3, fluorescent molecule B are fluorescent molecule Cy5.
4. the described in any item fluorescence resonance energy transfer biosensors of claims 1 to 3 are specifically resisting serum prostate
The application of the context of detection of former PSA.
5. application as claimed in claim 4, which is characterized in that the application the following steps are included:
(1) it by 95 DEG C to 4 DEG C annealing after aptamer1 stock solution and block1 stock solution addition buffer, obtains first and combines spy
Needle solution；By 95 DEG C to 4 DEG C annealing after aptamer2 stock solution addition buffer, the second bonding probes solution is obtained；
(2) PSA solution is added after mixing the first bonding probes solution, the second bonding probes solution, is uniformly mixed, room temperature is protected from light
Reaction surveyed fluorescence after 1 hour.
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Cited By (3)
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| CN111789961A (en) * | 2020-08-26 | 2020-10-20 | 西南大学 | Nano probe for nucleolin cross-linking induction of tumor cell apoptosis and preparation method and application thereof |
| CN111789961B (en) * | 2020-08-26 | 2022-03-29 | 西南大学 | Nano probe for nucleolin cross-linking induction of tumor cell apoptosis and preparation method and application thereof |
| CN114395558A (en) * | 2022-01-17 | 2022-04-26 | 南通大学 | Magnetic bead-DNA probe, MC-LR detection biosensor, preparation method and application |
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