CN105606580B - Ratio-type FRET probes of nanometer human lymph node and preparation method and application - Google Patents
Ratio-type FRET probes of nanometer human lymph node and preparation method and application Download PDFInfo
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- 239000000523 sample Substances 0.000 title claims abstract description 67
- 238000002866 fluorescence resonance energy transfer Methods 0.000 title claims abstract description 45
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 210000001165 lymph node Anatomy 0.000 title abstract 2
- 108010081589 Becaplermin Proteins 0.000 claims abstract description 45
- 238000001514 detection method Methods 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 14
- 150000007523 nucleic acids Chemical group 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 51
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 50
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 37
- 239000011780 sodium chloride Substances 0.000 claims description 25
- 239000007853 buffer solution Substances 0.000 claims description 22
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 19
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 18
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 18
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 18
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 18
- 101500021084 Locusta migratoria 5 kDa peptide Proteins 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 11
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 10
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 claims description 9
- 229930064664 L-arginine Natural products 0.000 claims description 9
- 235000014852 L-arginine Nutrition 0.000 claims description 9
- 238000005119 centrifugation Methods 0.000 claims description 9
- 229910001961 silver nitrate Inorganic materials 0.000 claims description 9
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 9
- 239000012279 sodium borohydride Substances 0.000 claims description 9
- 239000001509 sodium citrate Substances 0.000 claims description 9
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 9
- 239000012498 ultrapure water Substances 0.000 claims description 9
- 239000002244 precipitate Substances 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims description 5
- 230000005284 excitation Effects 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 4
- 239000013049 sediment Substances 0.000 claims description 4
- 239000013642 negative control Substances 0.000 claims description 3
- 238000002372 labelling Methods 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 235000011083 sodium citrates Nutrition 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 7
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 230000001678 irradiating effect Effects 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000001917 fluorescence detection Methods 0.000 description 3
- 239000004332 silver Substances 0.000 description 3
- 229910052709 silver Inorganic materials 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- 108010017843 platelet-derived growth factor A Proteins 0.000 description 2
- 108010000685 platelet-derived growth factor AB Proteins 0.000 description 2
- 238000002331 protein detection Methods 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000013076 target substance Substances 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 1
- 101710103494 Platelet-derived growth factor subunit B Proteins 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000010206 sensitivity analysis Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
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Abstract
The invention discloses the Ratio-type FRET probes of nanometer human lymph node, and, using decahedral nanometer ping-pong ball as kernel, ping-pong ball outer surface is bonded with three kinds of different nucleic acid chains and forms FRET probe for it.The invention also discloses the preparation method and application of above-mentioned probe.The characteristics of this probe is when object be present, and the fluorescence intensity at 515nm and 565nm can change simultaneously, and the ratio of fluorescence intensity and the concentration of object have certain linear relationship under two wavelength.Such a mode quantitatively detected can not only improve the sensitivity of detection but also can avoid the interference of false positive signal and external environment.Application the invention also discloses the kit comprising above-mentioned probe and its in PDGF BB are detected.This method detection PDGF BB range of linearity is 3.12ng/mL 200ng/mL, and test limit is low, and specificity is good.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a nano-silver enhanced ratio type FRET probe, a preparation method thereof and application thereof in detection of PDGF-BB.
Background
Fluorescence detection has received a wide range of attention and applications due to its sensitive detection properties. Fluorescence Resonance Energy Transfer (FRET) is a very sensitive and widely used Fluorescence technique, and can be used for target molecule concentration determination, molecule interaction determination, catalytic activity determination and the like. The FRET-based sensing method is simpler and more convenient than the traditional immunoassay method, and does not need complicated steps such as cleaning and separation, and does not need to add a catalyst or other auxiliary reagents. Compared with spherical nano silver (CN103525815A, CN104569420A), the decahedral nano silver has better fluorescence enhancement property, and can obviously improve the sensitivity of fluorescence detection. It is very significant to develop a new FRET-based sensing analysis method by utilizing the characteristic of fluorescence enhancement of decahedral nano-silver to realize sensitive and accurate detection of protein. The current protein detection based on FRET probe has very rare literature, and we have developed a single channel FRET technology based on nano silver for protein detection (anal. chem.2013,85, 4492-4499). In this patent, to further increase the accuracy and immunity of FRET technology, we developed FRET sensing probes based on a dual channel mode (i.e., ratiometric FRET probes) for analytical detection of proteins.
Disclosure of Invention
The invention aims to solve the technical problem of providing a nano-silver enhanced ratio type FRET probe for sensitive and accurate detection of PDGF-BB.
The present invention also provides a method for preparing a FRET probe of the ratiometric type.
The invention also aims to solve the technical problem of providing the application of the probe in detecting PDGF-BB.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the nano-silver enhanced ratio type FRET probe takes decahedral nano-silver with the diameter of 50nm as an inner core, and three nucleic acid chains are bonded on the outer surface of the decahedral nano-silver;
wherein,
the A chain is:
A-FITC: 5'-SH C6-AAAAAAAAAAAGGTACGGATCTGCG-FITC-3'; or
A-Alex Fluor 488：5'-SH C6-AAAAAAAAAAAGGTACGGATCTGCG-Alex Fluor 488-3'；
The chain B is as follows: 5'-GTGCCGTAGCCTGCGCAGATCCGTACCT-3'
The C chain is: 5'-Cy 3-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-BHQ-2-3'.
The probe is characterized in that when a target object exists, the fluorescence intensities at 515nm and 565nm change simultaneously, and the ratio of the fluorescence intensities at two wavelengths has a certain linear relation with the concentration of the target object. The quantitative detection mode can not only improve the detection sensitivity, but also avoid the interference of false positive signals and the external environment.
The preparation method of the nano-silver enhanced ratio type FRET probe comprises the following steps:
(1) adding sodium citrate, polyvinylpyrrolidone (PVP), L-arginine and silver nitrate into high-purity water, adding sodium borohydride, and mixing well, whereinThe molar ratio of sodium citrate to polyvinylpyrrolidone to L-arginine to silver nitrate to sodium borohydride is (1-99): 0.1-9): 1-99, the mass of high-purity water is 100-999 times of that of polyvinylpyrrolidone, and the high-purity water is placed under a 12W-120W blue LED lamp and irradiated for 6-94h to obtain a decahedral nano-silver Ag10NPs solution;
(2) adding the A chain, the B chain and the C chain into the Ag obtained in the step (1) according to the mol ratio of (1-99) to (1-99)10In the NPs solution, the total volume of the three chains of the A chain, the B chain and the C chain and Ag10The volume ratio of the NPs is 1 (10-100);
(3) adding 1-5 mol/L sodium chloride solution into the mixed system obtained in the step (2), standing for 2-4 hours, and repeating the steps of adding the sodium chloride solution and standing for 1-4 times to enable the final concentration of the sodium chloride to be 0.1-0.5 mol/L;
(4) standing the mixed system obtained in the step (3) for 6-24 hours;
(5) centrifuging the solution obtained in the step (4) to remove supernatant, adding the precipitate into 1 × PBS buffer solution for resuspension, repeating the centrifuging and resuspending steps for 2-4 times (preferably 2 times), wherein the adding volume of the 1 × PBS buffer solution is 0.1-5 times of the volume of the mixed system to be added each time; finally, the sediment obtained by centrifugation is resuspended by 1 XPBSM buffer solution, and then the standby probe is obtained.
In the step (1), the preferable molar ratio of sodium citrate, polyvinylpyrrolidone (PVP), L-arginine, silver nitrate and sodium borohydride is 30:0.9:1:4: 16.
In step (1), the preferred mass ratio of water to PVP is 194: 1.
In step (1), generally, the higher the power of the LED lamp, the shorter the time required, and the half of the illumination time is reduced for each doubling of the power. Preferably under a 24W blue LED lamp for 48 h.
In the step (2), the A chain, the B chain and the C chain are preferably added to the Ag obtained in the step (1) according to the molar ratio of 1:1:110The concentration of the three chains in the NPs solution is 1-50. mu.M, respectivelyThe optimum concentration is 10. mu.M.
In the step (2), the total volume of the three chains of the chain A, the chain B and the chain C and Ag10The ratio of the volumes of the NPs is preferably 1: 13.
In the step (3), preferably, 2mol/L sodium chloride solution is added into the mixed system obtained in the step (2), and the mixture is allowed to stand for 2 hours.
In the step (3), the step of adding the sodium chloride solution and standing is preferably repeated 3 times so that the final concentration of sodium chloride is 0.3 mol/L.
In the step (4), the mixed system obtained in the step (3) is preferably left to stand for 12 hours.
In the step (5), the formula of the 1 × PBS buffer solution is as follows: 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4·12H2O，2mM KH2PO4The solvent is water.
In step (5), the addition volume of 1 XPBS buffer per time is preferably 1 time the volume of the mixed system to be added.
In the step (5), the 1 XPBSM formula is 1 XPBSM and 1mmol/L MgCl2A mixture of (a).
In the step (5), the centrifugation is carried out for 8-20 minutes under the condition of 6000-15000 rpm, and the preferred centrifugation condition is 12000rpm for 12 minutes.
The application of the nano-silver enhanced ratiometric FRET probe in the preparation of a reagent for detecting PDGF-BB is also within the protection scope of the invention.
A kit for detecting PDGF-BB, the kit comprising the following reagents: the nanosilver-enhanced ratiometric FRET probe of claim 1, phosphate buffer, PDGF-BB protein, bovine serum albumin, 96-well fluorescent plate.
The application of the kit for detecting PDGF-BB in the preparation of a reagent for detecting PDGF-BB is also within the protection scope of the invention.
The specific method for detecting the PDGF-BB content by using the PDGF-BB detection kit sequentially comprises the following steps of:
(1) preparing a solution:
buffer solution: diluting 20 XPBS with secondary water to 20 times to 1 XPBS, and adding 1mM magnesium chloride to prepare 1 XPBS; diluting 1 XPBS by 10 times to 0.1 XPBS, and adding 0.3M sodium chloride and 1mM magnesium chloride to prepare 0.3 MPBSM; dissolving 0.1g bovine serum albumin in 100mL of 1 XPBS solution to obtain 1mg/mLBSA, and adding 1mM magnesium chloride to obtain 1mg/mL of BSAM solution; adding 1mg/mL BSA into 0.3M PBSM solution to obtain 0.3M BSAM;
negative control: 1 mg/mLBSA;
the nano-silver enhanced ratiometric FRET probe;
(2) mixing the ratio type FRET probe with PDGF-BB solutions with different concentrations and a sample to be detected respectively for reacting for 50 minutes;
(3) directly scanning by a BioTeK enzyme-labeling instrument, wherein the excitation wavelength is 470nm, the emission wavelength is 515nm and 565nm, quantitatively analyzing the concentration of the target protein by simultaneously calculating the fluorescence intensity ratio of 515nm to 565nm, obtaining a PDGF-BB standard curve by corresponding different ratios of PDGF-BB solutions with different concentrations, and calculating the concentration of PDGF-BB in the sample by using the standard curve.
Has the advantages that: the ratiometric FRET probe of the invention can present a change by directly adding an analyte without using an antibody or forming a sandwich structure, thereby qualitatively or quantitatively analyzing the target substance. The PDGF-BB can be sensitively and accurately detected by using the FRET technology, the PDGF-BB analysis range is 3.12ng/mL-200ng/mL, the specificity is good, the generation of false positive signals is avoided, the interference of the external environment on the detection is reduced, and the signal acquisition mode is simple and quick.
Drawings
FIG. 1 is a schematic diagram of the synthesis and detection of PDGF-BB for ratiometric FRET probes.
FIG. 2 fluorescence spectra of Ag-Alex probe and silver enhanced FRET probe.
FIG. 3 is a graph showing the results of PDGF-BB detection using the ratiometric FRET probe, wherein (a) shows the specificity of PDGF-BB detection using the ratiometric FRET probe, and (b) shows the results of PDGF-BB detection using three different probes.
FIG. 4 is a graph showing the relationship between the fluorescence intensity and PDGF-BB at different concentrations, wherein the FRET probe (a) is 12.5. mu.L and the ratiometric FRET probe (b) is 6.2. mu.L.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.
Example 1: synthesis of silver enhanced FRET probes
(1) Adding sodium citrate (15mL,50mM), polyvinylpyrrolidone (PVP,1.5mM,15mL), L-arginine (2.5mL,10mM) and silver nitrate (10mL,10mM) into high-purity water (175mL), adding sodium borohydride (0.8mL,500mM), uniformly mixing, placing under a 24W blue LED lamp, and irradiating for 48h to obtain decahedral nano-silver Ag10NPs solution;
(2) adding the chain A, the chain B and the chain C into the Ag obtained in the step (1) according to the mol ratio of 1:1:110In the NPs solution, the concentrations and the volumes of three chains of an A chain, a B chain and a C chain are respectively 10 mu M and 12.5 mu L, and the volume of the nano silver is 0.5 mL.
The chain A, the chain B and the chain C are respectively as follows:
the A chain is:
A-Alex Fluor 488：5'-SH C6-AAAAAAAAAAAGGTACGGATCTGCG-Alex Fluor 488-3'；
the chain B is as follows: 5'-GTGCCGTAGCCTGCGCAGATCCGTACCT-3'
The C chain is: 5'-Cy 3-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-BHQ-2-3'.
(3) Adding 2mol/L sodium chloride solution into the mixed system obtained in the step (2), standing for 2 hours, and repeating the steps of adding the sodium chloride solution and standing for 2 times to ensure that the final concentration of the sodium chloride is 0.3 mol/L;
(4) standing the mixed system obtained in the step (3) for 12 hours;
(5) centrifuging the solution obtained in the step (4) to remove supernatant, adding the precipitate into 1 × PBS buffer solution for resuspension, repeating the steps of centrifuging and resuspending for 2 times, wherein the adding volume of the 1 × PBS buffer solution is 1 time of the volume of the mixed system to be added; and finally, resuspending the precipitate obtained by centrifugation by using a 1 XPBSM buffer solution to obtain the standby probe Ag-Alex/Cy 3/BHQ-2. The fluorescence spectrum of the probe is shown in FIG. 2.
Example 2: synthesis of silver enhanced FRET probes
(1) Adding sodium citrate (15mL,50mM), polyvinylpyrrolidone (PVP,1.5mM,15mL), L-arginine (2.5mL,10mM) and silver nitrate (10mL,10mM) into high-purity water (175mL), adding sodium borohydride (0.8mL,500mM), uniformly mixing, placing under a 24W blue LED lamp, and irradiating for 48h to obtain decahedral nano-silver Ag10NPs solution;
(2) adding the chain A, the chain B and the chain C into the Ag obtained in the step (1) according to the mol ratio of 1:1:110In the NPs solution, the concentrations and the volumes of three chains of an A chain, a B chain and a C chain are respectively 10 mu M and 12.5 mu L, and the volume of the nano silver is 0.5 mL.
The chain A, the chain B and the chain C are respectively as follows:
the A chain is:
A-FITC：5'-SH C6-AAAAAAAAAAAGGTACGGATCTGCG-FITC-3'；
the chain B is as follows: 5'-GTGCCGTAGCCTGCGCAGATCCGTACCT-3'
The C chain is: 5'-Cy 3-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-BHQ-2-3'.
(3) Adding 2mol/L sodium chloride solution into the mixed system obtained in the step (2), standing for 2 hours, and repeating the steps of adding the sodium chloride solution and standing for 2 times to ensure that the final concentration of the sodium chloride is 0.3 mol/L;
(4) standing the mixed system obtained in the step (3) for 12 hours;
(5) centrifuging the solution obtained in the step (4) to remove supernatant, adding the precipitate into 1 × PBS buffer solution for resuspension, repeating the steps of centrifuging and resuspending for 2 times, wherein the adding volume of the 1 × PBS buffer solution is 1 time of the volume of the mixed system to be added; and finally, resuspending the precipitate obtained by centrifugation by using a 1 XPBSM buffer solution to obtain the standby probe Ag-FITC/Cy 3/BHQ-2.
Example 3:
(1) adding sodium citrate (15mL,500mM), polyvinylpyrrolidone (PVP,15mM,15mL), L-arginine (2.5mL,100mM), and silver nitrate (10mL,100mM) into high purity water (175mL), adding sodium borohydride (0.8mL,500mM), mixing, placing under 24W blue LED lamp, and irradiating for 94h to obtain decahedral nano-silver Ag10NPs solution;
(2) adding the chain A, the chain B and the chain C into the Ag obtained in the step (1) according to the mol ratio of 1:1:110In the NPs solution, the concentration and volume of three chains of A chain, B chain and C chain are 10 MuM and 5 MuL, and the volume of nano silver is 0.5 mL.
The chain A, the chain B and the chain C are respectively as follows:
the A chain is:
A-Alex Fluor 488：5'-SH C6-AAAAAAAAAAAGGTACGGATCTGCG-Alex Fluor 488-3'；
the chain B is as follows: 5'-GTGCCGTAGCCTGCGCAGATCCGTACCT-3'
The C chain is: 5'-Cy 3-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-BHQ-2-3'.
(3) Adding 2mol/L sodium chloride solution into the mixed system obtained in the step (2), standing for 2 hours, and repeating the steps of adding the sodium chloride solution and standing for 2 times to ensure that the final concentration of the sodium chloride is 0.3 mol/L;
(4) standing the mixed system obtained in the step (3) for 24 hours;
(5) centrifuging the solution obtained in the step (4) to remove supernatant, adding the precipitate into 1 × PBS buffer solution for resuspension, repeating the steps of centrifuging and resuspending for 2 times, wherein the adding volume of the 1 × PBS buffer solution is 2 times of the volume of the mixed system to be added; finally, the sediment obtained by centrifugation is resuspended by 1 XPBSM buffer solution, and then the standby probe is obtained.
Example 4:
(1) adding sodium citrate (15mL,5mM), polyvinylpyrrolidone (PVP,0.15mM,15mL), L-arginine (2.5mL,1mM) and silver nitrate (10mL,1mM) into high-purity water (175mL), adding sodium borohydride (0.8mL,50mM), uniformly mixing, placing under a 24W blue LED lamp, and irradiating for 24h to obtain decahedral nano-silver Ag10NPs solution;
(2) adding the chain A, the chain B and the chain C into the Ag obtained in the step (1) according to the mol ratio of 1:1:110In the NPs solution, the concentration and volume of three chains of A chain, B chain and C chain are 10 mu M,7.5 mu L and the volume of nano silver is 2 mL.
The chain A, the chain B and the chain C are respectively as follows:
the A chain is:
A-Alex Fluor 488：5'-SH C6-AAAAAAAAAAAGGTACGGATCTGCG-Alex Fluor 488-3'；
the chain B is as follows: 5'-GTGCCGTAGCCTGCGCAGATCCGTACCT-3'
The C chain is: 5'-Cy 3-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-BHQ-2-3'.
(3) Adding 2mol/L sodium chloride solution into the mixed system obtained in the step (2), standing for 4 hours, and repeating the steps of adding the sodium chloride solution and standing for 2 times to ensure that the final concentration of the sodium chloride is 0.3 mol/L;
(4) standing the mixed system obtained in the step (3) for 12 hours;
(5) centrifuging the solution obtained in the step (4) to remove supernatant, adding the precipitate into 1 × PBS buffer solution for resuspension, repeating the steps of centrifuging and resuspending for 2 times, wherein the adding volume of the 1 × PBS buffer solution is 1 time of the volume of the mixed system to be added; finally, the sediment obtained by centrifugation is resuspended by 1 XPBSM buffer solution, and then the standby probe is obtained.
Example 5: specificity detection
Synthesis of probes synthesized according to examples 1-3,
(1) preparing a solution:
buffer solution: diluting 20 XPBS with secondary water to 20 times to 1 XPBS, and adding 1mM magnesium chloride to prepare 1 XPBS; diluting 1 XPBS by 10 times to 0.1 XPBS, and adding 0.3M sodium chloride and 1mM magnesium chloride to prepare 0.3 MPBSM; dissolving 0.1g bovine serum albumin in 100mL of 1 XPBS solution to obtain 1mg/mLBSA, and adding 1mM magnesium chloride to obtain 1mg/mL of BSAM solution; adding 1mg/mL BSA into 0.3M PBSM solution to obtain 0.3M BSAM;
negative control: 1 mg/mLBSA;
the nano-silver enhanced ratiometric FRET probe;
(2) mixing the ratio type FRET probe with different analytes such as PDGF-AB, PDGF-BB, PDGF-AA and 2% serum respectively for 50 minutes;
(3) direct scanning with BioTeK microplate reader, excitation wavelength 470nm, emission wavelength 515nm and 565 nm. The concentration of the target substance is quantified by simultaneously calculating the ratio of the fluorescence intensities at 515nm and 565nm, the reaction diagram is shown in FIG. 1, and the experimental result is shown in FIG. 3 a. The FRET probe prepared as in example 1 has a low non-specific adsorption to PDGF-AA, PDGF-AB and serum, and a high affinity for PDGF-BB.
Example 6: sensitivity analysis
Preparation method of control probe Ag-Alex/Cy 3:
(1) adding the A chain, the B chain and the C control chain into the Ag obtained in the step (1) according to the molar ratio of 1:1:110In the NPs solution, the concentration and volume of three chains of the A chain, the B chain and the C control chain are 10 mu M,12.5 mu L and the volume of nano silver is 0.5 mL.
The A chain, the B chain and the C control chain are respectively as follows:
the A chain is:
A-Alex Fluor 488：5'-SH C6-AAAAAAAAAAAGGTACGGATCTGCG-Alex Fluor 488-3'；
the chain B is as follows: 5'-GTGCCGTAGCCTGCGCAGATCCGTACCT-3'
The C control strand is: 5'-Cy 3-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3'.
Other procedures different probes were synthesized according to examples 1-3.
A preparation method of a control probe Alex/Cy3/BHQ-2 comprises the following steps:
mix A chain, B chain and C contrast chain in the molar ratio of 1:1:1 to react for 1h, the concentration and volume of three chains of A chain, B chain and C contrast chain are 10 μ M,12.5 μ L, dilute them to the same concentration of the probe of this patent after reaction.
The A chain, the B chain and the C control chain are respectively as follows:
the A chain is:
A-Alex Fluor 488：5'-SH C6-AAAAAAAAAAAGGTACGGATCTGCG-Alex Fluor 488-3'；
the chain B is as follows: 5'-GTGCCGTAGCCTGCGCAGATCCGTACCT-3'
The C control strand is: 5'-Cy 3-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3'.
The sensitivity of the three probes, Ag-Alex/Cy3, Ag-Alex/Cy3/BHQ-2 and Alex/Cy3/BHQ-2, for detecting the protein PDGF-BB was analyzed.
Following the procedure of example 4, the procedure was followed,
(1) mixing different FRET probes with PDGF-BB to be detected for 50 minutes;
(2) direct scanning with BioTeK microplate reader, excitation wavelength 470nm, emission wavelength 515nm and 565 nm. The sensitivity of the different probes was analyzed by simultaneously calculating the ratio of the fluorescence intensities at 515nm and 565nm, and the experimental results are shown in FIG. 3 b. The result shows that the Ag-Alex/Cy3/BHQ-2 probe prepared by the method has higher sensitivity.
Example 7: method for detecting PDGF-BB content
Synthesis of probes synthesized according to examples 1-3,
(1) mixing the ratio type FRET probe with PDGF-BB at different concentrations for 50 minutes;
(2) direct scanning with BioTeK microplate reader, excitation wavelength 470nm, emission wavelength 515nm and 565 nm. The concentration of the target protein is quantitatively analyzed by simultaneously calculating the fluorescence intensity ratio of 515nm and 565nm, PDGF-BB solutions with different concentrations correspond to different ratios to obtain a PDGF-BB standard curve, and the concentration of PDGF-BB in the sample is calculated by the standard curve. Standard curves for PDGF-BB obtained with different volumes of probe are shown in FIG. 4.
The invention applies the ratio type FRET probe to PDGF-BB analysis and detection, when the PDGF-BB concentration is between 3.12ng/mL and 200ng/mL, the fluorescence signal intensity has good correlation with the concentration, and the correlation coefficient is 0.989. The method can greatly improve the sensitivity of fluorescence detection, not only can avoid the generation of false positive signals, but also can prevent the interference of external environment, and the signal acquisition mode is simple and quick.
Claims (7)
1. The nano-silver enhanced ratio type FRET probe is characterized in that decahedral nano-silver with the diameter of 50nm is used as an inner core, and three nucleic acid chains are bonded on the outer surface of the decahedral nano-silver;
wherein,
the A chain is:
A-FITC: 5'-SH C6-AAAAAAAAAAAGGTACGGATCTGCG-FITC-3'; or
A-Alex Fluor 488：5′-SH C6-AAAAAAAAAAAGGTACGGATCTGCG-Alex Fluor 488-3′；
The chain B is as follows: 5'-GTGCCGTAGCCTGCGCAGATCCGTACCT-3'
The C chain is: 5'-Cy 3-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-BHQ-2-3'.
2. The method for preparing a nanosilver enhanced ratiometric FRET probe of claim 1, comprising the steps of:
(1) adding sodium citrate, polyvinylpyrrolidone, L-arginine and silver nitrate into high-purity water, adding sodium borohydride and uniformly mixing, wherein the molar ratio of the sodium citrate to the polyvinylpyrrolidone to the L-arginine to the silver nitrate to the sodium borohydride is (1-99) to (0.1-9) to (1-99), the mass of the high-purity water is 100-999 times of that of the polyvinylpyrrolidone, and placing the mixture under a 12-120W blue LED lamp to irradiate for 6-94 hours to obtain decahedral nano-silver Ag10NPs solution;
(2) adding the chain A, the chain B and the chain C into the Ag obtained in the step (1) according to the molar ratio of (1-99) to (1-99)10In the NPs solution, the total volume of the three chains of the A chain, the B chain and the C chain and Ag10The volume ratio of the NPs is 1: 10-100;
(3) adding 1-5 mol/L sodium chloride solution into the mixed system obtained in the step (2), standing for 2-4 hours, and repeating the steps of adding the sodium chloride solution and standing for 1-4 times to enable the final concentration of the sodium chloride to be 0.1-0.3 mol/L;
(4) standing the mixed system obtained in the step (3) for 6-24 hours;
(5) centrifuging the solution obtained in the step (4) to remove supernatant, adding the precipitate into 1 × PBS buffer solution for resuspension, repeating the steps of centrifuging and resuspending for 2-4 times, wherein the adding volume of the 1 × PBS buffer solution is 0.1-5 times of the volume of the mixed system to be added each time; finally, the sediment obtained by centrifugation is resuspended by 1 XPBSM buffer solution, and then the standby probe is obtained.
3. The method for preparing a nano-silver enhanced ratiometric FRET probe according to claim 2, wherein in the step (5), the centrifugation is performed at 6000 to 15000rpm for 8 to 20 minutes.
4. Use of the nanosilver-enhanced ratiometric FRET probe of claim 1 in the preparation of a reagent for the detection of PDGF-BB.
5. A kit for detecting PDGF-BB, the kit comprising the following reagents: the nanosilver-enhanced ratiometric FRET probe of claim 1, phosphate buffer, PDGF-BB protein, bovine serum albumin, 96-well fluorescent plate.
6. Use of the kit of claim 5 for the detection of PDGF-BB.
7. The use of claim 6, wherein the specific method for detecting PDGF-BB content using a PDGF-BB detection kit comprises the following steps in the following order:
(1) preparing a solution:
buffer solution: diluting 20 XPBS with secondary water to 20 times to 1 XPBS, and adding 1mM magnesium chloride to prepare 1 XPBS; diluting 1 XPBS by 10 times to 0.1 XPBS, and adding 0.3M sodium chloride and 1mM magnesium chloride to prepare 0.3 MPBSM; dissolving 0.1g bovine serum albumin in 100mL of 1 XPBS solution to obtain 1mg/mLBSA, and adding 1mM magnesium chloride to obtain 1mg/mL of BSAM solution; adding 1mg/mL BSA into 0.3M PBSM solution to obtain 0.3M BSAM;
negative control: 1 mg/mLBSA;
the nanosilver enhanced ratiometric FRET probe of claim 1;
(2) mixing the ratio type FRET probe with PDGF-BB solutions with different concentrations and a sample to be detected respectively for reacting for 50 minutes;
(3) directly scanning by a BioTeK enzyme-labeling instrument, wherein the excitation wavelength is 470nm, the emission wavelength is 515nm and 565nm, quantitatively analyzing the concentration of the target protein by simultaneously calculating the fluorescence intensity ratio of 515nm to 565nm, obtaining a PDGF-BB standard curve by corresponding different ratios of PDGF-BB solutions with different concentrations, and calculating the concentration of PDGF-BB in the sample by using the standard curve.
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