CN113670887A - Method for detecting mercury ions by using fluorescent molecular probe based on nucleic acid aptamer - Google Patents
Method for detecting mercury ions by using fluorescent molecular probe based on nucleic acid aptamer Download PDFInfo
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- -1 mercury ions Chemical class 0.000 title claims abstract description 67
- 239000003068 molecular probe Substances 0.000 title claims abstract description 57
- 229910052753 mercury Inorganic materials 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 34
- 108091008104 nucleic acid aptamers Proteins 0.000 title claims abstract description 28
- 239000002105 nanoparticle Substances 0.000 claims abstract description 79
- 238000006243 chemical reaction Methods 0.000 claims abstract description 47
- 238000001514 detection method Methods 0.000 claims abstract description 42
- 229920002125 Sokalan® Polymers 0.000 claims abstract description 35
- 239000004584 polyacrylic acid Substances 0.000 claims abstract description 35
- 239000007864 aqueous solution Substances 0.000 claims abstract description 15
- 238000007112 amidation reaction Methods 0.000 claims abstract description 4
- 239000003446 ligand Substances 0.000 claims abstract description 3
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 claims description 54
- 108091023037 Aptamer Proteins 0.000 claims description 51
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 claims description 47
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 claims description 47
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 claims description 47
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 claims description 47
- 239000005642 Oleic acid Substances 0.000 claims description 47
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 claims description 47
- 239000011259 mixed solution Substances 0.000 claims description 35
- CCCMONHAUSKTEQ-UHFFFAOYSA-N octadec-1-ene Chemical compound CCCCCCCCCCCCCCCCC=C CCCMONHAUSKTEQ-UHFFFAOYSA-N 0.000 claims description 34
- 238000003756 stirring Methods 0.000 claims description 31
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 30
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- BQPIGGFYSBELGY-UHFFFAOYSA-N mercury(2+) Chemical compound [Hg+2] BQPIGGFYSBELGY-UHFFFAOYSA-N 0.000 claims description 30
- 239000008055 phosphate buffer solution Substances 0.000 claims description 29
- 239000006185 dispersion Substances 0.000 claims description 25
- 239000007788 liquid Substances 0.000 claims description 25
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- 239000000243 solution Substances 0.000 claims description 22
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- 230000009471 action Effects 0.000 claims description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 238000005406 washing Methods 0.000 claims description 15
- 239000007850 fluorescent dye Substances 0.000 claims description 14
- 238000003760 magnetic stirring Methods 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 239000008367 deionised water Substances 0.000 claims description 13
- 229910021641 deionized water Inorganic materials 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 11
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 10
- 238000005119 centrifugation Methods 0.000 claims description 10
- 238000001816 cooling Methods 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 9
- DDFHBQSCUXNBSA-UHFFFAOYSA-N 5-(5-carboxythiophen-2-yl)thiophene-2-carboxylic acid Chemical compound S1C(C(=O)O)=CC=C1C1=CC=C(C(O)=O)S1 DDFHBQSCUXNBSA-UHFFFAOYSA-N 0.000 claims description 7
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 5
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 5
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 5
- 229910052691 Erbium Inorganic materials 0.000 claims description 5
- 229910052769 Ytterbium Inorganic materials 0.000 claims description 5
- HWJJKWDAKXZNEA-UHFFFAOYSA-N [Na].ON1C(CCC1=O)=S Chemical compound [Na].ON1C(CCC1=O)=S HWJJKWDAKXZNEA-UHFFFAOYSA-N 0.000 claims description 5
- 229910052786 argon Inorganic materials 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 5
- UYAHIZSMUZPPFV-UHFFFAOYSA-N erbium Chemical compound [Er] UYAHIZSMUZPPFV-UHFFFAOYSA-N 0.000 claims description 5
- UJBPGOAZQSYXNT-UHFFFAOYSA-K trichloroerbium;hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Cl-].[Er+3] UJBPGOAZQSYXNT-UHFFFAOYSA-K 0.000 claims description 5
- LEYFXTUKPKKWMP-UHFFFAOYSA-K trichloroytterbium;hexahydrate Chemical compound O.O.O.O.O.O.Cl[Yb](Cl)Cl LEYFXTUKPKKWMP-UHFFFAOYSA-K 0.000 claims description 5
- IINACGXCEZNYTF-UHFFFAOYSA-K trichloroyttrium;hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Cl-].[Y+3] IINACGXCEZNYTF-UHFFFAOYSA-K 0.000 claims description 5
- 238000002604 ultrasonography Methods 0.000 claims description 5
- NAWDYIZEMPQZHO-UHFFFAOYSA-N ytterbium Chemical compound [Yb] NAWDYIZEMPQZHO-UHFFFAOYSA-N 0.000 claims description 5
- 229910052727 yttrium Inorganic materials 0.000 claims description 5
- VWQVUPCCIRVNHF-UHFFFAOYSA-N yttrium atom Chemical compound [Y] VWQVUPCCIRVNHF-UHFFFAOYSA-N 0.000 claims description 5
- 239000002994 raw material Substances 0.000 claims description 2
- 238000005979 thermal decomposition reaction Methods 0.000 claims description 2
- 238000001132 ultrasonic dispersion Methods 0.000 claims description 2
- 230000008901 benefit Effects 0.000 abstract description 5
- 150000002500 ions Chemical class 0.000 abstract description 5
- 238000011895 specific detection Methods 0.000 abstract description 5
- 229910001385 heavy metal Inorganic materials 0.000 abstract description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 238000010521 absorption reaction Methods 0.000 description 8
- 238000010586 diagram Methods 0.000 description 6
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 4
- 238000002189 fluorescence spectrum Methods 0.000 description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 4
- 239000001103 potassium chloride Substances 0.000 description 4
- 235000011164 potassium chloride Nutrition 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 238000000862 absorption spectrum Methods 0.000 description 3
- 238000002329 infrared spectrum Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000002441 X-ray diffraction Methods 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 description 1
- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical compound [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 description 1
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
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- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 description 1
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- 239000011572 manganese Substances 0.000 description 1
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- 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"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- 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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Abstract
The invention discloses a method for detecting mercury ions by a fluorescent molecular probe based on a nucleic acid aptamer, and belongs to the technical field of detection of heavy metal ions. The method comprises the steps of firstly preparing polyacrylic acid modified up-conversion nanoparticles by utilizing a surface ligand exchange reaction, then connecting a nucleic acid aptamer to the surface of the up-conversion nanoparticles by adopting an amidation reaction, preparing a nucleic acid aptamer fluorescent molecular probe, and finally drawing a standard working curve of fluorescence intensity-mercury ion concentration to establish a linear working equation. The detection method provided by the invention can realize the specific detection of mercury ions in the aqueous solution, has the advantages of wide linear detection range and low detection limit, and has wide application prospect in the fields of food, medical treatment and environment.
Description
Technical Field
The invention belongs to the technical field of heavy metal ion detection, and particularly relates to a method for detecting mercury ions by using a fluorescence molecular probe based on a nucleic acid aptamer.
Background
Mercury is a typical heavy metal pollutant, and has attracted wide attention all over the world due to its high toxicity, strong pollution persistence, bioaccumulation in the environment, and the like. Divalent mercury ions (Hg) are the most stable form of inorganic mercury2+) Even at low concentrations, can cause severe stressA serious human health problem. Mercury ions in the environment, water and food accumulate in the body through blood circulation when Hg is contained2+When the concentration of (A) is up to 0.025 mmol/L, various degrees of damage may occur to various organs in the human body, such as the heart, kidney, brain, stomach and intestinal tract. Therefore, Hg is carried out2+The method has very important significance for human health and ecosystem stability. Conventional Hg2+The detection method comprises atomic fluorescence spectroscopy, gas chromatography-mass spectrometry, electrochemical method, spectrophotometry, inductively coupled plasma mass spectrometry and the like. However, these methods often have the problems of expensive instruments, long analysis period, complex sample pretreatment, expensive detection cost and the like, and are increasingly not suitable for the requirements of convenience, rapidness, high sensitivity and the like of mercury ion detection. Therefore, it is urgently needed to develop and establish a method for detecting mercury ions quickly, in trace quantity, with high sensitivity and specificity.
Disclosure of Invention
Aiming at the defects of the existing method for detecting mercury ions, the invention provides a method for detecting mercury ions by a fluorescent molecular probe based on a nucleic acid aptamer. The detection method can realize the specific detection of the mercury ions in the aqueous solution, has the advantages of wide linear detection range, low detection limit, convenience and quickness, and has wide application prospect in the fields of food, medical treatment and environment.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting mercury ions by a fluorescent molecular probe based on a nucleic acid aptamer comprises the steps of taking polyacrylic acid modified up-conversion nanoparticles as an energy donor, and taking a fluorescent dye Cy5 as an energy acceptor; the method specifically comprises the following steps:
s1: preparing polyacrylic acid modified up-conversion nanoparticles:
preparing oleic acid modified upconversion nanoparticles by using rare earth chloride, oleic acid, 1-octadecene, ammonium fluoride and sodium hydroxide as raw materials through a high-temperature thermal decomposition method, and preparing polyacrylic acid modified upconversion nanoparticles by using a surface ligand exchange reaction;
s2: preparation of the aptamer fluorescent molecular probe:
adopting amidation reaction, connecting aptamer on the surface of the polyacrylic acid modified upconversion nano particle, and preparing a nucleic acid aptamer fluorescent molecular probe;
s3: drawing a standard working curve of fluorescence intensity-mercury ion concentration, and establishing a linear working equation:
adding mercury ion aqueous solutions with different concentrations into the nucleic acid aptamer fluorescent molecular probe in the step S2 to prepare a detection solution; and detecting the fluorescence intensity of the detection solution with different mercury ion concentrations under the action of 980 nm laser, drawing a standard working curve of the fluorescence intensity-mercury ion concentration, and establishing a linear working equation.
In the method for detecting mercury ions by using the aptamer-based fluorescent molecular probe, the step S1 is as follows:
adding 1 mmol of rare earth chloride into a mixed solution of 3-9 mL of oleic acid and 10-20 mL of 1-octadecene, magnetically stirring for 10-30 min at 90-130 ℃ under the protection of argon, then heating to 140-180 ℃, and continuing to magnetically stir for 20-40 min to prepare a mixed solution of the rare earth chloride, the oleic acid and the 1-octadecene; under magnetic stirring, naturally cooling the mixed solution of the rare earth chloride, oleic acid and 1-octadecene to room temperature, dropwise adding 5-15 mL of methanol solution dissolved with 0.1-0.2 g of ammonium fluoride and 0.05-0.2 g of sodium hydroxide, heating to 40-60 ℃, magnetically stirring for 20-40 min, heating to 60-80 ℃, magnetically stirring for 5-20 min, heating to 100-120 ℃, magnetically stirring for 5-20 min, and finally heating to 280-320 ℃, and magnetically stirring for 60-120 min; after the reaction is finished, under the magnetic stirring, naturally cooling the reaction mixed solution to room temperature, and washing the reaction mixed solution by using a mixed solution of ethanol and cyclohexane in a volume ratio of 1: 1-3: 1 through centrifugation to prepare the oleic acid modified up-conversion nanoparticles;
adding 20-60 mg of oleic acid modified up-conversion nanoparticles into 3-5 mL of chloroform, and performing ultrasonic dispersion for 5-20 min at room temperature to prepare an oleic acid modified up-conversion nanoparticle chloroform dispersion liquid; adding the oleic acid modified upconversion nanoparticle chloroform dispersion liquid into 5-20 mL of ethanol dispersion liquid dissolved with 100-300 mg of polyacrylic acid, magnetically stirring at room temperature for 12-20 h, and washing by using a mixed solution of ethanol and deionized water in a volume ratio of 1: 1-3: 1 through centrifugation to obtain the polyacrylic acid modified upconversion nanoparticle.
The rare earth chloride in the step I comprises yttrium chloride hexahydrate, ytterbium chloride hexahydrate and erbium chloride hexahydrate, wherein the molar ratio of yttrium, ytterbium and erbium elements is (60-80): (20-30): 1-3).
In the method for detecting mercury ions by using the aptamer-based fluorescent molecular probe, the step S2 is as follows:
dispersing 5-10 mg of polyacrylic acid modified up-conversion nanoparticles in 4-10 mL of dimethyl sulfoxide under the action of ultrasound, adding 10-20 mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 30-40 mg of N-hydroxy thiosuccinimide sodium salt, and finally adding 0.04-0.08 mL of PBS buffer solution dissolved with aptamer to obtain mixed dispersion liquid;
and secondly, incubating the mixed dispersion liquid obtained in the step I at 30-40 ℃ for 12-24 h, centrifuging the product, washing the product with PBS (phosphate buffer solution), and ultrasonically dispersing the product in 4 mL of PBS at room temperature to prepare the aptamer fluorescent molecular probe.
The PBS buffer solution in the step S2 is prepared by dissolving 8 g of sodium chloride, 0.24 g of potassium dihydrogen phosphate, 1.44 g of disodium hydrogen phosphate and 0.2 g of potassium chloride in 800 mL of deionized water, adjusting the pH value to 7.4 by using 0.1 mol/L hydrochloric acid, adding 200 mL of deionized water, and performing ultrasonic action.
The complete sequence of the aptamer in the step S2 is 5' -cysteine-Cy 5-TTGTTTGTCCCCTCTTTCTTA- (CH)2)6-NH2-3'; the concentration of the aptamer in the PBS buffer was 0.1 mmol/L.
In the method for detecting mercury ions by using the aptamer-based fluorescent molecular probe, the step S3 is as follows:
adding 0.1 mL of mercury ion aqueous solutions with different concentrations into 0.1 mL of nucleic acid aptamer fluorescent molecular probes respectively, and incubating for 40 min at room temperature to prepare a detection solution;
secondly, under the action of 980 nm laser, testing the fluorescence intensity of the detection solution prepared in the first step at 654 nm, drawing a standard working curve of fluorescence intensity-mercury ion concentration by taking the mercury ion concentration as a horizontal coordinate and the fluorescence intensity as a vertical coordinate, and establishing a linear working equation.
Preferably, the concentration of the mercury ions in the mercury ion aqueous solution in the step S3 is 0.1 nmol/L, 0.5 nmol/L, 1nmol/L, 5 nmol/L, 50 nmol/L, 100 nmol/L, and 500 nmol/L.
The application of the method in mercury ion detection.
The invention has the following remarkable advantages:
(1) according to the invention, polyacrylic acid modified up-conversion nanoparticles are used as an energy donor, a fluorescent dye Cy5 is used as an energy acceptor, and a fluorescence resonance energy transfer system is constructed, so that the advantage of low detection background can be achieved by utilizing laser-induced up-conversion fluorescence emission, the detection sensitivity is obviously improved, separation is not required in the detection process, and the operation is simple and convenient.
(2) The invention adopts amidation reaction to prepare aptamer 5' -Cytosine-Cy5-TTGTTTGTCCCCTCTTTCTTA- (CH) containing fluorescent dye Cy52)6-NH2The 3' -is connected to the surface of the upconversion nano particle, and the accuracy and the stability of detecting the mercury ions are improved by utilizing the specificity recognition of the nucleic acid aptamer to the mercury ions.
(3) In the invention, the up-conversion nanoparticles and the fluorescent dye Cy5 are arranged at two ends of the aptamer, so that complementary chains are avoided, the preparation steps of the fluorescent molecular probe are simplified, the preparation time of the fluorescent molecular probe is shortened, and the preparation cost of the fluorescent molecular probe is reduced.
(4) The detection method disclosed by the invention can realize specific detection on mercury ions in an aqueous solution, and has the advantages of wide linear detection range, low detection limit, convenience and quickness, wherein the linear detection range is 0.1-500 nmol/L, the linear correlation coefficient is 0.9508-0.9647, and the detection limit is 1.2575-1.2965 nmol/L, so that the detection method has a wide application prospect in the fields of food, medical treatment and environment.
Drawings
FIG. 1 is a schematic diagram of the mechanism of detecting mercury ions by the aptamer-based fluorescent molecular probe of the present invention.
Fig. 2 is an infrared spectrum of the oleic acid-modified upconversion nanoparticles and polyacrylic acid-modified upconversion nanoparticles prepared in example 1 of the present invention.
FIG. 3 is a diagram of the UV-VIS absorption spectrum of the polyacrylic acid modified upconversion nanoparticle and aptamer fluorescent molecular probe prepared in example 1 of the present invention.
FIG. 4 is an X-ray diffraction pattern of oleic acid modified upconversion nanoparticles prepared in example 1 of the present invention.
Fig. 6 is a scanning electron microscope photograph of the oleic acid-modified upconversion nanoparticles prepared in example 1 of the present invention.
Fig. 5 is a fluorescence emission spectrum of the oleic acid-modified upconversion nanoparticle prepared in example 1 of the present invention and an ultraviolet-visible absorption spectrum of a fluorescent dye Cy 5.
FIG. 7 is a fluorescence emission spectrum of the aptamer fluorescence molecular probe prepared in example 1 of the present invention at different mercury ion concentrations.
FIG. 8 is a standard working curve of "fluorescence intensity vs. mercury ion concentration" obtained in example 1 of the present invention.
FIG. 9 is a diagram showing the results of specific detection of the aptamer fluorescent molecular probe prepared in example 1 of the present invention.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
Example 1
A method for detecting mercury ions by a fluorescent molecular probe based on a nucleic acid aptamer specifically comprises the following steps:
s1: preparation of polyacrylic acid modified up-conversion nanoparticles
Adding 1 mmol of rare earth chloride into a mixed solution of 6 mL of oleic acid and 15 mL of 1-octadecene, magnetically stirring for 20 min at 110 ℃ under the protection of argon, then heating to 160 ℃, and continuing to magnetically stir for 30 min to prepare a mixed solution of the rare earth chloride, the oleic acid and the 1-octadecene; under magnetic stirring, naturally cooling the mixed solution of rare earth chloride, oleic acid and 1-octadecene to room temperature, dropwise adding 10 mL of methanol solution dissolved with 0.15 g of ammonium fluoride and 0.1 g of sodium hydroxide, heating to 50 ℃, magnetically stirring for 30 min, heating to 70 ℃, magnetically stirring for 10 min, heating to 110 ℃, magnetically stirring for 10 min, and finally heating to 300 ℃, magnetically stirring for 90 min; after the reaction is finished, under the magnetic stirring, naturally cooling the reaction mixed solution to room temperature, and washing the reaction mixed solution by using a mixed solution of ethanol and cyclohexane in a volume ratio of 2:1 through centrifugation to prepare the oleic acid modified up-conversion nanoparticles;
adding 40 mg of oleic acid modified upconversion nanoparticles into 4 mL of chloroform, and ultrasonically dispersing for 10 min at room temperature to prepare an oleic acid modified upconversion nanoparticle chloroform dispersion liquid; adding the oleic acid modified upconversion nanoparticle chloroform dispersion liquid into 10 mL of ethanol dispersion liquid dissolved with 200 mg of polyacrylic acid, magnetically stirring for 16 h at room temperature, and washing by using a mixed solution of ethanol and deionized water in a volume ratio of 2:1 through centrifugation to obtain polyacrylic acid modified upconversion nanoparticles;
the rare earth chloride comprises yttrium chloride hexahydrate, ytterbium chloride hexahydrate and erbium chloride hexahydrate, wherein the molar ratio of yttrium to ytterbium to erbium is 70:25: 2.
S2: preparation of nucleic acid aptamer fluorescent molecular probe
Dispersing 8 mg of polyacrylic acid modified upconversion nanoparticles in 7 mL of dimethyl sulfoxide under the action of ultrasound, adding 15 mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 35 mg of N-hydroxy thiosuccinimide sodium salt, and finally adding 0.06 mL of PBS buffer solution dissolved with aptamer to obtain mixed dispersion liquid;
and secondly, incubating the mixed dispersion liquid obtained in the step I at 35 ℃ for 18 h, centrifuging the product, washing the product by using a PBS (phosphate buffer solution), and ultrasonically dispersing the product in 4 mL of the PBS at room temperature to prepare the aptamer fluorescent molecular probe.
The PBS buffer solution is prepared by dissolving 8 g of sodium chloride, 0.24 g of potassium dihydrogen phosphate, 1.44 g of disodium hydrogen phosphate and 0.2 g of potassium chloride in 800 mL of deionized water, adjusting the pH value to 7.4 by using 0.1 mol/L hydrochloric acid, adding 200 mL of deionized water, and performing ultrasonic action.
The sequence of the aptamer is as follows:
5’-Cytosine-Cy5-TTGTTTGTCCCCTCTTTCTTA-(CH2)6-NH2-3’；
the concentration of the aptamer in the PBS buffer was 0.1 mmol/L.
S3: drawing standard working curve of 'fluorescence intensity-mercury ion concentration' and establishing linear working equation
Adding 0.1 mL of mercury ion aqueous solutions with different concentrations into 0.1 mL of nucleic acid aptamer fluorescent molecular probes respectively, and incubating for 40 min at room temperature to prepare a detection solution;
secondly, under the action of 980 nm laser, testing the fluorescence intensity of the detection solution prepared in the first step at 654 nm, drawing a standard working curve of fluorescence intensity-mercury ion concentration by taking the mercury ion concentration as a horizontal coordinate and the fluorescence intensity as a vertical coordinate, and establishing a linear working equation.
The concentration of mercury ions in the mercury ion aqueous solution is 0.1 nmol/L, 0.5 nmol/L, 1nmol/L, 5 nmol/L, 50 nmol/L, 100 nmol/L and 500 nmol/L.
FIG. 1 is a schematic diagram of the mechanism of detecting mercury ions by the aptamer-based fluorescent molecular probe of the present invention. As shown in FIG. 1, the fluorescent molecular probe of the present invention uses the upconversion nanoparticle as an energy donor, the fluorescent dye Cy5 as an energy acceptor, and the upconversion nanoparticle and the fluorescent dye are connected through a nucleic acid aptamer. As shown in FIG. 1 (left), when no mercury ion is added into the solution, the upconversion nanoparticles have an emission peak at a wavelength of 540 nm and at a wavelength of 654 nm under the excitation of 980 nm light. As shown in fig. 1 (right), when mercury ions are added into the solution, specific binding occurs between the mercury ions and the aptamer, and the aptamer folds to form a hairpin-shaped structure, resulting in a shorter distance between the upconversion nanoparticles and the fluorescent dye Cy 5. At this time, fluorescence energy resonance transfer occurs between the upconversion nanoparticles and the fluorescent dye Cy5, so that the fluorescence of the upconversion nanoparticles at the wavelength of 654 nm is quenched, and the quenched degree of the fluorescence and the concentration of the added mercury ions form a linear relationship, thereby achieving the purpose of quantitatively detecting the mercury ions.
Fig. 2 is an infrared spectrum of the oleic acid-modified upconversion nanoparticle and the polyacrylic acid-modified upconversion nanoparticle prepared in this example. As can be seen from the infrared spectrogram of the oleic acid modified up-conversion nanoparticles, the wave number is 2928 cm-1And 2857 cm-1Absorption peaks corresponding to asymmetric and symmetric stretching vibration of long chain alkyl methylene respectively, 1563 cm-1And 1467 cm-1Respectively, the asymmetric vibration absorption peak and the symmetric vibration absorption peak of carboxyl, which show that oleic acid is successfully modified on the surface of the upconversion nanoparticles. As can be seen from the infrared spectrum of the polyacrylic acid modified up-conversion nanoparticles, the wave number is 1723 cm-1A new strong carbonyl stretching vibration absorption peak is observed, which indicates that the content of carboxyl in the product is increased, and meanwhile, the methylene has the wave number of 2928 cm-1And 2857 cm-1The strength of the stretching vibration absorption peak is weakened, and the results show that polyacrylic acid is successfully used for replacing oleic acid to modify the surface of the upconversion nanoparticles.
FIG. 3 is a diagram of the ultraviolet-visible absorption spectrum of the polyacrylic acid modified upconversion nanoparticle and aptamer fluorescent molecular probe prepared in this example. As can be seen from the figure, the polyacrylic acid modified upconversion nanoparticles do not show a distinct absorption peak in the tested wavelength range, and the aptamer fluorescent molecular probes respectively show characteristic absorption peaks of the aptamer and the fluorescent dye Cy5 at the wavelengths of 260 nm and 654 nm, which indicates that the aptamer is successfully modified on the surface of the upconversion nanoparticles.
Fig. 4 is an X-ray diffraction pattern of the oleic acid-modified upconversion nanoparticle prepared in this example. From the figure, it can be found that the diffraction peak of the oleic acid modified upconversion nanoparticle prepared in the embodiment is consistent with the diffraction peak shown in the standard card JCPDS No.28-1192, which indicates that the oleic acid modified upconversion nanoparticle of the embodiment is hexagonal phase beta-NaYF4。
Fig. 5 is a scanning electron microscope photograph of the oleic acid-modified upconversion nanoparticles prepared in this example. From the results in the figure, it can be seen that the size of the oleic acid modified upconversion nanoparticles is uniform, and the particle size is about 24.5 nm.
FIG. 6 is a fluorescence emission spectrum of the oleic acid-modified upconversion nanoparticle and an ultraviolet-visible absorption spectrum of a fluorescent dye Cy5 in this example. As can be seen from the figure, the fluorescence emission peak of the oleic acid modified upconversion nanoparticle at 654 nm overlaps with the ultraviolet-visible absorption peak of the fluorescent dye Cy5, and the fluorescence resonance energy transfer condition is met, so that the fluorescence of the upconversion nanoparticle at 654 nm is quenched by the fluorescent dye Cy 5.
FIG. 7 is a fluorescence emission spectrum of the aptamer fluorescent molecular probe prepared in this example at mercury ion concentrations of 0.1 nmol/L, 0.5 nmol/L, 1nmol/L, 5 nmol/L, 50 nmol/L, 100 nmol/L and 500 nmol/L, respectively. As can be seen from the figure, the intensity of the fluorescence emission peak of the aptamer fluorescence molecular probe is reduced along with the increase of the concentration of mercury ions, and when the concentration of mercury ions is 500 nmol/mL, the intensity of the fluorescence emission peak of the aptamer fluorescence molecular probe is reduced to about 15% of the initial value, which indicates that the aptamer fluorescence molecular probe prepared by the invention can be used for high-sensitivity detection of trace mercury ions.
FIG. 8 is a standard operating curve of "fluorescence intensity vs. mercury ion concentration" obtained in this example. As can be seen from the figure, the concentration of mercury ions is in the range of 0.1-500 nmol/L, the intensity of the fluorescence emission peak of the aptamer fluorescence molecular probe prepared in the embodiment and the concentration of mercury ions have a good linear relationship, the linear working equation is Y = -46944.1Ln (X) +213167.7, the linear correlation coefficient is 0.9647, and the detection limit is 1.2575 nmol/L.
FIG. 9 is a diagram showing the results of specific detection of the fluorescent molecular probe for aptamer prepared in this example. Calcium ion (Ca) with a concentration of 10 mmol/L2+) Sodium ion (Na)+) Manganese ion (Mn)2+) Potassium ion (K)+) Iron ion (Fe)3+) Aluminum ion (Al)3+) Magnesium ion (Mg)2+) Zinc ion (Zn)2+) Plasma interference ions and mercury ions (Hg) with a concentration of 500 nmol/L2+) The interfering ions are respectively added into the detection system of the aptamer fluorescent molecular probe prepared in the embodiment, and the result shows that the interfering ions have almost no influence on the fluorescence emission peak intensity of the aptamer fluorescent molecular probe, which indicates thatThe nucleic acid aptamer fluorescent molecular probe prepared by the invention has high specificity detection on mercury ions.
Example 2
A method for detecting mercury ions by a fluorescent molecular probe based on a nucleic acid aptamer specifically comprises the following steps:
s1: preparation of polyacrylic acid modified up-conversion nanoparticles
Adding 1 mmol of rare earth chloride into a mixed solution of 3 mL of oleic acid and 10 mL of 1-octadecene, magnetically stirring for 30 min at 90 ℃ under the protection of argon, heating to 140 ℃, and continuously magnetically stirring for 40 min to prepare a mixed solution of the rare earth chloride, the oleic acid and the 1-octadecene; under magnetic stirring, naturally cooling the mixed solution of rare earth chloride, oleic acid and 1-octadecene to room temperature, dropwise adding 5 mL of methanol solution dissolved with 0.1 g of ammonium fluoride and 0.05 g of sodium hydroxide, heating to 40 ℃, magnetically stirring for 40 min, heating to 60 ℃, magnetically stirring for 20 min, heating to 100 ℃, magnetically stirring for 20 min, and finally heating to 280 ℃, and magnetically stirring for 120 min; after the reaction is finished, under the magnetic stirring, naturally cooling the reaction mixed solution to room temperature, and washing the reaction mixed solution by using a mixed solution of ethanol and cyclohexane with the volume ratio of 1:1 through centrifugation to prepare the oleic acid modified up-conversion nanoparticles;
adding 20 mg of oleic acid modified upconversion nanoparticles into 3 mL of chloroform, and ultrasonically dispersing for 5 min at room temperature to prepare a chloroform dispersion liquid of oleic acid modified upconversion nanoparticles; adding the oleic acid modified upconversion nanoparticle chloroform dispersion liquid into 5 mL of ethanol dispersion liquid dissolved with 100 mg of polyacrylic acid, magnetically stirring for 12 hours at room temperature, and washing by using a mixed solution of ethanol and deionized water in a volume ratio of 1:1 through centrifugation to obtain the polyacrylic acid modified upconversion nanoparticles.
The rare earth chloride comprises yttrium chloride hexahydrate, ytterbium chloride hexahydrate and erbium chloride hexahydrate, wherein the molar ratio of yttrium to ytterbium to erbium is 60:20: 1.
S2: preparation of nucleic acid aptamer fluorescent molecular probe
Dispersing 5 mg of polyacrylic acid modified up-conversion nanoparticles in 4 mL of dimethyl sulfoxide under the action of ultrasound, adding 10 mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 30 mg of N-hydroxy thiosuccinimide sodium salt, and finally adding 0.04 mL of PBS buffer solution dissolved with aptamer to obtain mixed dispersion liquid;
and secondly, incubating the mixed dispersion liquid obtained in the step I at 30 ℃ for 24 hours, centrifuging the product, washing the product by using a PBS (phosphate buffer solution), and ultrasonically dispersing the product in 4 mL of the PBS at room temperature to prepare the aptamer fluorescent molecular probe.
The PBS buffer solution is prepared by dissolving 8 g of sodium chloride, 0.24 g of potassium dihydrogen phosphate, 1.44 g of disodium hydrogen phosphate and 0.2 g of potassium chloride in 800 mL of deionized water, adjusting the pH value to 7.4 by using 0.1 mol/L hydrochloric acid, adding 200 mL of deionized water, and performing ultrasonic action.
The sequence of the aptamer is as follows:
5’-Cytosine-Cy5-TTGTTTGTCCCCTCTTTCTTA-(CH2)6-NH2-3’；
the concentration of the aptamer in the PBS buffer was 0.1 mmol/L.
S3: drawing standard working curve of 'fluorescence intensity-mercury ion concentration' and establishing linear working equation
Adding 0.1 mL of mercury ion aqueous solutions with different concentrations into 0.1 mL of nucleic acid aptamer fluorescent molecular probes respectively, and incubating for 40 min at room temperature to prepare a detection solution;
secondly, under the action of 980 nm laser, testing the fluorescence intensity of the detection solution prepared in the first step at 654 nm, drawing a standard working curve of fluorescence intensity-mercury ion concentration by taking the mercury ion concentration as a horizontal coordinate and the fluorescence intensity as a vertical coordinate, and establishing a linear working equation.
The concentration of mercury ions in the mercury ion aqueous solution is 0.1 nmol/L, 0.5 nmol/L, 1nmol/L, 5 nmol/L, 50 nmol/L, 100 nmol/L and 500 nmol/L.
The fluorescence emission peak intensity of the aptamer fluorescence molecular probe prepared in the example and the mercury ion concentration have a good linear relationship, the linear working equation is Y = -45980.1Ln (X) +205257.5, the linear correlation coefficient is 0.9543, and the detection limit is 1.2839 nmol/L.
Example 3
A method for detecting mercury ions by a fluorescent molecular probe based on a nucleic acid aptamer specifically comprises the following steps:
s1: preparation of polyacrylic acid modified up-conversion nanoparticles
Adding 1 mmol of rare earth chloride into a mixed solution of 9 mL of oleic acid and 20 mL of 1-octadecene, magnetically stirring for 10 min at 130 ℃ under the protection of argon, heating to 180 ℃, and continuing to magnetically stir for 20 min to prepare a mixed solution of the rare earth chloride, the oleic acid and the 1-octadecene; under magnetic stirring, naturally cooling the mixed solution of rare earth chloride, oleic acid and 1-octadecene to room temperature, dropwise adding 15 mL of methanol solution dissolved with 0.2 g of ammonium fluoride and 0.2 g of sodium hydroxide, heating to 60 ℃, magnetically stirring for 20 min, heating to 80 ℃, magnetically stirring for 5 min, heating to 120 ℃, magnetically stirring for 5 min, and finally heating to 320 ℃, and magnetically stirring for 60 min; after the reaction is finished, under the magnetic stirring, naturally cooling the reaction mixed solution to room temperature, and washing the reaction mixed solution by using a mixed solution of ethanol and cyclohexane in a volume ratio of 3:1 through centrifugation to prepare the oleic acid modified up-conversion nanoparticles;
adding 60 mg of oleic acid modified up-conversion nanoparticles into 5 mL of chloroform, and ultrasonically dispersing for 20 min at room temperature to prepare a chloroform dispersion liquid of oleic acid modified up-conversion nanoparticles; adding the oleic acid modified upconversion nanoparticle chloroform dispersion liquid into 20 mL of ethanol dispersion liquid dissolved with 300 mg of polyacrylic acid, magnetically stirring for 20 hours at room temperature, and washing by using a mixed solution of ethanol and deionized water in a volume ratio of 3:1 through centrifugation to obtain the polyacrylic acid modified upconversion nanoparticles.
The rare earth chloride comprises yttrium chloride hexahydrate, ytterbium chloride hexahydrate and erbium chloride hexahydrate, wherein the molar ratio of yttrium to ytterbium to erbium is 80:30: 3.
S2: preparation of nucleic acid aptamer fluorescent molecular probe
Firstly, dispersing 10 mg of polyacrylic acid modified up-conversion nanoparticles in 10 mL of dimethyl sulfoxide under the action of ultrasound, then adding 20 mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 40 mg of N-hydroxy thiosuccinimide sodium salt, and finally adding 0.08 mL of PBS buffer solution dissolved with aptamer to obtain mixed dispersion liquid;
and secondly, incubating the mixed dispersion liquid obtained in the step I at 40 ℃ for 12 hours, centrifuging the product, washing the product by using a PBS (phosphate buffer solution), and ultrasonically dispersing the product in 4 mL of the PBS at room temperature to prepare the aptamer fluorescent molecular probe.
The PBS buffer solution is prepared by dissolving 8 g of sodium chloride, 0.24 g of potassium dihydrogen phosphate, 1.44 g of disodium hydrogen phosphate and 0.2 g of potassium chloride in 800 mL of deionized water, adjusting the pH value to 7.4 by using 0.1 mol/L hydrochloric acid, adding 200 mL of deionized water, and performing ultrasonic action.
The complete sequence of the aptamer is:
5’-Cytosine-Cy5-TTGTTTGTCCCCTCTTTCTTA-(CH2)6-NH2-3’；
the concentration of the aptamer in the PBS buffer was 0.1 mmol/L.
S3: drawing standard working curve of 'fluorescence intensity-mercury ion concentration' and establishing linear working equation
Adding 0.1 mL of mercury ion aqueous solutions with different concentrations into 0.1 mL of nucleic acid aptamer fluorescent molecular probes respectively, and incubating for 40 min at room temperature to prepare a detection solution;
secondly, under the action of 980 nm laser, testing the fluorescence intensity of the detection solution prepared in the first step at 654 nm, drawing a standard working curve of fluorescence intensity-mercury ion concentration by taking the mercury ion concentration as a horizontal coordinate and the fluorescence intensity as a vertical coordinate, and establishing a linear working equation.
The concentration of mercury ions in the mercury ion aqueous solution is 0.1 nmol/L, 0.5 nmol/L, 1nmol/L, 5 nmol/L, 50 nmol/L, 100 nmol/L and 500 nmol/L.
The fluorescence emission peak intensity of the aptamer fluorescence molecular probe prepared in the example and the mercury ion concentration have a good linear relationship, the linear working equation is Y = -45530.5Ln (X) +201105.3, the linear correlation coefficient is 0.9508, and the detection limit is 1.2965 nmol/L.
While the invention has been described with respect to specific embodiments thereof, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention; those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and alterations of the above embodiments according to the spirit and techniques of the present invention are also within the scope of the present invention.
SEQUENCE LISTING
<110> Fuzhou university
<120> method for detecting mercury ions by using fluorescent molecular probe based on nucleic acid aptamer
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> Artificial sequence
<400> 1
ttgtttgtcc cctctttctt a 21
Claims (8)
1. A method for detecting mercury ions by a fluorescent molecular probe based on a nucleic acid aptamer is characterized by comprising the following steps: the method takes polyacrylic acid modified up-conversion nano particles as an energy donor and takes a fluorescent dye Cy5 as an energy acceptor; the method specifically comprises the following steps:
s1: preparing polyacrylic acid modified up-conversion nanoparticles:
preparing oleic acid modified upconversion nanoparticles by using rare earth chloride, oleic acid, 1-octadecene, ammonium fluoride and sodium hydroxide as raw materials through a high-temperature thermal decomposition method, and preparing polyacrylic acid modified upconversion nanoparticles by using a surface ligand exchange reaction;
s2: preparation of the aptamer fluorescent molecular probe:
adopting amidation reaction, connecting aptamer on the surface of the polyacrylic acid modified upconversion nano particle, and preparing a nucleic acid aptamer fluorescent molecular probe;
s3: drawing a standard working curve of fluorescence intensity-mercury ion concentration, and establishing a linear working equation:
adding mercury ion aqueous solutions with different concentrations into the nucleic acid aptamer fluorescent molecular probe in the step S2 to prepare a detection solution; and detecting the fluorescence intensity of the detection solution with different mercury ion concentrations under the action of 980 nm laser, drawing a standard working curve of the fluorescence intensity-mercury ion concentration, and establishing a linear working equation.
2. The method of detecting mercury ions according to claim 1, wherein: the step S1 is specifically as follows:
adding 1 mmol of rare earth chloride into a mixed solution of 3-9 mL of oleic acid and 10-20 mL of 1-octadecene, magnetically stirring for 10-30 min at 90-130 ℃ under the protection of argon, then heating to 140-180 ℃, and continuing to magnetically stir for 20-40 min to prepare a mixed solution of the rare earth chloride, the oleic acid and the 1-octadecene; under magnetic stirring, naturally cooling a mixed solution of rare earth chloride, oleic acid and 1-octadecene to room temperature, dropwise adding 5-15 mL of methanol solution dissolved with 0.1-0.2 g of ammonium fluoride and 0.05-0.2 g of sodium hydroxide, firstly carrying out magnetic stirring for 20-40 min at 40-60 ℃, then heating to 60-80 ℃, carrying out magnetic stirring for 5-20 min, then heating to 100-120 ℃, carrying out magnetic stirring for 5-20 min, and finally heating to 280-320 ℃, and carrying out magnetic stirring for 60-120 min; after the reaction is finished, under the magnetic stirring, naturally cooling the reaction mixed solution to room temperature, and washing the reaction mixed solution by using a mixed solution of ethanol and cyclohexane in a volume ratio of 1: 1-3: 1 through centrifugation to prepare the oleic acid modified up-conversion nanoparticles;
adding 20-60 mg of oleic acid modified up-conversion nanoparticles into 3-5 mL of chloroform, and performing ultrasonic dispersion for 5-20 min at room temperature to prepare an oleic acid modified up-conversion nanoparticle chloroform dispersion liquid; adding the oleic acid modified upconversion nanoparticle chloroform dispersion liquid into 5-20 mL of ethanol dispersion liquid dissolved with 100-300 mg of polyacrylic acid, magnetically stirring at room temperature for 12-20 h, and washing by using a mixed solution of ethanol and deionized water in a volume ratio of 1: 1-3: 1 through centrifugation to obtain the polyacrylic acid modified upconversion nanoparticle.
3. The method for preparing polyacrylic acid modified upconversion nanoparticles according to claim 2, wherein the polyacrylic acid modified upconversion nanoparticles are prepared by: the rare earth chloride in the step I comprises yttrium chloride hexahydrate, ytterbium chloride hexahydrate and erbium chloride hexahydrate, wherein the molar ratio of yttrium to ytterbium to erbium is 60-80: 20-30: 1-3.
4. The method of detecting mercury ions according to claim 1, wherein: the step S2 is specifically as follows:
dispersing 5-10 mg of polyacrylic acid modified up-conversion nanoparticles in 4-10 mL of dimethyl sulfoxide under the action of ultrasound, adding 10-20 mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 30-40 mg of N-hydroxy thiosuccinimide sodium salt, and finally adding 0.04-0.08 mL of PBS buffer solution dissolved with aptamer to obtain mixed dispersion liquid;
and secondly, incubating the mixed dispersion liquid obtained in the step I at 30-40 ℃ for 12-24 h, centrifuging the product, washing the product with PBS (phosphate buffer solution), and ultrasonically dispersing the product in 4 mL of PBS at room temperature to prepare the aptamer fluorescent molecular probe.
5. The method for preparing the aptamer fluorescent molecular probe according to claim 4, wherein the method comprises the following steps: the sequence of the aptamer is 5' -cysteine-Cy 5-TTGTTTGTCCCCTCTTTCTTA- (CH)2)6-NH2-3'; the concentration of the aptamer in the PBS buffer was 0.1 mmol/L.
6. The method of detecting mercury ions according to claim 1, wherein: the step S3 is specifically as follows:
adding 0.1 mL of mercury ion aqueous solutions with different concentrations into 0.1 mL of nucleic acid aptamer fluorescent molecular probes respectively, and incubating for 40 min at room temperature to prepare a detection solution;
secondly, under the action of 980 nm laser, testing the fluorescence intensity of the detection solution prepared in the first step at 654 nm, drawing a standard working curve of fluorescence intensity-mercury ion concentration by taking the mercury ion concentration as a horizontal coordinate and the fluorescence intensity as a vertical coordinate, and establishing a linear working equation.
7. The plotted standard "fluorescence intensity-mercury ion concentration" working curve of claim 6, wherein: in the mercury ion aqueous solution, the concentration of mercury ions is 0.1 nmol/L, 0.5 nmol/L, 1nmol/L, 5 nmol/L, 50 nmol/L, 100 nmol/L and 500 nmol/L.
8. Use of the method of claim 1 for mercury ion detection.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114657185A (en) * | 2022-03-28 | 2022-06-24 | 福州大学 | Aptamer ordered arrangement-based gold magnetic nanoprobe and application thereof in okadaic acid detection |
| WO2023160554A1 (en) * | 2022-02-28 | 2023-08-31 | 江苏大学 | Preparation method for and application of dye-functionalized flexible upconversion light-emitting solid-phase sensor |
| US11773322B2 (en) | 2022-02-28 | 2023-10-03 | Jiangsu University | Preparation and application of dye-functionalized flexible upconversion-luminescence solid-phase sensor |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102590170A (en) * | 2012-02-28 | 2012-07-18 | 江南大学 | Method for simultaneously detecting mercury ion and/or silver ion in water solution based on fluorescence resonance energy transfer |
| CN103773359A (en) * | 2014-02-11 | 2014-05-07 | 江苏大学 | Surface modification method for rare earth up-conversion fluorescent nanomaterial |
| CN105866101A (en) * | 2016-05-23 | 2016-08-17 | 中国科学院生态环境研究中心 | Heavy metal mercury ion detection method based on nucleic acid aptamer labeling |
| CN107436297A (en) * | 2017-07-19 | 2017-12-05 | 太原理工大学 | Mercury ion detecting method in water based on hairpin-type DNA probe |
| CN113237856A (en) * | 2021-04-29 | 2021-08-10 | 中国人民解放军军事科学院军事医学研究院 | Aptamer-based detection Tl+Fluorescent probe of |
-
2021
- 2021-09-28 CN CN202111139159.8A patent/CN113670887A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102590170A (en) * | 2012-02-28 | 2012-07-18 | 江南大学 | Method for simultaneously detecting mercury ion and/or silver ion in water solution based on fluorescence resonance energy transfer |
| CN103773359A (en) * | 2014-02-11 | 2014-05-07 | 江苏大学 | Surface modification method for rare earth up-conversion fluorescent nanomaterial |
| CN105866101A (en) * | 2016-05-23 | 2016-08-17 | 中国科学院生态环境研究中心 | Heavy metal mercury ion detection method based on nucleic acid aptamer labeling |
| CN107436297A (en) * | 2017-07-19 | 2017-12-05 | 太原理工大学 | Mercury ion detecting method in water based on hairpin-type DNA probe |
| CN113237856A (en) * | 2021-04-29 | 2021-08-10 | 中国人民解放军军事科学院军事医学研究院 | Aptamer-based detection Tl+Fluorescent probe of |
Non-Patent Citations (5)
| Title |
|---|
| E. RUGGIERO ET AL.: "Near infrared photolysis of a Ru polypyridyl complex by upconverting nanoparticles", 《CHEMICAL COMMUNICATIONS》 * |
| GONGKE WANG ET AL.: "DNA-functionalization gold nanoparticles based fluorescence sensor for sensitive detection of Hg2+in aqueous solution", 《SENSORS AND ACTUATORS B: CHEMICAL》 * |
| SHIJIA WU ET AL.: "Dual fluorescence resonance energy transfer assay between tunable", 《TALANTA》 * |
| 张扬: "喹诺酮类兽药和磺胺喹噁啉上转换荧光试纸条的研制", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
| 陈云 等: "《现代生物医学研究技术实验教程》", 31 January 2019, 武汉大学出版社 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023160554A1 (en) * | 2022-02-28 | 2023-08-31 | 江苏大学 | Preparation method for and application of dye-functionalized flexible upconversion light-emitting solid-phase sensor |
| US11773322B2 (en) | 2022-02-28 | 2023-10-03 | Jiangsu University | Preparation and application of dye-functionalized flexible upconversion-luminescence solid-phase sensor |
| CN114657185A (en) * | 2022-03-28 | 2022-06-24 | 福州大学 | Aptamer ordered arrangement-based gold magnetic nanoprobe and application thereof in okadaic acid detection |
| CN114657185B (en) * | 2022-03-28 | 2023-11-10 | 福州大学 | Gold magnetic nano probe based on ordered arrangement of aptamer and application of gold magnetic nano probe in field halichondrin acid detection |
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