CN110819695A - Method for detecting silver ions - Google Patents
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- CN110819695A CN110819695A CN201810915714.3A CN201810915714A CN110819695A CN 110819695 A CN110819695 A CN 110819695A CN 201810915714 A CN201810915714 A CN 201810915714A CN 110819695 A CN110819695 A CN 110819695A
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- 238000000034 method Methods 0.000 title claims abstract description 24
- 229910052709 silver Inorganic materials 0.000 title abstract description 7
- 239000004332 silver Substances 0.000 title abstract description 7
- -1 silver ions Chemical class 0.000 title abstract description 7
- 108060002716 Exonuclease Proteins 0.000 claims abstract description 36
- 102000013165 exonuclease Human genes 0.000 claims abstract description 36
- 238000001514 detection method Methods 0.000 claims abstract description 26
- 229910052737 gold Inorganic materials 0.000 claims abstract description 26
- 239000010931 gold Substances 0.000 claims abstract description 26
- 239000002131 composite material Substances 0.000 claims abstract description 18
- 239000011148 porous material Substances 0.000 claims abstract description 17
- 239000006228 supernatant Substances 0.000 claims abstract description 5
- 239000012488 sample solution Substances 0.000 claims abstract description 3
- FOIXSVOLVBLSDH-UHFFFAOYSA-N Silver ion Chemical compound [Ag+] FOIXSVOLVBLSDH-UHFFFAOYSA-N 0.000 claims description 70
- 239000000243 solution Substances 0.000 claims description 28
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 14
- 239000007993 MOPS buffer Substances 0.000 claims description 11
- 150000007523 nucleic acids Chemical class 0.000 claims description 11
- 102000039446 nucleic acids Human genes 0.000 claims description 11
- 108020004707 nucleic acids Proteins 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 238000007885 magnetic separation Methods 0.000 claims description 8
- 239000000047 product Substances 0.000 claims description 8
- 239000000523 sample Substances 0.000 claims description 8
- 229940104302 cytosine Drugs 0.000 claims description 7
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical group [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims description 7
- 229940043267 rhodamine b Drugs 0.000 claims description 7
- 230000009466 transformation Effects 0.000 claims description 5
- 239000011324 bead Substances 0.000 claims description 4
- 238000007865 diluting Methods 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 229920000371 poly(diallyldimethylammonium chloride) polymer Polymers 0.000 claims description 3
- PUAQLLVFLMYYJJ-UHFFFAOYSA-N 2-aminopropiophenone Chemical compound CC(N)C(=O)C1=CC=CC=C1 PUAQLLVFLMYYJJ-UHFFFAOYSA-N 0.000 claims description 2
- 238000001917 fluorescence detection Methods 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 abstract description 41
- 230000003321 amplification Effects 0.000 abstract description 22
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 22
- 239000002539 nanocarrier Substances 0.000 abstract description 19
- 125000004122 cyclic group Chemical group 0.000 abstract description 18
- 230000035945 sensitivity Effects 0.000 abstract description 14
- 239000000463 material Substances 0.000 abstract description 6
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 3
- 239000002114 nanocomposite Substances 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 abstract 1
- 238000000926 separation method Methods 0.000 abstract 1
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- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
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- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
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- 241000282412 Homo Species 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
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- 238000004458 analytical method Methods 0.000 description 1
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- PRAHCKGOTFKWKD-UHFFFAOYSA-N dimethyl(propyl)azanium;chloride Chemical compound Cl.CCCN(C)C PRAHCKGOTFKWKD-UHFFFAOYSA-N 0.000 description 1
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- 229910001385 heavy metal Inorganic materials 0.000 description 1
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- 238000002354 inductively-coupled plasma atomic emission spectroscopy Methods 0.000 description 1
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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
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
-
- 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"
-
- 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
- C12Q2304/00—Chemical means of detecting microorganisms
- C12Q2304/10—DNA staining
Abstract
The invention provides a nano-gold composite material for Ag based on exonuclease cyclic amplification technology and base mismatch identification technology+The detection method can be used in the fields of food, environment, medicine, health and the like. The invention utilizes nano gold with hollow and porous structure and identifiable Ag+The biological molecules are combined to construct the nano composite material with the pore cap. After the sample solution containing silver ions is added, the silver ions and the biomolecules on the surface of the nano carrier act to enable the biomolecules to be separated from the surface of the nano carrier, dye molecules in the nano carrier are released, after separation, supernatant liquid generates fluorescence emission under the irradiation of exciting light with a certain wavelength, and the detection of the silver ions is realized according to the strength of fluorescence emission signals. Meanwhile, in order to improve the sensitivity, the invention realizes the circular amplification of the fluorescence signal by using exonuclease. The method of the invention has the advantages of simplicity, high efficiency, sensitivity, good selectivity, convenience, rapidness and low cost,the application range is wide.
Description
Technical Field
The invention relates to a method for detecting Ag+In particular to Ag based on the exonuclease cyclic amplification technology and the base mismatch identification technology+The method of (1).
Background
Silver ion (Ag) as heavy metal ion+) With mercury ions (Hg)2+) Similarly, it is a metal ion with high toxicity and is also a widely distributed environmental pollutant. Even at low concentrations, can be severely and permanently toxic to the environment and humans. More seriously, through contaminated water source, Ag+Can be continuously accumulated in agricultural products and aquatic products and then enter the food chain of human beings. If exposed to Ag for a long time+In the existing environment, it may cause the slow occurrence of degenerative diseases in human body and nervous system. Thus, highly efficient, sensitive and economical Ag was established+The detection method has very important significance in the fields of environmental monitoring, food safety, clinical diagnosis and the like.
At present, for Ag+The traditional methods for detection mainly include plasma mass spectrometry (ICP-AES), atomic absorption/emission spectrometry, polarography and the like. However, these methods often require cumbersome operations, time-consuming analyses, and expensive, complicated equipment, etc., making them unusable in resource-limited environments, and in addition, they are still subject to improvement in detection sensitivity and selectivity. To overcome these disadvantages, there is an urgent need to develop a simple, sensitive, economical and efficient assay method to satisfy the demands of Ag in biological, medical and environmental fields+The detection requirement of (1).
The metal ions can form coordinate bonds with base pairs and can replace hydrogen bonds in conventional Watson-Crick base pairs to form metal-base pairs, and the effect has important significance for quickly and efficiently detecting the metal ions, so that the metal ions are widely concerned. Wherein cytosine (C) may be combined with Ag+Formation of C-Ag+The combination of-C base pair is stable, and the generation of the special structure can be used for Ag+Detection of (3). Furthermore, due to a C-C base mismatchRecognition of Ag+To form Ag+Qiao-linked base pairing of Ag+Has very high specificity and selectivity. According to the invention, Ag+The reaction characteristic combines the nucleic acid biomolecule rich in cytosine, is skillfully applied to the field of nano materials with hollow and porous structures, and combines the shearing action of biological enzyme on the basis of a base mismatch identification technology to improve the detection sensitivity, so that the Ag is Ag+Provides a novel, specific and efficient detection technology. Adopts nano gold with a hollow and porous structure as a nano carrier and utilizes C-C base mismatching Ag+Specific biological recognition function for constructing nano composite material to detect Ag+The technology of (A) has not been reported in the literature.
Disclosure of Invention
Aiming at overcoming the defects of the prior art, the method aims at detecting Ag by using the nano-gold composite material based on the exonuclease cyclic amplification technology and the base mismatch identification technology+The technique of (a) has not been reported, and therefore, the first object of the present invention: provides and constructs a novel nano-gold composite material based on exonuclease cyclic amplification technology and base mismatch identification technology, specifically, hollow and porous nano-gold is used as a nano-carrier to design and synthesize a nano-gold composite material capable of being coated with Ag+The identified biological molecules are assembled on the surface of the nano carrier and are used as a 'pore cap' to plug the orifice of the nano carrier and prevent substances in the pore from leaking; on the other hand as Ag+The recognition probe of (A) can be reacted with Ag+Specific base mismatch recognition reaction occurs to form C-Ag+Conformation transformation occurs at the same time of-C base pair to separate from the surface of the nanocapsule, so that the blocked 'pore cap' is opened, dye molecules in the nanocapsule are released, supernatant is separated, fluorescence emission is generated under the irradiation of exciting light with certain wavelength, and the Ag is realized according to the strength of a fluorescence emission signal+Detection of (3). Meanwhile, in order to further increase the sensitivity, the invention realizes the cyclic amplification of the fluorescent signal by utilizing the shearing action of the exonuclease on the basis of the base mismatch recognition.
Due to the coreThe exonuclease can be used for preparing the biological molecule-Ag with double-chain structure+Shearing the combination, after shearing, Ag+Is released, these Ag+Can be subjected to recognition reaction with other recognition probes again and then be sheared … … again, resulting in Ag+The materials are recycled, more 'pore caps' are opened, and more substances in pores are released. It is due to the action of the cleaving enzyme, Ag+Is recycled, so that the detection sensitivity is obviously enhanced. The detection system based on the exonuclease cyclic amplification technology and the base mismatch identification technology can be used for detecting trace Ag+The sample realizes high-sensitivity and high-selectivity detection. Even if the sample contains a very small amount of Ag+Satisfactory detection results can also be obtained; second object of the invention: provides a method for detecting trace Ag+The preparation method of the nano-gold composite material based on the exonuclease cycle amplification technology and the base mismatch identification technology; the third object of the present invention: provides a method for detecting trace Ag+The method of (1).
The invention achieves the purpose through the following technical scheme. The invention provides the detection of Ag+The nano-gold composite material takes nano-gold with a hollow and porous structure as a nano-carrier, and utilizes the characteristics of the hollow and porous structure to load guest molecules such as fluorescent dye, preferably rhodamine B. In order to prevent the leakage of fluorescent dye, the invention designs and synthesizes the silver-coated+The identified biological molecules are assembled on the surface of the nano carrier to form a 'pore cap' for plugging an orifice, so that the effect of preventing substances in the pore from leaking is achieved; wherein said may be Ag+The identified biomolecule is a specially designed and synthesized cytosine-rich nucleic acid biomolecule with a certain base length, the base sequence of the nucleic acid biomolecule is 5'-TCCTCC CTC CTTAAG GAACCA CCCACC A-3', and the biomolecule is used as Ag+The recognition probe is assembled on the surface of the hollow and porous nanogold to form a 'pore cap', and the assembly of the 'pore cap' is realized by a method of modifying a positive charge modifier on the surface of a nano carrier in advance, preferably, the positive charge modifier is polydiallylbisMethyl ammonium chloride. In order to open more "pore caps" and release more fluorescent dye, the Ag coated glass designed and synthesized by the invention+Recognized biomolecules with Ag+The combined product can be acted by exonuclease, namely the invention utilizes the exonuclease to react with the biomolecule-Ag with double-chain structure+Shearing the combination to obtain Ag+From mismatched base pairs C-Ag+-C is released and recycled back into solution and is mixed with other Ag+The recognition probe is combined, so that more rhodamine B is released, the cyclic amplification of a fluorescence signal is realized, and preferably, the exonuclease is exonuclease ExoIII.
Preparation of the trace Ag detection material provided by the invention+The preparation method of the nano-gold composite material based on the exonuclease cycle amplification technology and the base mismatch identification technology comprises the following steps:
(1) designed and synthesized to be capable of reacting with Ag+Cytosine-rich nucleic acid biomolecules of a certain base length recognized by base mismatches, which are capable of reacting with Ag+A specific base mismatch recognition reaction occurs, and the binding product of the silver ion and the silver ion can be cut by exonuclease;
(2) mixing the magnetic beads with a nano-gold carrier solution with a hollow and porous structure, adding a poly (diallyldimethylammonium chloride) solution, carrying out magnetic separation after 10-12h, and cleaning with an MOPS buffer solution;
(3) adding dye molecule solution, adding recognizable Ag after 10-12h+Performing magnetic separation after 10-12h, and washing with MOPS buffer solution;
wherein, the recognizable Ag+The biomolecule of (1), having a base sequence of 5'-TCC TCC CTC CTTAAG GAACCACCCACCA-3', which is mixed with Ag+The binding product of (A) can be cleaved by exonuclease to release Ag+。
The invention has the beneficial effects that: the invention provides a nano-gold composite material based on exonuclease cyclic amplification technology and base mismatch identification technology, which can identify Ag+And a nano-molecule having a hollow, porous structureThe gold materials are combined, and can be coated with Ag by design and synthesis+Recognized biological molecules are assembled on the surface of the nano carrier to form a 'pore cap', and Ag can be recognized by using the recognized biological molecules+With Ag+The base mismatch recognition reaction occurs to form C-Ag+Conformation transformation is carried out at the same time of-C base pair to separate from the surface of the nano carrier, so that a blocked 'pore cap' is opened, dye molecules in the nano carrier are released, and in order to further increase the sensitivity, the invention also utilizes exonuclease to carry out double-chain structure on biomolecule-Ag+The shearing action of the conjugate realizes the cyclic amplification and detection of the fluorescence signal.
The method makes Ag+The detection sensitivity is obviously improved, and the detection on Ag can be realized+High sensitivity and high selectivity. The nanogold composite material based on the exonuclease cyclic amplification technology and the base mismatch identification technology has the advantages of simple structure, easiness in synthesis, excellent performance, stability, economy, high efficiency, sensitivity and the like, and cannot be subjected to other common interference substances such as Cd2+，Hg2+，Pb2+，Cu2+，Fe3+，Zn2+The influence of plasma metal ions has high specificity and selectivity. The experimental result shows that compared with other common technical methods, the nanogold composite material based on the exonuclease cyclic amplification technology and the base mismatch identification technology provided by the invention shows high sensitivity and excellent selectivity at 1.0 x 10-13～8.0×10- 11Detecting Ag within the mol/L concentration range+The logarithm of the concentration and the fluorescence signal intensity present a good linear relation, and the detection limit is as low as 1.0 multiplied by 10-13mol/L. Compared with the literature value, the invention is applied to Ag+The detection sensitivity of (2) is improved by nearly 100 times. The invention provides a nano-gold composite material based on an exonuclease cyclic amplification technology and a base mismatch identification technology, a preparation method and a detection technology thereof, and the nano-gold composite material has huge medical application potential and wide application prospect, and can play an important role in the fields of early diagnosis and treatment of major diseases, food, biomedicine, medicine, environment and the like.
Drawings
FIG. 1.Ag+Log concentration versus fluorescence signal intensity.
Detailed Description
The following are specific examples related to the present invention, and further description is made on the technical solutions of the present invention, but the scope of the present invention is not limited to these examples. All changes, modifications and equivalents that do not depart from the spirit of the invention are intended to be included within the scope thereof.
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.
An experimental instrument: THZ-82A gas bath constant temperature oscillator (gold jar medical instrument factory); f-4600 Fluorospectrophotometer (Hitachi, Japan); magnetic separation rack (Tianjin double Si le chromatographic development center).
The experimental reagents include exonuclease Exo Ш (Thermo Scientific, USA), polydiallyl propyl dimethyl ammonium chloride (Shanghai-Arlatin Biotech Co., Ltd.), 3-4 μm sulfhydryl magnetic bead (Tianjin Shuangsi-Si-le chromatography technical development center), rhodamine B (Shanghai-Arlatin Biotech Co., Ltd.), and Agents for testing Ag+The identified biomolecules are specially designed and synthesized cytosine-rich nucleic acid biomolecules with a certain base length, wherein the base sequence of the nucleic acid biomolecules is 5'-TCC TCCCTC CTTAAG GAACCACCCACCA-3' (Shanghai Biotechnology, Inc.), and the MOPS buffer solution is 0.01M (pH 7.0, Shanghai Aladdin Biotechnology, Inc.).
Example 1:
the invention provides a method for preparing a nanogold composite material based on an exonuclease cyclic amplification technology and a base mismatch identification technology, which comprises the following steps of:
(1) designed and synthesized to be capable of reacting with Ag+Cytosine-rich nucleic acid biomolecules of a certain base length recognized by base mismatches, which are capable of reacting with Ag+A specific base mismatch recognition reaction occurs, and the binding product of the silver ion and the silver ion can be cut by exonuclease;
(2) washing thiol magnetic beads (20 mu L) with MOPS buffer solution, mixing with hollow and porous nano-gold carrier solution (400 mu L), adding poly (diallyl dimethyl ammonium chloride) solution (200 mu L, 11.664mg/mL), performing magnetic separation at 37 ℃ for 10h, and washing with MOPS buffer solution;
(3) adding rhodamine B solution (2 mu L, final concentration 1.0X 10)-4mol/L), diluting to 100 μ L with MOPS buffer solution with pH 7.0, adding identifiable Ag after 10h at 37 deg.C+10. mu.L of a biomolecule solution (final concentration of 1.0X 10)-6mol/L), performing magnetic separation after 10 hours at 37 ℃, and cleaning by using MOPS buffer solution;
wherein, the recognizable Ag+The nucleic acid biomolecule of (1), whose base sequence is 5'-TCC TCC CTC CTTAAGGAACCACCCACC A-3', which is mixed with Ag+The binding product of (A) can be cleaved by exonuclease Exo III to release Ag+(ii) a The nanogold material with the hollow and porous structure is obtained according to a literature method (W.Wang, C.Chen, X.X.Li, S.Y.WangandX.L.Luo.Chem.Commun.,2015,51, 9109-one 9112.).
Example 2:
the invention provides a method for detecting Ag by adopting the nano-gold composite material based on the exonuclease cyclic amplification technology and the base mismatch identification technology+The method comprises the following steps:
(1) adding 10 μ L of Ag+Adding the sample solution to be detected into the nanogold composite material based on the exonuclease cyclic amplification technology and the base mismatch identification technology, diluting the solution to 100 mu L by using MOPS buffer solution (pH 7.0), adding exonuclease Exo III (20U), oscillating the solution at the constant temperature of 37 ℃ for 2h, and identifying Ag on the surface of the nanogold carrier with a hollow and porous structure+With Ag+A specific recognition reaction occurs to form C-Ag+Conformation transformation occurs at the same time of the-C base pair to separate from the surface of the nano carrier, so that a blocked 'pore cap' is opened, and a dye molecule rhodamine B in the nano carrier is released; meanwhile, the exonuclease Exo III pairs to generate a biomolecule-Ag with a double-chain structure+Shearing the combination to obtain Ag+From mismatchesBase pair of (C-Ag)+-C is released, recycled back into solution and can recognize Ag with other+The fluorescent dye is combined with the biological molecules, so that more rhodamine B is released, and the fluorescent signal is obviously enhanced;
(2) performing magnetic separation, taking supernatant, diluting the supernatant to 2.0mL by using secondary water, and performing fluorescence detection under the following detection conditions: the excitation wavelength and emission wavelength were 530, 575nm, respectively.
FIG. 1 shows Ag+The logarithm of the concentration and the intensity of the fluorescence signal are plotted linearly, and the result shows that Ag+The concentration is 1.0 × 10-13～8.0×10-11At mol/L, the intensity of fluorescence signal and Ag+The logarithm of the concentration shows a good linear relationship, and the linear equation is as follows: FL 354.1304+121.7803lgCAg +(10-13) The linear correlation coefficient is 0.9935.
The invention combines the exonuclease circulation amplification technology with the base mismatch identification technology and the nano-carrier technology, and can be Ag by design and synthesis+Recognized biological molecules are assembled on the surface of a nano carrier to form a 'pore cap', and C-C base mismatch and Ag are utilized+To form C-Ag+Conformation transformation is carried out at the same time of-C base pair to separate from the surface of the nano carrier, so that a blocked 'pore cap' is opened, dye molecules in the nano carrier are released, and in order to further increase the sensitivity, the invention also utilizes exonuclease to carry out double-chain structure on biomolecule-Ag+The shearing action of the conjugate realizes the cyclic amplification and detection of the fluorescence signal.
The nanogold composite material based on the exonuclease cyclic amplification technology and the base mismatch identification technology has the advantages of simple structure, easiness in synthesis, excellent performance, stability, economy, sensitivity, high efficiency and the like, and cannot be subjected to other common interference substances such as Cd2+，Hg2+，Pb2+，Cu2+，Fe3+，Zn2+The influence of plasma metal ions has high specificity and selectivity, and can be used for low-content or even extremely low-content Ag in a trace sample+Is particularly important for high-sensitivity and high-selectivity detectionThe nano-gold composite material based on the exonuclease cyclic amplification technology and the base mismatch identification technology, the preparation method and the detection technology thereof have great medical application potential and wide application prospect, and can play an important role in the fields of early diagnosis and treatment of major diseases, food, biomedicine, medicine, environment and the like.
Sequence listing
<110> Qingdao university of science and technology
<120> a method for detecting silver ions
<141>2018-08-13
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
tcctccctcc ttaaggaacc acccacca 28
Claims (4)
1. Detect Ag+The method is characterized by utilizing designed and synthesized Ag+The biomolecule identified by base mismatch is assembled on the surface of a nano-gold carrier with hollow and porous structure characteristics, and on one hand, the biomolecule is used as a 'hole cap' for plugging the hole opening of the nano-gold carrier and preventing dye molecules in the hole from leaking; on the other hand as Ag+The recognition probe of (A) can be reacted with Ag+Specific base mismatch recognition reaction occurs to form C-Ag+Conformation transformation occurs at the same time of the-C base pair to separate from the surface of the nano-gold carrier, so that a blocked 'pore cap' is opened, and dye molecules in the nano-gold carrier are released; at the same time, designed and synthesized can be mixed with Ag+Biomolecules recognized by base mismatches with Ag+The conjugate can be cleaved by exonuclease to cleave Ag+From mismatched base pairs C-Ag+-C is released and recycled back into the solution and optionally Ag+By base mismatch recognitionOther biological molecules are combined, so that more dye molecules are released, and finally the Ag is realized by using the circularly amplified fluorescent signal+The detection method comprises the following steps:
(1) designed and synthesized to be capable of reacting with Ag+Cytosine-rich nucleic acid biomolecules of a certain base length recognized by base mismatches, which are capable of reacting with Ag+A specific base mismatch recognition reaction occurs, and the binding product of the silver ion and the silver ion can be cut by exonuclease;
(2) mixing the magnetic beads with a nano-gold carrier solution with a hollow and porous structure, adding a poly (diallyldimethylammonium chloride) solution, carrying out magnetic separation after 10-12h, and cleaning with an MOPS buffer solution;
(3) adding dye molecule solution, adding the Ag which is designed and synthesized in the step (1) and can be identified after 10-12h+The nucleic acid biomolecule solution is magnetically separated after 10 to 12 hours, and is washed by MOPS buffer solution;
(4) will contain Ag+Adding the sample solution to be tested into the nano-gold composite material prepared in the previous step, diluting with MOPS buffer solution (pH is 7.0), adding exonuclease, and oscillating at the constant temperature of 37 ℃ for 2-3 h;
(5) magnetic separation, taking supernatant, and performing fluorescence detection.
2. A detecting Ag according to claim 1+The method of (2), characterized by: the said may be reacted with Ag+A nucleic acid biomolecule recognized by a base mismatch, which has a base sequence of 5'-TCC TCC CTC CTT AAG GAA CCA CCC ACC A-3'.
3. A detecting Ag according to claim 1+The method of (2), characterized by: the dye molecule is rhodamine B.
4. A detecting Ag according to claim 1+The method of (2), characterized by: the exonuclease is Exo III.
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|---|---|---|---|
| CN201810915714.3A CN110819695B (en) | 2018-08-13 | 2018-08-13 | Method for detecting silver ions |
Applications Claiming Priority (1)
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