CN110819695B - Method for detecting silver ions - Google Patents
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- CN110819695B CN110819695B CN201810915714.3A CN201810915714A CN110819695B CN 110819695 B CN110819695 B CN 110819695B CN 201810915714 A CN201810915714 A CN 201810915714A CN 110819695 B CN110819695 B CN 110819695B
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- 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 biomolecules in the porous material 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 is simple, efficient, sensitive, good in selectivity, convenient and fast, low in cost and wide in application range.
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 (4).
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 prepared byThe food enters the food chain of human beings after being continuously accumulated in agricultural products and aquatic products. 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, environmental and other fields + The detection requirement of (2).
As the metal ions can form coordination bonds with base pairs and can replace hydrogen bonds in the conventional Watson-Crick base pairs to form metal-base pairs, the effect has important significance on the rapid and efficient detection of the metal ions, and thus the metal-base pairs 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 + The detection of (3). Furthermore, only Ag is recognized due to C-C base mismatch + To form Ag + Cross 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 (2) 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 serve as a 'pore cap' to plug the orifice of the nano-carrier and prevent substances in the pores from leaking; on the other hand as Ag + The recognition probe of (A) can be reacted with Ag + A 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.
The exonuclease can be applied to 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 sheared again for 82308230while Ag is obtained + 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 isContains trace 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 detection of Ag provided by the invention + 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 an object molecule such as a fluorescent dye in the nano-gold composite material, preferably, the fluorescent dye is rhodamine B. In order to prevent the leakage of the fluorescent dye, the invention designs and synthesizes the silver-coated silver + 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 biomolecule is 5 + 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, wherein the positive charge modifier is preferably poly diallyl dimethyl ammonium chloride. In order to open more "caps", release more fluorochrome, ag-coated beads designed and synthesized in the present 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 circulating 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 a binding product of the specific base mismatch recognition reaction and silver ions 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 + After 10-12h, carrying out magnetic separation on the biomolecule solution, and washing the biomolecule solution by using MOPS buffer solution;
wherein, the recognizable Ag + The base sequence of the biomolecule of (1) is 5 'TCC CTTAAG GAA CCACCCACCA-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 + The biological molecules are combined with nano-gold materials with hollow and porous structures, and can be coated with Ag through 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 Cd 2+ ，Hg 2+ ，Pb 2+ ，Cu 2+ ，Fe 3+ ，Zn 2+ 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 - 11 Detecting 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 -13 mol/L. Compared with literature value, the invention is to Ag + The detection sensitivity of (2) is improved by nearly 100 times. The invention can be used for preparing the nano-gold composite material based on the exonuclease cyclic amplification technology and the base mismatch identification technology, and the preparation method and the detection technology thereof have huge medical application potential and wide application prospect, and can play an important role in the fields of early diagnosis and treatment of serious diseases, food, biomedicine, medicine, environment and the like.
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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).
Experimental reagent: exonuclease Exo III (Thermo Scientific, USA); poly-dienylpropyldimethylammonium chloride (shanghai aladine biochemistry science and technology, ltd); 3-4 μm mercapto magnetic beads (Tianjin Shuanle chromatography development center); rhodamine B (Shanghai Aladdin Biotechnology Ltd.); can be covered with Ag + The identified biomolecule is a specially designed and synthesized cytosine-rich nucleic acid biomolecule with a certain base length, the base sequence is 5.
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 nucleic acid biomolecules are capable of binding to 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 (diallyldimethylammonium chloride) solution (200 mu L,11.664 mg/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) -4 mol/L), diluted to 100 μ L with MOPS buffer solution pH =7.0, added recognizable Ag after 10h at 37 ℃ + 10. Mu.L of a biomolecule solution (final concentration of 1.0X 10) -6 mol/L), performing magnetic separation after 10 hours at 37 ℃, and cleaning by using MOPS buffer solution;
wherein, the identifiable Ag + The nucleic acid biomolecule of (1), whose base sequence is 5 'TCC TCC CTTAAG GAACCACCACCCCACC 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.Wang and X.L.Luo.chem.Commun.,2015,51, 9109-9112.).
Example 2:
the invention provides a method for detecting Ag by adopting a nano-gold composite material based on an exonuclease cyclic amplification technology and a 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 mismatched base pairs 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 -11 At mol/L, the intensity of fluorescence signal and Ag + The logarithm of the concentration shows a good linear relationship, and the linear equation is: FL =354.1304+121.7803lgC Ag + (10 -13 ) The linear correlation coefficient was 0.9935.
The invention combines the exonuclease circulating amplification technology with the base mismatch identification technology and the nano-carrier technology, and can be used for synthesizing Ag through design + Recognized biological molecules are assembled on the surface of a nano carrier to form a 'pore cap', and the mismatch of C-C base and Ag is 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 Cd 2+ ，Hg 2+ ，Pb 2+ ，Cu 2+ ，Fe 3+ ，Zn 2+ 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 + The invention provides a nano-gold composite material based on an exonuclease cyclic amplification technology and a base mismatch identification technology, and a preparation method and a detection technology thereof, which have great medical application potential and wide application prospect and 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 (2)
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 + Through the combination of biomolecules subjected to base mismatch recognition, more dye molecules are released, and finally the Ag is realized by utilizing a 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 a binding product of the specific base mismatch recognition reaction and silver ions 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 diallyl dimethyl ammonium 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 composite material prepared in the step (3), diluting with MOPS buffer solution with pH =7.0, adding exonuclease, and oscillating at constant temperature of 37 ℃ for 2-3 h;
(5) Performing magnetic separation, and taking supernatant for fluorescence detection;
said can react with Ag + The nucleic acid biomolecule identified by base mismatch has a base sequence of 5.
2. A detecting Ag according to claim 1 + The method is characterized in that: the dye molecule is rhodamine B.
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| CN113295656A (en) * | 2020-02-22 | 2021-08-24 | 青岛科技大学 | Intracellular As3+、Pb2+And Hg2+Simultaneous fluorescence imaging method |
| CN113295857A (en) * | 2020-02-22 | 2021-08-24 | 青岛科技大学 | Detect As simultaneously3+、Pb2+And Hg2+Method (2) |
| CN113295657A (en) * | 2020-02-22 | 2021-08-24 | 青岛科技大学 | Detect As simultaneously3+、Pb2+And Hg2+The nanocapsule-nucleic acid molecule compound and the preparation method thereof |
| CN112094886A (en) * | 2020-08-05 | 2020-12-18 | 青岛科技大学 | Method for detecting urine biomarkers of tumor patients |
| CN114317820A (en) * | 2021-12-20 | 2022-04-12 | 西南大学 | Single-particle visual sensor for multiple hepatitis virus markers constructed based on different scattered light nanoparticles |
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Effective date of registration: 20230828 Address after: 230000 B-2705, wo Yuan Garden, 81 Ganquan Road, Shushan District, Hefei, Anhui. Patentee after: HEFEI LONGZHIYUN PHARMACEUTICAL TECHNOLOGY Co.,Ltd. Address before: 230000 floor 1, building 2, phase I, e-commerce Park, Jinggang Road, Shushan Economic Development Zone, Hefei City, Anhui Province Patentee before: Dragon totem Technology (Hefei) Co.,Ltd. Effective date of registration: 20230828 Address after: 230000 floor 1, building 2, phase I, e-commerce Park, Jinggang Road, Shushan Economic Development Zone, Hefei City, Anhui Province Patentee after: Dragon totem Technology (Hefei) Co.,Ltd. Address before: 266000 Songling Road, Laoshan District, Qingdao, Shandong Province, No. 99 Patentee before: QINGDAO University OF SCIENCE AND TECHNOLOGY |