CN113984726B - Method for detecting mercury ions by amino phenylboronic acid functionalized magnetic beads/glyoxal modified DNA - Google Patents
Method for detecting mercury ions by amino phenylboronic acid functionalized magnetic beads/glyoxal modified DNA Download PDFInfo
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- LEQAOMBKQFMDFZ-UHFFFAOYSA-N glyoxal Chemical compound O=CC=O LEQAOMBKQFMDFZ-UHFFFAOYSA-N 0.000 title claims abstract description 70
- 229910052753 mercury Inorganic materials 0.000 title claims abstract description 59
- -1 mercury ions Chemical class 0.000 title claims abstract description 59
- 238000000034 method Methods 0.000 title claims abstract description 37
- 229940015043 glyoxal Drugs 0.000 title claims abstract description 35
- 239000011324 bead Substances 0.000 title claims abstract description 29
- ILOJFJBXXANEQW-UHFFFAOYSA-N aminooxy(phenyl)borinic acid Chemical compound NOB(O)C1=CC=CC=C1 ILOJFJBXXANEQW-UHFFFAOYSA-N 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- 108020004414 DNA Proteins 0.000 claims description 68
- 238000006243 chemical reaction Methods 0.000 claims description 63
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- 238000001514 detection method Methods 0.000 claims description 31
- 239000000243 solution Substances 0.000 claims description 31
- BQPIGGFYSBELGY-UHFFFAOYSA-N mercury(2+) Chemical compound [Hg+2] BQPIGGFYSBELGY-UHFFFAOYSA-N 0.000 claims description 26
- 102000053602 DNA Human genes 0.000 claims description 25
- 238000004140 cleaning Methods 0.000 claims description 14
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- 238000004458 analytical method Methods 0.000 claims description 12
- JMZFEHDNIAQMNB-UHFFFAOYSA-N m-aminophenylboronic acid Chemical compound NC1=CC=CC(B(O)O)=C1 JMZFEHDNIAQMNB-UHFFFAOYSA-N 0.000 claims description 11
- 239000000523 sample Substances 0.000 claims description 11
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims description 9
- 238000002189 fluorescence spectrum Methods 0.000 claims description 9
- 108700004991 Cas12a Proteins 0.000 claims description 8
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 8
- 239000008367 deionised water Substances 0.000 claims description 7
- 229910021641 deionized water Inorganic materials 0.000 claims description 7
- 239000008055 phosphate buffer solution Substances 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 6
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 4
- 238000004364 calculation method Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
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- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
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- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 9
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- 238000010586 diagram Methods 0.000 description 4
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 3
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- 125000003277 amino group Chemical group 0.000 description 2
- 238000009739 binding Methods 0.000 description 2
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- UTHULKKJYXJZLV-UHFFFAOYSA-N (3-aminophenoxy)boronic acid Chemical compound NC1=CC=CC(OB(O)O)=C1 UTHULKKJYXJZLV-UHFFFAOYSA-N 0.000 description 1
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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"
- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6417—Spectrofluorimetric devices
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Abstract
The invention provides a method for detecting mercury ions by amino phenylboronic acid functionalized magnetic beads/glyoxal modified DNA, which comprises the following steps: s1, preparing CMB@APBA; s2. Preparation of dna; s3, establishing a standard curve; s4, detecting a sample. The method has the advantages of convenient operation, high selectivity, high sensitivity, short time and the like.
Description
Technical Field
The invention relates to the technical field of mercury ions, in particular to a method for detecting mercury ions by using amino phenylboronic acid functionalized magnetic beads/glyoxal modified DNA.
Background
Mercury ion (Hg) 2+ ) Is a widely existing toxic metal pollutant, and has wide application in the industrial production of dye textile manufacturing industry, mining industry, smelting industry, battery manufacturing industry, fertilizer and chemical fertilizer. Wherein the soil, air and water are polluted by mercury ions to different degrees. Crops can accumulate mercury ions to different degrees under the conditions of absorbing nutrients in polluted soil and drinking polluted water by livestock. Because of its non-biodegradability and bioaccumulation in the body, people can have deleterious effects on human health, such as nucleic acid dysfunction, anemia, and cardiovascular disease, even at low concentrations, if they eat food contaminated with mercury ions for a long period of time. Therefore, the development of a method for detecting mercury ions in foods with high sensitivity and high selectivity has very important significance.
At present, various conventional methods and novel analytical means have been developed to efficiently detect Hg 2+ . Traditional methods include inductively coupled plasma mass spectrometry, atomic absorption/emission spectrometry, cold vapor atomic fluorescence spectrometry, and the like, which have high sensitivity and accuracy, but some inherent disadvantages include complicated sample pretreatment operations, high cost, and difficulty in meeting the requirements of on-site rapid detection by professional operators and large-scale equipment. Nucleic acid aptamers have been developed in an effort to develop various bases due to their ease of synthesis and modification, small size, lack of immunogenicity, and low costMethods for detecting mercury ions in nucleic acid aptamers are often combined with colorimetric methods, electrochemical methods, fluorescent methods, and the like. The colorimetry generally needs the assistance of nano materials or catalysts, quantification is carried out through color change, and then the problems of troublesome material synthesis, poor stability of a catalytic system due to environmental interference such as pH and the like, low sensitivity and the like can be faced in the practical application. Thus, efficient and rapid establishment of a suitable Hg 2+ The method of field detection is imperative.
Patent number CN103884669B discloses a method for detecting mercury ions by using a nano silver probe, and detection of mercury ions is realized by using the change of ultraviolet absorption spectra before and after adding mercury ions by using a synthesized nano silver probe. The method has high sensitivity, but the synthesized nano silver probe particles are uneven in size and are easy to be influenced by environment to cause aggregation, precipitation and other phenomena, so that the detection result is influenced.
Disclosure of Invention
The invention aims to provide a method for detecting mercury ions by amino phenylboronic acid functionalized magnetic beads/glyoxal modified DNA, which aims to solve the problems of complex sample processing operation, high cost, professional technical requirements of operators, complex material synthesis, environmental influence on a system, poor stability and the like in the existing analysis method.
The technical scheme of the invention is realized as follows: the invention provides a method for detecting mercury ions by amino phenylboronic acid functionalized magnetic beads/glyoxal modified DNA, which comprises the following steps:
preparation of CMB@APBA: connecting 3-aminophenylboronic acid to activated carboxyl magnetic beads in a manner of combining amino groups with carboxyl groups to obtain CMB@APBA;
s2. Preparation of DNA × (glyoxal modified DNA): under the heating condition, reacting the DNA solution with a mixture of glyoxal solution, dimethyl sulfoxide and deionized water to obtain glyoxal modified DNA, and regulating the pH value to 3 by using dilute hydrochloric acid, namely the DNA;
s3, establishing a standard curve: removing supernatant after magnetically separating CMB@APBA, respectively immersing in mercury ion solution with determined concentration, performing first reaction, cleaning, magnetically separating to remove supernatant, adding DNA, performing second reaction, cleaning, magnetically separating to remove supernatant, adding phosphate buffer solution and CRISPR-Cas12a system, performing third reaction, then performing fourth reaction, terminating reaction, detecting spectrum of a reference reagent by using a fluorescence spectrum analysis device, and making a linear graph of mercury ions and fluorescence intensity of a target substance;
s4, sample detection: under the same conditions of the step S3, removing supernatant after magnetically separating CMB@APBA, immersing the supernatant in a liquid to be detected, performing first reaction, cleaning, magnetically separating the supernatant, adding DNA, performing second reaction, cleaning, magnetically separating the supernatant, adding phosphate buffer solution and CRISPR-Cas12a system, performing third reaction, then performing fourth reaction, terminating the reaction, detecting the spectrum of the supernatant by using a fluorescence spectrum analysis device, establishing a calculation module of a linear relation between the mercury ion concentration of the mercury ion sample solution to be detected and the fluorescence intensity value of the reagent system at a specific wavelength, comparing the characteristic spectrum of the reference reagent obtained in the step S3 by using the analysis system, and determining the content level of the detected mercury ions by using a spectrum comparison result.
As a further improvement of the present invention, the specific steps of step S1 are as follows:
s101, dissolving N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide and N-N-hydroxysuccinimide in water to obtain an activated solution; magnetic washing the carboxyl magnetic bead solution twice, removing the supernatant, adding the activated solution into the magnetic beads, and reacting for 10-20min at room temperature;
s102, cleaning the product obtained in the step S101, removing supernatant, then adding the supernatant into 2-10mg/mL of 3-aminophenylboronic acid solution, reacting for 1-5 hours at the temperature of 35-40 ℃ and the speed of 300-700rpm, washing to remove supernatant, and adding deionized water to obtain CMB@APBA with the concentration of 2-10mg/mL.
As a further improvement of the invention, the mass ratio of the N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide to the N-N-hydroxysuccinimide is (15-20): (2-5).
As a further improvement of the present invention, the concentration of the DNA solution in step S2 is 80 to 120. Mu. Mol/L; the concentration of the glyoxal solution is 30-50wt%; the reaction condition is that the reaction is carried out for 30-50min at 40-60 ℃; the volume ratio of the DNA solution, the glyoxal solution, the dimethyl sulfoxide and the deionized water is (1-5): (10-17): (40-60): (25-35).
As a further improvement of the invention, the first reaction condition in the step S3 or the step S4 is that the reaction is carried out for 20-40min at room temperature; the second reaction condition is that in a system with pH=3, the reaction is carried out for 0.5-2h at 35-40 ℃, and the third reaction condition is that the reaction is carried out for 0.5-2h at 35-40 ℃; the fourth reaction condition is that the reaction is carried out for 5-20min at 60-70 ℃.
As a further improvement of the present invention, the volume ratio of phosphate buffer and CRISPR-Cas12a system in step S3 or step S4 is (3-5): 1, a step of; the phosphate buffer ph=7-7.7; the CRISPR-Cas12a system is prepared by mixing 8. Mu.L buffer, 6.4. Mu.L DEPC water, 3.2. Mu.L, 5. Mu.M Cas12a, 0.8. Mu.L, 20. Mu.M crRNA and 1.6. Mu.L, 10. Mu.M ssDNA.
As a further improvement of the invention, the concentration of the mercury ion solution in step S3 is in the range of 20nM to 400nM.
As a further improvement of the invention, the DNA sequence is shown as SEQ ID No. 1; the crRNA sequence is shown as SEQ ID No.2; the ssDNA sequence is shown as SEQ ID No.3.
As a further improvement of the invention, the linear equation of the method is: fl= -2263.767C Hg2+ +1203.3967(R 2 ＝0.989)。
As a further improvement of the present invention, the linear detection range is: 20nM-400nM, limit of detection: 5.46nM.
The invention has the following beneficial effects:
1. according to the invention, through the specific combination of mercury ions and 3-aminophenylboronic acid, the analysis method has good specificity, and the interference of other substances is eliminated;
2. the invention develops a new mode for capturing DNA by utilizing the characteristic that stable diamino adducts formed on three bases (A, G, C) of glyoxal modified DNA can react with 3-aminophenylboric acid to generate boric acid ester, and the DNA is fixed on an interface, so that the reaction system required in different reaction steps can be changed by magnetic washing;
3. the CRISPR-Cas12a is adopted to perform signal output in a mode of trans-cutting ssDNA, so that signal amplification can be performed, and the sensitivity of the sensor is enhanced;
4. the invention is based on a fluorescence spectrum analysis method, has the advantages of high sensitivity, stable signal, simple detection mode and the like, and can efficiently, sensitively and rapidly detect mercury ions in food.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is a schematic diagram of a method for detecting mercury ions by using amino phenylboronic acid functionalized magnetic beads/glyoxal modified DNA;
FIG. 2 is a bar graph of pH condition optimization for a DNA (glyoxal modified DNA) ligation reaction with CMB@APBA;
FIG. 3 is a graph of fluorescence spectrum of mercury ion concentration gradient detection provided by the invention;
FIG. 4 is a graph showing the linear relationship between fluorescence intensity values at 520nm in the mercury ion detection method provided by the invention;
fig. 5 is a schematic diagram of a selective test result of the method for detecting mercury ions provided by the invention.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1: method for detecting mercury ions based on amino phenylboronic acid functionalized magnetic beads/glyoxal modified DNA
The invention provides a method for detecting mercury ions based on amino phenylboronic acid functionalized magnetic beads/glyoxal modified DNA, which comprises the following steps:
preparation of CMB@APBA: connecting 3-aminophenylboronic acid to activated carboxyl magnetic beads in a manner of combining amino groups with carboxyl groups to obtain CMB@APBA;
s2, under the heating condition, reacting the DNA solution with a mixture of glyoxal solution, dimethyl sulfoxide and deionized water to obtain glyoxal modified DNA, and regulating the pH value to 3 by using dilute hydrochloric acid, namely the DNA;
s3, establishing a standard curve: removing supernatant after magnetically separating CMB@APBA, respectively immersing in mercury ion solution with determined concentration, performing first reaction, cleaning, magnetically separating to remove supernatant, adding DNA, performing second reaction, cleaning, magnetically separating to remove supernatant, adding phosphate buffer solution and CRISPR-Cas12a system, performing third reaction, then performing fourth reaction, terminating reaction, detecting spectrum of a reference reagent by using a fluorescence spectrum analysis device, and making a linear graph of mercury ions and fluorescence intensity of a target substance;
s4, sample detection: under the same conditions of the step S3, removing supernatant after magnetically separating CMB@APBA, immersing the supernatant in a liquid to be detected, performing first reaction, cleaning, magnetically separating the supernatant, adding DNA, performing second reaction, cleaning, magnetically separating the supernatant, adding phosphate buffer solution and CRISPR-Cas12a system, performing third reaction, then performing fourth reaction, terminating the reaction, detecting the spectrum of the supernatant by using a fluorescence spectrum analysis device, establishing a calculation module of a linear relation between the mercury ion concentration of the mercury ion sample solution to be detected and the fluorescence intensity value of the reagent system at a specific wavelength, comparing the characteristic spectrum of the reference reagent obtained in the step S3 by using the analysis system, and determining the content level of the detected mercury ions by using a spectrum comparison result.
Referring to fig. 1, a schematic diagram of a detection principle of detecting mercury ions based on the combination of the aminophenylboronic acid functionalized magnetic beads and glyoxal modified DNA is provided. When no mercury ions exist in the reaction system, DNA reacts with 3-aminophenylboronic acid to generate boric acid ester, so that CMB@APBA successfully captures the DNA, after the Cas12a/crRNA complex is added, the DNA is combined with the Cas12a/crRNA complex to obtain active Cas12a, so that ssDNA is cut in a trans mode, and stronger fluorescence intensity is obtained; when mercury ions exist in a reaction system, the mercury ions are specifically combined with 3-aminophenylboronic acid, and DNA cannot be captured on the surface of the magnetic beads after a magnetic washing process, so that Cas12a is still inactive after Cas12a/crRNA complex is added, ssDNA cannot be cut, and lower fluorescence intensity is caused.
The optimal experimental conditions for the combination of the two were obtained by adjusting the pH of the DNA x and cmb@apba reaction system as follows, obtaining example 2
Example 2: optimized screening of pH of CMB@APBA reaction system
Reaction of DNA with cmb@apba the pH of the system was adjusted to 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, respectively, by addition of dilute hydrochloric acid. Adding mercury ion with the concentration of 1 mu M; the present embodiment has substantially the same parameters as the first embodiment, and is characterized in that:
using DNA of 3' end marked Alexa 488 fluorophore, after glyoxal modification, adjusting the system to different pH values, measuring the fluorescence intensity; alexa 488-DNA of the same concentration and treatment mode was reacted with CMB@APBA under different pH conditions, and the fluorescence of the supernatant was measured after magnetic separation. The Alexa 488-DNA sequence is as follows: 5'-AATCCCCTCCAATGCAGGCCTAACGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-Alexa 488-3'.
Fluorescence value detection: and placing the obtained supernatant into a fluorescent cuvette, and measuring the spectrum in the wavelength range of 500-600 nm.
As shown in FIG. 2, a bar graph of fluorescence intensity at 520nm measured at different pH conditions for DNA binding reaction with CMB@APBA is shown. As can be seen from fig. 2, when the pH of the reaction system is 3, the corresponding fluorescence intensity difference value reaches the maximum. Therefore, under the condition, cmb@apba can capture more DNA, and the detection sensitivity is higher when mercury ions are detected.
In summary, in the method for detecting mercury ions provided by the invention, the optimal pH value of the reaction of DNA and CMB@APBA is 3.
Example 3: drawing a standard curve
The reaction system was 100. Mu.L in volume, the pH value of the binding reaction of DNA with CMB@APBA was 3, the mercury ion concentration of the solution to be measured was 0. Mu.M, 0.002. Mu.M, 0.004. Mu.M, 0.01. Mu.M, 0.02. Mu.M, 0.03. Mu.M, 0.04. Mu.M, 0.05. Mu.M, 0.1. Mu.M, 0.15. Mu.M, 0.2. Mu.M, 0.25. Mu.M, 0.3. Mu.M, 0.35. Mu.M, 0.4. Mu.M, 0.5. Mu.M, 1. Mu.M, 2. Mu.M, 4. Mu.M, respectively, and the remaining parameters were as described in example 1. By measuring the fluorescence of the supernatant, a linear plot of mercury ions and fluorescence intensity is made, and the level of mercury ions detected is determined.
FIG. 3 and FIG. 4, wherein FIG. 3 is a graph of fluorescence spectra of mercury ion reaction systems of different concentrations; fig. 4 is a standard graph of the method for detecting mercury ions provided by the invention. As can be seen in conjunction with fig. 3 and 4, the fluorescence intensity value of the reaction product solution at a wavelength of 520nm decreases with increasing concentration of mercury ions. As can be seen from fig. 4, the method for detecting mercury ions provided by the present invention has the following linear equation: fl= -2263.767C Hg2+ +1203.3967(R 2 =0.989), the linear relationship is good, the linear detection range is 20nM-400nM, and the detection limit is 5.46nM. Therefore, the mercury ion detection method provided by the invention has a lower detection limit and can meet the actual detection requirement.
Example 4: specificity test of the detection method of the present invention
For testing the specificity of the mercury ion detection method provided by the invention, several common metal ions Cr are selected 3+ 、Ca 2+ 、pb 2+ 、Mg 2+ 、Zn 2+ 、Fe 2+ 、Ni 2+ Verifying the specificity of the method according to the procedure of example one; similarly, cr 3+ 、Ca 2+ 、pb 2+ 、Mg 2+ 、Zn 2+ 、Fe 2+ 、Ni 2+ Hg and Hg 2+ Adding the two components together into a detection system, and verifying the anti-interference performance of the method according to the operation steps of the first embodiment; wherein Cr is 3+ 、Ca 2+ 、pb 2+ 、Mg 2+ 、Zn 2+ 、Fe 2+ 、Ni 2+ Is 2mM Hg in concentration 2+ The concentration is 10 mu M; and adding blank groups for comparison experiments;
fig. 5 is a schematic diagram of a specific experimental result of the method for detecting mercury ions provided by the invention. As can be seen from FIG. 5, even Cr 3+ 、Ca 2+ 、pb 2+ 、Mg 2+ 、Zn 2+ 、Fe 2+ 、Ni 2+ Is 200 times higher than the mercury ion concentration, and still cannot cause obvious change of fluorescence intensity value; and other metal ions to Hg 2+ The detection of the mercury ions is basically not interfered, and the main reason is the high specificity of the reaction of the mercury ions and the 3-aminophenylboronic acid, so that the detection method of the mercury ions provided by the invention has good selectivity and anti-interference performance.
Application examples: actual sample detection study
The concentration of mercury ions in milk and tea beverages was determined by the fluorescence method of this example using the sample recovery method, and all samples were averaged three times, see table below. It can be seen that the fluorescence sensor of this embodiment can accurately determine the concentration of mercury ions in an actual sample. The results are shown in Table 1.
TABLE 1
As can be seen from table 1, the detection method of mercury ions provided by the invention can prevent the glyoxal modified DNA from being combined with 3-aminophenylboronic acid by utilizing the specific combination of mercury ions and 3-aminophenylboronic acid, so that the DNA cannot be captured on the magnetic beads, ssDNA in the system cannot be trans-cut after Cas12a/crRNA is added, and the final product is analyzed by a fluorescence spectrometer and mercury ion detection is realized. The mercury ion detection method provided by the invention has the advantages of convenience in operation, high selectivity, high sensitivity, short time and the like.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
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<110> Shanghai university
<120> method for detecting mercury ions by amino phenylboronic acid functionalized magnetic beads/glyoxal modified DNA
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 55
<212> DNA
<213> none (unknown)
<400> 1
aatcccctcc aatgcaggcc taacgttttt tttttttttt tttttttttt ttttt 55
<210> 2
<211> 41
<212> RNA
<213> none (unknown)
<400> 2
uaauuucuac uaaguguaga uaaaaaaaaa aaaaaaaaaa a 41
<210> 3
<211> 5
<212> DNA
<213> none (unknown)
<400> 3
ttatt 5
Claims (9)
1. The method for detecting mercury ions by using the amino phenylboronic acid functionalized magnetic beads/glyoxal modified DNA is characterized by comprising the following steps of:
preparation of CMB@APBA: 3-aminophenylboronic acid is connected to activated carboxyl magnetic beads in a mode of combining amino and carboxyl to obtain CMB@APBA;
s2. Preparation of dna: under the heating condition, reacting the DNA solution with a mixture of glyoxal solution, dimethyl sulfoxide and deionized water to obtain glyoxal modified DNA, and regulating the pH value to 3 by using dilute hydrochloric acid, namely the DNA;
s3, establishing a standard curve: removing supernatant after magnetically separating CMB@APBA, respectively immersing in mercury ion solution with determined concentration, performing first reaction, cleaning, magnetically separating to remove supernatant, adding DNA, performing second reaction, cleaning, magnetically separating to remove supernatant, adding phosphate buffer solution and CRISPR-Cas12a system, performing third reaction, then performing fourth reaction, terminating reaction, detecting spectrum of a reference reagent by using a fluorescence spectrum analysis device, and making a linear graph of mercury ions and fluorescence intensity of a target substance;
s4, sample detection: under the same conditions of the step S3, removing supernatant after magnetically separating CMB@APBA, immersing the supernatant in a liquid to be detected, performing first reaction, cleaning, magnetically separating the supernatant, adding DNA, performing second reaction, cleaning, magnetically separating the supernatant, adding phosphate buffer solution and CRISPR-Cas12a system, performing third reaction, then performing fourth reaction, terminating the reaction, detecting the spectrum of the supernatant by using a fluorescence spectrum analysis device, establishing a calculation module of a linear relation between the mercury ion concentration of the mercury ion sample solution to be detected and the fluorescence intensity value of the reagent system at a specific wavelength, comparing the characteristic spectrum of the reference reagent obtained in the step S3 by using an analysis system, and determining the content level of the detected mercury ions by using a spectrum comparison result;
the first reaction condition in the step S3 or the step S4 is that the reaction is carried out for 20-40min at room temperature; the second reaction condition is that in a system with pH=3, the reaction is carried out for 0.5-2h at 35-40 ℃, and the third reaction condition is that the reaction is carried out for 0.5-2h at 35-40 ℃; the fourth reaction condition is that the reaction is carried out for 5-20min at 60-70 ℃.
2. The method for detecting mercury ions by using the amino phenylboronic acid functionalized magnetic beads/glyoxal modified DNA according to claim 1, wherein the specific steps of the step S1 are as follows:
s101, dissolving N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide and N-N-hydroxysuccinimide in water to obtain an activated solution; magnetic washing the carboxyl magnetic bead solution twice, removing the supernatant, adding the activated solution into the magnetic beads, and reacting for 10-20min at room temperature;
s102, cleaning the product obtained in the step S101, removing supernatant, then adding the supernatant into 2-10mg/mL of 3-aminophenylboronic acid solution, reacting for 1-5 hours at the temperature of 35-40 ℃ and the speed of 300-700rpm, washing to remove supernatant, and adding deionized water to obtain CMB@APBA with the concentration of 2-10mg/mL.
3. The method for detecting mercury ions by using the aminophenylboronic acid functionalized magnetic bead/glyoxal modified DNA according to claim 2, wherein the mass ratio of the N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide to the N-hydroxysuccinimide is (15-20): (2-5).
4. The method for detecting mercury ions by using the amino phenylboronic acid functionalized magnetic beads/glyoxal modified DNA according to claim 1, wherein the concentration of the DNA solution in the step S2 is 80-120 mu mol/L; the concentration of the glyoxal solution is 30-50wt%; the reaction condition is that the reaction is carried out for 30-50min at 40-60 ℃; the volume ratio of the DNA solution, the glyoxal solution, the dimethyl sulfoxide and the deionized water is (1-5): (10-17): (40-60): (25-35).
5. The method for detecting mercury ions by using the aminophenylboronic acid functionalized magnetic bead/glyoxal modified DNA according to claim 1, wherein the volume ratio of the phosphate buffer solution to the CRISPR-Cas12a system in step S3 or step S4 is (3-5): 1, a step of; the phosphate buffer ph=7-7.7;
the CRISPR-Cas12a system is prepared by mixing 8. Mu.L buffer, 6.4. Mu.L DEPC water, 3.2. Mu.L, 5. Mu.M Cas12a, 0.8. Mu.L, 20. Mu.M crRNA and 1.6. Mu.L, 10. Mu.M ssDNA.
6. The method for detecting mercury ions by using the amino phenylboronic acid functionalized magnetic bead/glyoxal modified DNA according to claim 1, wherein the concentration of the mercury ion solution in the step S3 is in the range of 20nM to 400nM.
7. The method for detecting mercury ions by using the amino phenylboronic acid functionalized magnetic beads/glyoxal modified DNA (deoxyribonucleic acid) according to claim 1, wherein the DNA sequence is shown in SEQ ID No. 1; the crRNA sequence is shown as SEQ ID No.2; the ssDNA sequence is shown as SEQ ID No.3.
8. The method for detecting mercury ions by using the amino phenylboronic acid functionalized magnetic bead/glyoxal modified DNA according to claim 1, wherein a linear equation of the method is as follows: fl= -2263.767
C Hg2+ +1203.3967(R 2 =0.989), wherein FL is fluorescence intensity, C Hg2+ Is the concentration of mercury ions.
9. The method for detecting mercury ions by using the amino phenylboronic acid functionalized magnetic bead/glyoxal modified DNA according to claim 8, wherein the linear detection range is as follows: 20nM-400nM, limit of detection: 5.46nM.
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