CN109444397B - Mercury ion detection method - Google Patents
Mercury ion detection method Download PDFInfo
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- CN109444397B CN109444397B CN201811285305.6A CN201811285305A CN109444397B CN 109444397 B CN109444397 B CN 109444397B CN 201811285305 A CN201811285305 A CN 201811285305A CN 109444397 B CN109444397 B CN 109444397B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
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- G—PHYSICS
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- 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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
Abstract
The invention provides a mercury ion detection method combining an enzyme-free amplification technology for forming DNA enzyme by fragment DNA and a heme-graphene oxide compound catalytic color reaction. Wherein, the surface of the heme-graphene oxide compound adsorbs a plurality of DNA fragments to prevent aggregation, the DNA fragments are formed by cutting nucleic acid molecule hairpin loops through circulation of DNase, and the DNase is formed by two divided DNase sequences passing through T-Hg 2+ -T interaction formation. The large amount of heme-graphene oxide complexes catalyzes the color reaction so that the supernatant exhibits a dark blue color. Hg can be calculated by ultraviolet and visible absorption spectrum measurement 2+ And (4) concentration. This method shows a linear range of 50pM to 1200 pM. The limit detection can be as low as 33pM. The method can rapidly and sensitively detect Hg 2+ 。
Description
Technical Field
The invention relates to the field of mercury ion detection, in particular to the field of a mercury ion detection method combining an enzyme-free amplification technology for forming DNA enzyme by fragment DNA and a heme-graphene oxide compound catalytic color reaction.
Background
Mercury has attracted public attention as one of the most toxic heavy metals, adversely affecting environmental safety and human health concerns. Mercury pollution is prevalent in various natural and human activities. The maximum pollutant level (MCL) of mercury in drinking water was set by the World Health Organization (WHO) at 30nM. Description of the Prior Art 2+ T-Hg is formed by intercalation of thymine-thymine (T-T) mismatch 2+ A T base pair, various Hg is established 2+ Detection and analysis methods include colorimetric methods, fluorescence, surface-enhanced Raman scattering and electrochemical detection methods. However, the detection limit is more than 30nM, and the detection sensitivity is not high. To increase sensitivity, enzyme-free amplification techniques are used for signal amplification, such as hybridization strand reactions and hairpin-catalyzed self-assembly of nucleic acid molecules. Conditions such as isothermal conditions, low cost and no need for enzymes have attracted much attention. However, most non-enzymatic amplification techniques require complex designs of the auxiliary nucleusAn acid molecule hairpin.
It is difficult to directly apply heme as a catalyst due to its low solubility in aqueous solutions and high molecular aggregation. In the prior art, a heme-graphene oxide compound is synthesized through pi-pi interaction between heme and GO. GO serves as a carrier for heme, providing electrical conductivity for heme. Thus, it exhibits peroxidase activity, catalyzing a color reaction of a peroxidase substrate. However, the heme-graphene oxide complex is easy to aggregate and settle in a salt solution, so that the catalytic effect of the centrifuged supernatant is not obvious. In addition, the heme-graphene oxide complex exhibits different dispersion characteristics for single-stranded or double-stranded DNA sequences in the presence of sodium chloride. And thus are used to detect hydrogen peroxide, glucose, enzyme activity, bisphenol a and DNA damage.
There is no report that the enzyme-free amplification technology for forming DNA enzyme by fragmented DNA and the heme-graphene oxide compound catalytic color reaction are combined to detect mercury ions.
Disclosure of Invention
In order to solve the problems, the invention provides a mercury ion detection method combining an enzyme-free amplification technology for forming DNA enzyme by fragmented DNA and a heme-graphene oxide compound catalytic color reaction. The "heme-graphene oxide complex" described above is hereinafter abbreviated as H-GNs.
The detection principle of the invention is as follows: in Hg 2+ In the presence of two DNA enzyme sequence fragments can pass through T-Hg 2+ The T interaction forms a duplex section in the middle, becoming two complete DNA enzyme chains at both ends. The DNA polymerase chain combines with the substrate chain sequence of the hairpin loop portion of the nucleic acid molecule to produce a DNA enzyme structure. Dnazymes can cyclically cleave the loop of nucleic acid molecule hairpins, producing a large number of DNA fragments. The resulting DNA fragments can be adsorbed onto the surface of H-GNs to prevent aggregation of H-GNs in saline solutions. Thus, the supernatant after centrifugation contained more dispersed H-GNs, and after the color reaction, the supernatant showed a dark blue color. However, in the absence of Hg 2+ In the case of (2), the H-GNs aggregate without protection of the DNA fragment, and a small amount of H-GNs remains in the supernatant after centrifugation, resulting in a bluish color.Hg is caused by circular cleavage of nucleic acid molecule hairpin 2+ The detected significant signal is amplified. The method provides a color development and enzyme-free amplification mode, and can rapidly and sensitively detect Hg 2+ Without the need for complex auxiliary hairpin designs.
The invention comprises the following steps:
(1) Synthesizing H-GNs by adopting the prior art;
(2) Heating the nucleic acid molecule hairpin sequence 5 '-CACCAAATTCTCTCTrAGGACAAAAAAAGT GGTG-3' to 90 ℃ for 5 minutes, and then cooling for 2 hours to room temperature to form a nucleic acid molecule hairpin structure;
(3) Formation of DNase 50nM fragment DNase sequence 1 and fragment DNase sequence 2 were obtained in 10mM Tris-HCl (pH 7.5) buffer with Hg 2+ Mixing the solution to be detected and the nucleic acid molecule hairpin obtained in the step (2) of 200nM for reaction to form a DNA enzyme structure,
the fragment DNase sequence 1 is 5'-TTTTGTCAGCGATCCGGAATTGTGGTTGGTGCGGCACCCATGTGAG AGAA-3',
the fragment DNase sequence 2 is 5'-TTTTGTCAGCGATCCGGAACTCCTTCCTCTTCGGCACCCATGTGAG AGAA-3';
(4) Shearing the hairpin loop of the nucleic acid molecule, mixing the solution obtained in the step (3) with 10mM Mg 2+ Mixing the solution and reacting for 15 minutes;
(5) Forming an H-GNs catalyst, diluting and mixing the H-GNs obtained in the step (1) with a Tris-HCl solution, incubating and adding a proper amount of NaCl, centrifuging and taking a supernatant, wherein the supernatant is the H-GNs catalyst;
(6) Developing and determining, the H-GNs catalyst obtained in the step (5) is used for catalyzing TMB (3,3 ',5,5' -tetramethyl benzidine) and H 2 O 2 The color reaction of the Tris-HCl buffer solution of (1), the UV-vis absorption spectrum thereof was measured, and Hg was calculated by the standard curve method 2+ And (4) concentration.
Preferably, the step (1) is specifically: the method specifically comprises the following steps: sonicating 20mL of water containing 10mg of graphene oxide (hereinafter referred to as GO) for 1 hour, and then subjecting 20mL of water containing 0.5mg of GO to sonication -1 The heme solution was mixed with the above GO dispersion and shaken for a few minutes, then added sequentially with 200. Mu.lL of an aqueous ammonia solution and 30. Mu.L of hydrazine hydrate were stirred at 60 ℃ for 3.5 hours, centrifuged for 30 minutes, and the precipitate was washed with ultrapure water several times. Diluted to 0.3mg mL with ultrapure water -1 And (5) standby.
Preferably, the Tris-HCl solution used in step (5) has a pH of 7.5 and the Tris-HCl solution used in step (6) has a pH of 5.
Preferably, the UV-vis absorption spectrum determined in step (6) is in the range of 500 to 800 nm.
The invention creatively combines the enzyme-free amplification technology for forming DNA enzyme by fragment DNA and H-GNs catalytic color reaction to detect mercury ions, which are taken as an inseparable whole, not only can specifically identify the mercury ions and amplify detection signals, but also saves the design of complex auxiliary nucleic acid molecule hairpins and solves the problem of poor catalytic effect of H-GNs easy aggregation.
Drawings
FIG. 1 is a schematic diagram of the detection process of the present invention.
FIG. 2 (A) UV-visible absorption spectra of GO, hemoglobin and H-GNs. FIG. 2 (B) AFM images of synthesized H-GNs. FIG. 2 (C) is an AFM image of GO.
FIG. 3 is a comparison of absorbance for different detection methods.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The present invention will be described in further detail with reference to examples.
Example 1
The mercury ion detection method combining the enzyme-free amplification technology for forming the DNA enzyme by fragment DNA and the H-GNs catalytic color reaction is provided, the detection schematic diagram is shown in figure 1, and the method comprises the following specific steps:
(1) Synthesis of H-GNs 20mL of water containing 10mg GO were sonicated for 1 hour, then 20mL of 0.5mg mL -1 The heme solution was mixed with the above GO dispersion and shaken for a few minutes, then 200 μ L of aqueous ammonia solution and 30 μ L of hydrazine hydrate were added in sequence, stirred at 60 ℃ for 3.5 hours, centrifuged for 30 minutes, and the precipitate was rinsed several times with ultrapure water.
The synthesized graphene composite was characterized by UV-vis absorption spectroscopy. There is a strong absorption peak near 230nm (fig. 2A), which corresponds to the pi-pi transition of aromatic C = C, and a shoulder at 290-300nm, which corresponds to the n-pi transition. C = O bond. In addition, hemoglobin shows two characteristic absorptions, a B band peak at 386nm and a Q band peak near 480-670 nm. The synthesized graphene complex showed a GO peak red-shifted to 265nm at 35nm and an absorption peak at 418nm corresponding to the B absorption band of heme with a 32nm red-shift. The red shift represents the pi-pi interaction between GO and heme. These results are consistent with previous reports that the interaction of cationic porphyrin derivatives with chemically converted graphene leads to a red-shift of the porphyrin Soret band.
AFM was used to characterize the surface of graphene composite nanoplatelets (fig. 2B). The average thickness of H-GN is about 1.2nm. The thickness increase was about 0.25nm compared to bare GO (fig. 2C). This may be caused by a 0.2nm monolayer of hemoglobin that absorbs on the GO surface. Uv-vis spectroscopy and AFM showed successful attachment of heme molecules to the GO surface.
(2) Heating a nucleic acid molecule hairpin sequence 5 '-CACCAAATTCTCTCTrAGGACAAAAAAAGT GGTG-3' to 90 ℃ for 5 minutes, and then cooling for 2 hours to room temperature to form a nucleic acid molecule hairpin structure;
(3) Forming DNase, mixing 50nM fragment DNase sequence 1 and fragment DNase sequence 2 obtained from the step (2) with Hg2+ solution to be tested and 200nM nucleic acid molecule obtained from the step (2) in 10mM Tris-HCl (pH 7.5) buffer for reaction to form DNase structure,
the fragment DNase sequence 1 is 5'-TTTTGTCAGCGATCCGGAATTGTGGTTGGTGCGGCACCCATGTGAG AGAA-3',
the fragment DNase sequence 2 is 5'-TTTTGTCAGCGATCCGGAACTCCTTCCTCTTCGGCACCCATGTGAG AGAA-3';
(4) Shearing the hairpin loop of the nucleic acid molecule, namely shearing the solution obtained in the step (3), the hairpin of the nucleic acid molecule obtained in the step (2) of 200nM and Mg 2+ Mixing the solution and reacting for 15 minutes;
(5) Forming the H-GNs catalyst, diluting and mixing a proper amount of H-GNs obtained in the step (1) and 40 mu L of solution obtained in the step (4) by using a Tris-HCl solution (pH7.5), incubating and adding a proper amount of NaCl, and centrifuging to obtain a supernatant, wherein the supernatant is the H-GNs catalyst;
(6) Developing and measuring, using 30 mu L of H-GNs catalyst obtained in the step (5)In the presence of catalyst 800 μm TMB (3,3 ',5,5' -tetramethylbenzidine) and 10mM H 2 O 2 And 760. Mu.L of Tris-HCl solution (pH 5), and Hg was measured by recording UV-vis absorption spectrum at 500 to 800nm using a spectrophotometer 2+ And (4) quantifying the concentration.
Example 2
The method is used for detecting Hg in Atractylodis rhizoma 2+ . Briefly, 1g of white atractylodes rhizome was pulverized and passed through a 100 mesh sieve. The powder was dried at 120 ℃ for 10 hours. Then, 100mL of HNO was added 3 And heated. Then, H was added dropwise 2 O 2 Until the solution became clear. The solution was diluted to 200mL with ultrapure water and filtered through a 0.45 micron filter. The pH of the solution was adjusted to 7.5 before testing as the test solution. The rest of the detection process was the same as in example 1. The results show Hg in two different samples of Atractylodis rhizoma 2+ The concentrations of (A) and (B) were 68.7. Mu.g/kg, respectively -1 And 72.1. Mu.g.kg -1 . Adding 10 and 20 mu g/kg of label -1 The post recovery was 91% and 97%.
Comparative example
In order to embody the technical effects of the above embodiments, the following comparative examples are provided: comparative example 1: the H-GNs were discarded and the rest of the test procedure was the same as in example 1; comparative example 2: the H-GNs were replaced with GO and the rest of the test procedure was the same as in example 1; comparative example 3: same as example 1; comparative example 4: a blank sample, namely the liquid to be tested does not contain mercury ions, and the rest of the test process is the same as that of the example 1; comparative example 5: the molar ratio of fragment dnase sequence to nucleic acid molecule hairpin decreases to 1:2, the rest of the test procedure is the same as in example 1; comparative example 6: the shear reaction time was reduced to 5 minutes and the rest of the test procedure was the same as in example 1.
The results are shown in FIG. 3, where 1 is the absorbance signal of comparative example 1, and there is no color reaction due to the absence of H-GN. The absorbance signal of comparative example 2, no. 2, shows similar absorbance as comparative example 1, which means that heme has a significant influence on the color reaction, which induces the formation of the entire dnase and the catalytic cleavage of nucleic acid molecule hairpins in the presence of Hg2+, producing a large number of DNA fragments and the color reaction is dark blue, as shown in comparative example 3, i.e., the absorbance signal of example 1, no. 3. No. 4 is the absorbance signal of comparative example 4, i.e., a blank signal, no catalytic cleavage reaction could be induced without Hg2+, resulting in a small amount of DNA fragments and a color development reaction in light blue, indicating that the method of the present invention is specific for recognition of Hg2 +. No. 5 is the absorbance signal of comparative example 5, when the molar ratio of fragment DNase sequence to nucleic acid molecule hairpin decreased to 1:2, when the pressure is lower; this means that the molar ratio of dnase to nucleic acid molecule hairpin is 1:1 and does not initiate a cyclic shear amplification reaction. No. 6 is the absorbance signal of comparative example 6, and the intensity of. DELTA. Absorbance was relatively low due to a small amount of DNA fragments caused by the cleavage reaction. When the shear reaction time was reduced to 5 minutes, the delta absorbance intensity decreased. This can be attributed to one-third of the incubation time resulting in incomplete cyclic shear amplification reaction.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (4)
1. A mercury ion detection method comprises the following steps:
(1) Synthesizing a heme-graphene oxide compound, namely synthesizing H-GNs by adopting the prior art, wherein the heme-graphene oxide compound is called H-GNs for short;
(2) Heating the nucleic acid molecule hairpin sequence 5 '-CACCAAATTCTCTCTrAGGACAAAAAAAGTGGTG-3' to 90 ℃ for 5 minutes, and then cooling for 2 hours to room temperature to form a nucleic acid molecule hairpin structure;
(3) Formation of DNase 50nM fragment DNase sequence 1 and fragment DNase sequence 2 were purchased in 10mM Tris-HCl buffer pH7.5 together with Hg 2+ Mixing the solution to be detected and the nucleic acid molecule hairpin obtained in the step (2) of 200nM for reaction to form a DNA enzyme structure,
the fragment DNase sequence 1 is 5'-TTTTGTCAGCGATCCGGAATTGTGGTTGGTGCGGCACCCATGTGAGAGAA-3',
the fragment DNase sequence 2 is 5'-TTTTGTCAGCGATCCGGAACTCCTTCCTCTTCGGCACCCATGTGAGAGAA-3';
(4) Shearing the hairpin loop of the nucleic acid molecule, mixing the solution obtained in step (3) with 10mM Mg 2+ Mixing the solution and reacting for 15 minutes;
(5) Forming an H-GNs catalyst, mixing the H-GNs obtained in the step (1) and the solution obtained in the step (4), diluting with a Tris-HCl solution, incubating, adding a proper amount of NaCl, and centrifuging to obtain a supernatant, wherein the supernatant is the H-GNs catalyst;
(6) Developing and determining, using the H-GNs catalyst obtained in the step (5) to catalyze the H and the 3,3',5,5' -tetramethyl benzidine 2 O 2 The color reaction of the Tris-HCl buffer solution of (1), the UV-vis absorption spectrum thereof was measured, and Hg was calculated by the standard curve method 2+ The concentration of the above-mentioned 3,3',5,5' -tetramethylbenzidine is abbreviated as TMB.
2. The method according to claim 1, wherein step (1) is specifically: sonicate 20mL of water containing 10mg of graphene oxide for 1 hour, then sonicate 20mL of 0.5mg mL -1 Mixing the heme solution with the graphene oxide dispersion and shaking for several minutes, then sequentially adding 200 μ L of ammonia water solution and 30 μ L of hydrazine hydrate, stirring at 60 ℃ for 3.5 hours, centrifuging for 30 minutes, washing the precipitate with ultrapure water for several times, diluting with ultrapure water to 0.3mg mL -1 And (5) standby.
3. The method of claim 2, wherein the Tris-HCl solution used in step (5) has a pH of 7.5 and the Tris-HCl solution used in step (6) has a pH of 5.
4. A method according to claim 3, wherein the UV-vis absorption spectrum determined in step (6) is in the range of 500 to 800 nm.
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| CN104597240A (en) * | 2015-02-02 | 2015-05-06 | 广西医科大学 | Biosensing method for detecting leukemia by graphene/mimetic peroxidase double-signal amplification |
| CN106191042A (en) * | 2016-07-16 | 2016-12-07 | 湖南工程学院 | Two-way Cycle series signals based on exonuclease III auxiliary amplifies DNA combination probe compositions and preparation method and application |
| CN107314981A (en) * | 2017-07-31 | 2017-11-03 | 河南大学 | The method that detection PARP activity is analyzed based on hemin graphene composite materials |
| CN107941797A (en) * | 2017-12-11 | 2018-04-20 | 福州大学 | A kind of visual colorimetric determination sensor for detecting mercury ion |
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Patent Citations (4)
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| CN104597240A (en) * | 2015-02-02 | 2015-05-06 | 广西医科大学 | Biosensing method for detecting leukemia by graphene/mimetic peroxidase double-signal amplification |
| CN106191042A (en) * | 2016-07-16 | 2016-12-07 | 湖南工程学院 | Two-way Cycle series signals based on exonuclease III auxiliary amplifies DNA combination probe compositions and preparation method and application |
| CN107314981A (en) * | 2017-07-31 | 2017-11-03 | 河南大学 | The method that detection PARP activity is analyzed based on hemin graphene composite materials |
| CN107941797A (en) * | 2017-12-11 | 2018-04-20 | 福州大学 | A kind of visual colorimetric determination sensor for detecting mercury ion |
Non-Patent Citations (1)
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