CN107561064B

CN107561064B - Application of G-quadruplex DNA enzyme in ultra-weak chemiluminescence detection of sulfide ions

Info

Publication number: CN107561064B
Application number: CN201710608208.5A
Authority: CN
Inventors: 侯静; 王素华
Original assignee: North China Electric Power University
Current assignee: North China Electric Power University
Priority date: 2017-07-24
Filing date: 2017-07-24
Publication date: 2020-09-08
Anticipated expiration: 2037-07-24
Also published as: CN107561064A

Abstract

The invention belongs to the technical field of detection, and provides application of G-quadruplet DNA enzyme in ultra-weak chemiluminescence detection of sulfide ions, wherein the G-quadruplet DNA enzyme has peroxidase catalytic activity. The invention also provides a method for detecting the ultra-weak chemiluminescence of the sulfide ions by using the G-quadruplex DNA enzyme. The rapid detection method for the sulfur ions based on the ultra-weak chemiluminescence method provided by the invention establishes, perfects and develops a biosensing analysis method for sulfur ion pollutants, can provide reference for environmental health risk evaluation, develops a new rapid, high-sensitivity and low-cost detection method, and has important significance and better application value for perfecting the existing detection technical means.

Description

Application of G-quadruplex DNA enzyme in ultra-weak chemiluminescence detection of sulfide ions

Technical Field

The invention belongs to the technical field of detection, and particularly relates to a chemiluminescence detection method for sulfide ions.

Background

Pollutants in the environment directly affect the ecological environment on which human beings depend to live, and threaten the health of human beings. The monitoring and the prevention and the treatment of environmental pollutants become an important link for accelerating the modernization pace of the country and adhering to the strategy of sustainable development, and the detection of pollutants in industrial sewage is a key link in the link. In recent years, the discharge of sulfur-containing sewage has serious consequences on an environmental system and an environmental ecological environment, and sulfides contained in water not only directly affect the health of human bodies, but also can react with certain substances in the water or air to generate H₂S、SO₂And the harmful gases not only seriously pollute the atmosphere, but also cause a series of serious pollution such as acid rain and the like. Therefore, the sulfur-containing wastewater is analyzed and detected, and the method has important significance for strictly controlling the low-standard discharge of the sulfur-containing wastewater.

Research shows that the sulfur-containing compounds exist in nature in a complex form, the sulfur content of different samples is greatly different, and the sulfur content of different samples is difficult to adopt a single determination method. So far, high-sensitivity and selective detection of sulfur ions can be realized by applying instrument analysis methods such as fluorescence spectrophotometry, ultraviolet-visible spectrophotometry, ion selective electrode method, ion chromatography and the like. However, in industrial and agricultural production and environmental monitoring thereof, the instrument is required to react quickly and can perform continuous and on-site measurement, and the detection method by adopting a large-scale chromatographic instrument cannot meet the requirement of field on-site detection, so that the application of the instrument in the detection of the sulfur ions in the sewage is limited. Therefore, the method for detecting the sulfur ions more quickly, simply, accurately, sensitively and efficiently has important significance and research value.

The ultra-weak chemiluminescence method has the advantages of short sample preparation period, rapid detection, small sample dosage, strong response value and the like, and is an ideal method for rapidly detecting sulfur ion pollutants. Meanwhile, with the deep knowledge of DNA, researchers find that DNA with G-quadruplex result can be combined with hemin molecule to form G-quadruplex/hemin complex, called G-quadruplex DNase, which has strong catalytic activity and can catalyze oxydol to oxidize luminol to generate chemiluminescence (Kosman, J., Juskowiak, B.,2011. Peroxidase-hybridization DNAzymes for biosensing applications: A review, animal, Chim. acta 707, 7-17). Based on this property, G-quadruplex DNases have been widely used in the field of biosensing analysis. However, no research report for ultra-weak chemiluminescence detection of sulfur ion pollutants based on G-quadruplex DNA enzyme has been reported so far.

Disclosure of Invention

Aiming at the defects in the field, the invention aims to provide the application of G-quadruplex DNA enzyme in the ultra-weak chemiluminescence detection of sulfide ions.

The second purpose of the invention is to provide a method for detecting the ultra-weak chemiluminescence of the sulfur ions by using G-quadruplex DNA enzyme.

The technical scheme for realizing the above purpose of the invention is as follows:

the application of G-quadruplex DNA enzyme in the ultra-weak chemiluminescence detection of sulfur ions, wherein the G-quadruplex DNA enzyme has peroxidase catalytic activity.

Wherein the sequence of the G-quadruplex DNA enzyme DNA is one of sequences shown by PW17, PS2.M and PS2. M2.

The DNA powder can be PW17DNA, and the nucleotide sequence is as follows: GGGTAGGGCGGGTTGGG are provided.

In practice, other DNA sequences having a contiguous G sequence may be used for substitution, for example:

PS2.M:GTGGGTAGGGCGGGTTGG；

PS2.M2:GGGTAGGGCGGGTTGGGT。

a method for detecting ultra-weak chemiluminescence of sulfide ions by using G-quadruplex DNA enzyme comprises the following steps:

(1) mixing G-quadruplex DNA enzyme, luminol solution, and sulfide ion (S)^2-Ions) are mixed and homogenized by a vortex oscillator;

(2) placing the mixed solution prepared in the step (1) into a chemiluminescence detection pool, and then placing H into the chemiluminescence detection pool₂O₂And (4) quickly injecting the mixture into a chemiluminescence detection pool, and immediately detecting a chemiluminescence signal.

Preferably, in the mixed luminescent system obtained in the step (2), the concentration of the G-quadruplex DNA enzyme is 1-50nM, and the concentration of the luminol is 1-100 nM; step (2) H₂O₂The concentration of the solution is 1-100 mM.

More preferably, in the obtained mixed luminescent system, the molar concentration ratio of the G-quadruplex DNase to the luminol is 1: 5-10; the molar concentration of the sulfur ions is 0-1000 nM.

Wherein the G-quadruplex DNA enzyme is prepared by the following method:

s1: dissolving a DNA sample by using a buffer solution, treating the DNA sample in a water bath at the temperature of 80-90 ℃ for 1-60min, taking out the DNA sample, uniformly mixing the DNA sample by using a vortex oscillator, and naturally cooling the DNA sample at room temperature for later use;

s2: and (4) adding the DNA solution prepared in the step S1 into a centrifuge tube, adding a buffer solution into the centrifuge tube, incubating for 1-200min, adding hemin, uniformly mixing by using a vortex oscillator, and standing for 1-200min to obtain the G-quadruplex DNA enzyme.

Further, the molar concentration ratio of DNA to hemin in the DNA peroxide mimetic enzyme of step S1 is 1: 0.1 to 10; the concentration of DNA in the solution after the buffer solution is added in step S2 is 1-50. mu. mol/L.

Wherein the pH value of the constructed mixed luminescent system is 4-12; the buffer solution is prepared from Tris and KClO₄、KH₂PO₄，K₂HPO₄Two or three of phosphoric acid, potassium phosphate, citric acid, hydrochloric acid and potassium chloride.

Further, the pH value of the mixed luminescent system is 8.0-10.0, and the buffer solution is prepared from Tris and KClO₄Is prepared by the following steps.

The method for detecting the sulfur ion ultra-weak chemiluminescence further comprises the steps of preparing a mixed luminescent system by using sulfur ions with standard concentration, detecting ultra-weak chemiluminescence signals, constructing a standard curve, substituting the detection value of a sample to be detected into a calculation, and carrying out quantitative analysis on sulfur ion pollutants of the sample to be detected.

The invention has the beneficial effects that:

the rapid detection method for the sulfur ions based on the ultra-weak chemiluminescence method provided by the invention establishes, perfects and develops a biosensing analysis method for sulfur ion pollutants, can provide reference for environmental health risk evaluation, develops a new rapid, high-sensitivity and low-cost detection method, and has important significance and better application value for perfecting the existing detection technical means.

The invention establishes the ultra-weak chemiluminescence method for detecting the sulfur ion pollutants based on the G-quadruplet DNA enzyme-hydrogen peroxide-luminol chemiluminescence system, and the chemiluminescence method has the advantages of simple preparation, quick detection, low cost, small sample dosage, high sensitivity and the like. Provides a new detection method for the rapid detection of the sulfur ion pollutants in the environmental system, which has important significance in improving the existing detection technology of the sulfur ion pollutants and simultaneously widens the application of the G-quadruplet DNA enzyme in the field of analytical chemistry.

Drawings

FIG. 1 shows the measured chemiluminescence CL spectra of different systems in example 4 of the invention; a represents a compound containing luminol and H₂O₂G-quadruplet DNA enzyme buffer solution system; b represents a compound containing luminol and H₂O₂A sulfide ion and a G-quadruplex DNA enzyme buffer solution system.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

The invention is further described with reference to the following figures and specific embodiments.

EXAMPLE 1 preparation of G-quadruplex DNase

The solid DNA powder (PW17) was washed with 20mM Tris-HClO at pH 9.0₄Dissolving the buffer solution, mixing with vortex oscillator, placing in water bath at 85 deg.C for 10min, taking out, standing at room temperature, and naturally cooling.

An appropriate amount of the above DNA solution was put into a centrifuge tube, and 20mM Tris-KClO with pH 9.0 was added thereto₄Diluting the solution to 10 mu mol/L, incubating for 2h, adding 12 mu mol/L hemin, mixing uniformly by using a vortex oscillator, and standing for 2h to obtain a G-quadruplex/hemin compound with the concentration of 10 mu mol/L, namely G-quadruplex DNA enzyme.

Example 2

The detection equipment is a weak chemiluminescence measuring instrument, and the manufacturer is Beijing Apersus Tech Co.

With 20mM Tris-KClO, pH 9.0₄Preparing a mixed solution containing 100nM G-quadruplex DNA enzyme and 800nM luminol from the buffer solution, and marking as solution A; 400mM H was prepared₂O₂Solution, marked as solution B; a mixed solution of 100. mu.M of sulfide ions was prepared using a Tris-KClO4 buffer solution and designated as solution C.

1. 2940 μ L of the above buffer solution was added to the measurement cell, 30 μ L of solution A was added to the chemiluminescence detection cell, and 30 μ L of solution B was injected to the chemiluminescence detection cell to perform chemiluminescence measurement (line of FIG. 1 a).

2. 2910. mu.L of the above buffer solution was added to the measurement cell, 30. mu.L of solution A and 30. mu.L of solution C were added to the chemiluminescence detection cell, and 30. mu.L of solution B was injected into the chemiluminescence detection cell to carry out chemiluminescence measurement (FIG. 1B line).

As can be seen from fig. 1, when luminol, hydrogen peroxide and G-quadruplet dnase exist in the determination system, the chemiluminescence signal (the chemiluminescence intensity, CL intensity) is very strong (line a), which indicates that G-quadruplet dnase can effectively catalyze the luminol and hydrogen peroxide system to generate chemiluminescence; when sulfur ions exist, the chemiluminescence system chemiluminescence signal is reduced significantly (line b), so that the detection of the sulfur ions can be realized according to the change of the signal.

Example 3 Effect of pH on the chemiluminescent Signal

A mixed luminescence system having different pH values was set, and chemiluminescence detection was performed in the same manner as in example 2. Wherein the buffer solution is Tris-KClO with pH values of 4.0, 6.0, 8.0, 10.0 and 12.0₄And (4) a buffer solution.

When the pH value of the mixed luminescent system is 8.0-12.0, a distinct luminescent signal can be detected.

When the pH value of the mixed luminescent system is 8.0-10.0, the luminescent signal is stronger.

Example 4

1. Uniformly mixing the G-quadruplex DNA enzyme prepared in the example 1 with a luminol solution and sulfur ions with different concentrations by using a vortex oscillator;

2. placing the mixed solution obtained in the step 1 in a chemiluminescence detection pool, and then placing H₂O₂And (4) quickly injecting the mixture into a chemiluminescence detection pool to detect a chemiluminescence signal.

In order to verify the influence of the sulfur ions with different concentrations on the luminescence signal, the sulfur ion concentrations in the mixed system are respectively 0nmol/L, 10nmol/L, 50nmol/L, 100nmol/L, 500nmol/L and 1000nmol/L by adding sulfur ion solutions with different volumes. Simultaneous mixed light emitting system: the concentration of G-quadruplex DNase was 1nM, K⁺In a concentration of 20mM (K in buffer solution)⁺)，H₂O₂Is 4mM and luminol is 8 nM.

The chemiluminescence intensities of different mixed luminescence systems were measured and are shown in table 1.

TABLE 1 chemiluminescence intensity after interaction with different concentrations of sulfide ion

The test result shows the good linear relation between the concentration of the sulfur ions and the luminous intensity, and can be used for detecting unknown water samples.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

SEQUENCE LISTING

<110> North China university of electric Power

Application of <120> G-quadruplex DNA enzyme in ultra-weak chemiluminescence detection of sulfur ions

<130>KHP171114527.9

<160>3

<170>PatentIn version 3.3

<210>1

<211>17

<212>DNA

<213>PW17

<400>1

gggtagggcg ggttggg 17

<210>2

<211>18

<212>DNA

<213>PS2.M

<400>2

gtgggtaggg cgggttgg 18

<210>3

<211>18

<212>DNA

<213>PS2.M2

<400>3

gggtagggcg ggttgggt 18

Claims

1. A method for detecting ultra-weak chemiluminescence of sulfide ions by using G-quadruplex DNA enzyme is characterized by comprising the following steps:

(1) mixing G-quadruplet DNA enzyme, luminol solution and sulfur ions, and uniformly stirring by using a vortex oscillator; the sequence of the G-quadruplex DNA enzyme DNA is one of sequences shown by PW17, PS2.M and PS2. M2;

(2) placing the mixed solution prepared in the step (1) into a chemiluminescence detection pool, and then placing H into the chemiluminescence detection pool₂O₂Quickly injecting the mixture into a chemiluminescence detection pool, and immediately detecting a chemiluminescence signal;

in a mixed luminescent system, the concentration of G-quadruplex DNA enzyme is 1-50nM, and the concentration of luminol is 1-100 nM; the molar concentration ratio of the G-quadruplex DNase to the luminol is 1: 5-10; step (2) H₂O₂The concentration of the solution is 1-100 mM; the molar concentration of the sulfide ions is 0-1000 nM;

the pH value of the mixed luminescent system is 8.0-10.0, and the buffer solution is composed of Tris and KClO₄Is prepared by the following steps.

2. The method for detecting ultra-weak chemiluminescence of sulfide ion according to claim 1, wherein the G-quadruplex DNase is prepared by:

3. The method of claim 2, wherein the molar ratio of DNA to hemin in the G-quadruplex DNase of step S2 is 1: 0.1 to 10; the concentration of DNA in the solution after the buffer solution is added in step S2 is 1-50. mu. mol/L.

4. The method according to any one of claims 1 to 3, wherein a mixed luminescence system is prepared using sulfide ions of a standard concentration, ultra-weak chemiluminescence signal detection is performed, a standard curve is constructed, a detection value of a sample to be detected is calculated, and quantitative analysis is performed on sulfide ion contaminants in the sample to be detected.