阅读说明：本技术 一种基于硅量子点、荧光素标记的dna、剪切酶的汞离子荧光检测方法 (Mercury ion fluorescence detection method based on silicon quantum dots, fluorescein labeled DNA and shear enzyme ) 是由 由天艳 李文佳 刘�东 李玉叶 于 2020-05-08 设计创作，主要内容包括：本发明属于荧光生物检测技术领域,公开了一种基于硅量子点、荧光素标记的DNA、剪切酶的汞离子荧光检测方法。本发明中,我们利用硅量子点带正电并可以猝灭荧光素(Rox)的荧光信号的性质,将可以与汞离子特异性结合成双链的DNA连接在Rox上,结合exonuclease III酶剪切双链DNA,达到信号放大的目的,实现了对汞离子的高灵敏检测。本发明提出的基于硅量子点、荧光素标记的DNA、剪切酶的汞离子荧光检测方法对汞离子检测的线性范围宽(2×10<Sup>-11</Sup>–1×10<Sup>-</Sup><Sup>8</Sup>mol/L),检测限低(6.67×10<Sup>-12</Sup>mol/L),并且具有良好的选择性。通过标准加入法,对镇江古运河水和农田土壤中的汞离子进行分析,获得了满意的回收率。具有良好的实际应用前景。(The invention belongs to the technical field of fluorescence biological detection, and discloses a mercury ion fluorescence detection method based on silicon quantum dots, fluorescein labeled DNA and a shear enzyme.A DNA capable of being specifically combined with mercury ions into a double-chain is connected to Rox by utilizing the property that the silicon quantum dots are positively charged and can quench a fluorescence signal of the fluorescein (Rox), and the double-chain DNA is sheared by combining exonuclease III enzyme to achieve the purpose of signal amplification and realize high-sensitivity detection of mercury ions.A mercury ion fluorescence detection method based on the silicon quantum dots, the fluorescein labeled DNA and the shear enzyme, which is provided by the invention, has a wide linear range for mercury ion detection (2 × 10) ‑11 –1×10 ‑ 8 mol/L), low detection limit (6.67 × 10) ‑12 mol/L) and has good selectivity. The mercury ions in the ancient canal water and farmland soil of Zhenjiang were analyzed by a standard addition method to obtain the waterThe recovery rate is satisfactory. Has good practical application prospect.)

1. A mercury ion fluorescence detection method based on silicon quantum dots, fluorescein labeled DNA and a shear enzyme is characterized by comprising the following steps:

(1) adding the prepared SiQDs solution and Rox-DNA solution into a certain volume of Tris-HCl solution, and incubating for a certain time at a specific temperature;

(2) adding Hg with known concentration into the mixed solution prepared in the step (1)²⁺Mixing the solutions, and incubating for a certain time at a specific temperature;

(3) adding an ExoIII solution into the mixed solution prepared in the step (2), mixing, acting for a period of time at a specific temperature, then inactivating the ExoIII at a high temperature, cooling the mixed solution, and detecting the fluorescence intensity I of the solution at 414nm and 607nm respectively at room temperature by using a fluorescence spectrophotometer₄₁₄And I₆₀₇Obtaining the fluorescence intensity I₆₀₇/I₄₁₄A standard curve of the ratio to the logarithm of the concentration of mercury ions;

(4) the known Hg concentration is adjusted according to the procedures of the steps (1) to (3)²⁺Hg to be measured for solution²⁺Replacing the solution, and respectively detecting the fluorescence intensity I of the solution at 414nm and 607nm by a fluorescence spectrophotometer at room temperature₄₁₄And I₆₀₇Calculate the time I₆₀₇/I₄₁₄And (4) substituting the ratio into the standard curve in the step (3) to obtain the concentration of the mercury ions in the solution to be detected.

2. The fluorescence detection method for mercury ions based on silicon quantum dots, fluorescein labeled DNA and splicing enzyme as claimed in claim 1, wherein in the step (1), the concentration of SiQDs solution is 0.5mg/mL, the concentration of Rox-DNA solution is 1 μ M, the volume ratio of SiQDs solution, Rox-DNA solution and Tris-HCl is 10: 10: 53; the incubation conditions were: incubate at 37 ℃ for 15 min.

3. The method for fluorescence detection of mercury ions based on silicon quantum dots, fluorescein-labeled DNA and splicing enzyme according to claim 1, wherein in the step (2), Hg is used²⁺The concentration of the solution was 2 × 10^-11–1×10^-8mol/L; SiQDs solution: hg is a mercury vapor²⁺The volume ratio of the solution is 5: 2; the incubation conditions were: incubate at 37 ℃ for 40 min.

4. The fluorescence detection method for mercury ions based on silicon quantum dots, fluorescein-labeled DNA, and splicing enzyme according to claim 1, wherein in the step (3), the concentration of the ExoIII solution is 2U/μ L, and the concentration of the SiQDs solution: ExoIII solution volume ratio 10: 3; incubating at 37 deg.C for 65 min; ExoIII was inactivated by standing at 80 ℃ for 10 min.

5. The fluorescence detection method for mercury ions based on silicon quantum dots, fluorescein labeled DNA and splicing enzyme as claimed in claim 1, wherein in steps (3) and (4), the excitation wavelength of the fluorescence spectrophotometer is set to 350nm and 550nm, the excitation slit width is 3nm, and the emission slit width is 3 nm.

6. The fluorescence detection method for mercury ions based on silicon quantum dots, fluorescein labeled DNA and splicing enzyme as claimed in claim 1, wherein the concentration of Tris-HCl is 10mM, pH is 7.0, 10mM MgCl₂。

Technical Field

The invention belongs to the technical field of fluorescence biological detection, and particularly relates to a mercury ion fluorescence detection method based on silicon quantum dots, fluorescein labeled DNA and a shear enzyme.

Background

Mercury (Hg)²⁺) Contamination has been recognized as a significant worldwide problem for many years becauseFor which it is highly toxic at low concentrations and can produce a bio-accumulative effect in humans. Therefore, a trace amount of Hg²⁺The detection of (2) is necessary. The fluorescence method has been widely used for mercury ion (Hg) due to its advantages of fast analysis speed, good cost benefit, convenient operation, etc²⁺) Detection of (3). To avoid interference from other potential substances, researchers have utilized Hg²⁺The coordination interaction with thymine base improves the selectivity of detection, specifically, the mismatch of thymine-thymine (T-T) in DNA double strand attracts Hg in water solution²⁺Pairing with thymine (T) to form stable thymine-Hg²⁺-thymine (T-Hg)²⁺-T) DNA double strand.

In order to increase the couple of Hg of the fluorescence sensor²⁺The detection sensitivity, the amplification strategies of Hybrid Chain Reaction (HCR), Rolling Circle Amplification (RCA), enzyme-assisted amplification and the like are widely applied to Hg²⁺And (4) during detection. Among them, the enzyme-assisted amplification strategy mainly employs the property that exonuclease III (ExoIII) can efficiently degrade blunt ends and 5 '-protruding ends of dsDNA, but has low activity on ssDNA or 3' -protruding ends of dsDNA. It is an ideal candidate for signal amplification strategies since it does not require specific recognition sequences.

Disclosure of Invention

The invention aims to develop a mercury ion fluorescence detection method based on silicon quantum dots, fluorescein labeled DNA and a shear enzyme. The method is realized by the following technical scheme: by utilizing the property that the silicon quantum dots are positively charged and can quench the fluorescent signal of fluorescein (Rox), DNA which can be specifically combined with mercury ions into a double strand is connected to the Rox (Rox-DNA), and when no mercury ions exist, the silicon quantum dots quench the fluorescent signal of Rox through electrostatic adsorption; when mercury ions are present, Rox-DNA passes through T-Hg²⁺-T structure forms double-stranded structure, Exo III cleaves double-stranded DNA, releasing Rox and Hg²⁺The purpose of signal amplification is achieved, and high-sensitivity detection of mercury ions is realized.

A mercury ion fluorescence detection method based on silicon quantum dots, fluorescein labeled DNA and a shear enzyme comprises the following steps:

(1) adding the prepared SiQDs solution and Rox-DNA solution into a certain volume of Tris-HCl solution, and incubating for a certain time at a specific temperature;

(2) adding Hg with known concentration into the mixed solution prepared in the step (1)²⁺Mixing the solutions, and incubating for a certain time at a specific temperature;

(3) adding an ExoIII solution into the mixed solution prepared in the step (2), mixing, acting for a period of time at a specific temperature, then inactivating the ExoIII at a high temperature, cooling the mixed solution, and detecting the fluorescence intensity I of the solution at 414nm and 607nm respectively at room temperature by using a fluorescence spectrophotometer₄₁₄And I₆₀₇Obtaining the fluorescence intensity I₆₀₇/I₄₁₄A standard curve of the ratio to the logarithm of the concentration of mercury ions;

(4) the known Hg concentration is adjusted according to the procedures of the steps (1) to (3)²⁺Hg to be measured for solution²⁺Replacing the solution, and respectively detecting the fluorescence intensity I of the solution at 414nm and 607nm by a fluorescence spectrophotometer at room temperature₄₁₄And I₆₀₇Calculate the time I₆₀₇/I₄₁₄And (4) substituting the ratio into the standard curve in the step (3) to obtain the concentration of the mercury ions in the solution to be detected.

In the step (1), the specific method for synthesizing SiQDs comprises the following steps: 6.665mL of 60mM reducing agent L-glutathione (L-GSH) and 13.135mL of deionized water are added into a 100mL beaker, nitrogen is introduced for 10min, 0.2mL of silicon source N- [3- (trimethoxysilyl) propyl ] ethylenediamine (DAMO) is added, and nitrogen is continuously introduced for 10min to obtain a mixed solution. And (3) putting the beaker filled with the mixed solution into a beautiful household microwave oven, and carrying out microwave reaction for 8min under the condition that the microwave power is 700W to obtain a brown yellow solid. Adding 10mL of deionized water into a beaker to dissolve the obtained solid matter, transferring the solution into a centrifuge tube, placing the centrifuge tube into a centrifuge for centrifugation for 10min, wherein the centrifugation speed in the centrifugation process is 10000rpm, taking supernatant, and transferring the supernatant into a dialysis bag for dialysis treatment for 24 h. And freeze-drying and grinding the purified quantum dot solution to obtain yellowish silicon quantum dot powder, and storing at 4 ℃. The molecular weight cut-off of the dialysis bag is 1000.

In the step (1), the concentration of the SiQDs solution is 0.5mg/mL, the concentration of the Rox-DNA solution is 1 muM, and the volume ratio of the SiQDs solution, the Rox-DNA solution and the Tris-HCl is 10: 10: 53; the incubation conditions were: incubate at 37 ℃ for 15 min.

In step (2), Hg²⁺The concentration of the solution was 2 × 10^-11–1×10^-8mol/L; SiQDs solution: hg is a mercury vapor²⁺The volume ratio of the solution is 5: 2; the incubation conditions were: incubate at 37 ℃ for 40 min.

In the step (3), the concentration of the ExoIII solution is 2U/. mu.L, and the concentration of the SiQDs solution is as follows: ExoIII solution volume ratio 10: 3; incubating at 37 deg.C for 65 min; ExoIII was inactivated by standing at 80 ℃ for 10 min.

In the steps (3) and (4), the excitation wavelength of the fluorescence spectrophotometer is set to be 350nm and 550nm respectively, the width of the excitation slit is 3nm, and the width of the emission slit is 3 nm.

The concentration of Tris-HCl is 10mM, the pH value is 7.0, and 10mM MgCl is adopted₂。

The invention has the beneficial effects that:

(1) the invention uses the enzyme shearing amplification strategy to realize the signal amplification of the fluorescence detection method;

(2) the invention introduces DNA rich in T basic group and Hg²⁺Combined to form T-Hg²⁺-T double-chain structure realization for Hg²⁺Detecting the specificity of (1);

(3) the proposed detection method for Hg²⁺Shows satisfactory analytical performance and has a detection limit of 6.67 × 10^{- 12}mol/L (S/N is 3), linear range width 2 × 10^-11–1×10^-8mol/L。

Drawings

FIG. 1 is a schematic diagram of the principle of the fluorescence method of the present invention for detecting mercury ions.

FIG. 2 is a graph of fluorescence excitation and emission spectra of SiQDs.

FIG. 3A is a Zeta potential diagram of SiQDs, Rox-DNA and SiQDs + Rox-DNA; and B is a fluorescence spectrum of different solutions in the feasibility analysis of the fluorescence method of the invention.

FIG. 4A shows different Hg concentrations²⁺A fluorescence spectrum of the solution in the presence of a standard solution; b is Hg²⁺The linear relationship between the concentration logarithm and the ratio of the fluorescence intensity.

FIG. 5 is a graph showing the change in the ratio of fluorescence of a solution in the presence of various interferents.

Detailed Description

The invention is described in detail below with reference to the drawings and examples of the specification:

the detection process of the detection method is shown in fig. 1, and the basic principle is as follows:

SiQDs were first synthesized with a maximum excitation wavelength of 350nm and a maximum emission wavelength of 414nm, as shown in FIG. 2. The positively charged and negatively charged Rox-DNA of the silicon quantum dots are adsorbed by electrostatic interaction (as shown in figure 3A) and quench the fluorescence signal of fluorescein (Rox), and when mercury ions exist, the Rox-DNA passes through T-Hg²⁺-T structure forms double-stranded structure, Exo III cleaves double-stranded DNA, releasing Rox and Hg²⁺The purpose of signal amplification is achieved, and high-sensitivity detection of mercury ions is realized.

The feasibility analysis of the detection method is as follows:

to further verify the feasibility of the protocol, the changes in fluorescence before and after addition of various substances to the SiQDs solution and Rox-DNA solution were examined. As shown in FIG. 3B, SiQDs have strong fluorescence signal at 414nm, and Rox-DNA has strong fluorescence signal at 607 nm; when the two are mixed, the intensity of the fluorescence signal of SiQDs at 414nm is unchanged, and the fluorescence signal of Rox-DNA at 607nm can be quenched by the SiQDs; the fluorescent signal of Rox-DNA at 607nm is not recovered by adding mercury ions alone, and when the addition of Exo III is continued, the Exo III cleaves double-stranded DNA to release Rox, so that the fluorescent signal of Rox at 607nm is recovered. The method of the invention is proved to be applicable to the detection of mercury ions.