A fluorescent probe and its application in DNA detection

Abstract

This invention relates to a fluorescent probe and its application in DNA detection, achieved by simply mixing SiNDs and Ru(bpy)2(dppx). 2+ A method for ratiometric fluorescence detection of DNA in an aqueous phase was established. (Ru(bpy)2(dppx)) 2+ It can specifically and efficiently recognize DNA, and the interaction between the two does not affect Ru(bpy)2(dppx). 2+ The fluorescence of SiNDs was quenched. Based on this, a SiNDs-Ru(bpy)2(dppx) assay for ratiometric fluorescence detection of DNA was constructed. 2+ The probe possesses advantages such as high sensitivity, good selectivity, and strong anti-interference ability, enabling the visual detection of low-concentration DNA and the quantitative detection of target DNA in human serum.

Description

Technical Field

[0001] The present invention relates to the technical field of DNA detection, and in particular to a fluorescent probe and application thereof in DNA detection. Background Art

[0002] Deoxyribonucleic acid (DNA) is one of the most basic substances in living organisms. DNA abnormalities are an important cause of many diseases, including cancer. Therefore, the establishment and development of sensitive DNA detection methods are of great significance for clinical diagnosis, pathogenesis research and gene therapy. Currently, the methods for detecting DNA include surface enhanced Raman scattering, colorimetry, fluorescence, etc. Among them, the ratio fluorescence method has attracted more and more attention due to its self-correction advantage. Silicon dots (SiNDs) are a new type of fluorescent material. Due to their advantages such as green preparation, excellent optical properties and good biocompatibility, they are used in bioanalysis, fluorescence imaging, enantiomer recognition and anti-counterfeiting. The fluorescence of SiNDs synthesized by different methods can be selectively quenched by certain ions or fluorescent dyes. Therefore, SiNDs can be used for the detection of specific targets (such as Cr 6+ , Hg 2+ 、NO 2- ). However, there are few reports on the detection of analytes by quenching the fluorescence of SiNDs by complexes. Summary of the invention

[0003] The purpose of the present invention is to design a fluorescent probe for DNA detection and provide a new method for constructing a visual detection solution for trace DNA and quantitative analysis of target substances in human serum.

[0004] The solution adopted by the present invention to solve the above technical problems is:

[0005] A fluorescent probe comprising silicon dots and Ru(bpy)2(dppx) uniformly mixed in a buffer 2+ (polypyridine ruthenium (II) complex, where bpy = 2,2'-bipyridine, dppx = 7,8-dimethylpyridine [3,2-a: 2',3'c] phenazine).

[0006] Preferably, the silicon dots and Ru(bpy)2(dppx) in the buffer 2+ The ratio is 400μg / mL:7.5~112.5μmol / L.

[0007] Preferably, the buffer is a PBS buffer with a phosphate concentration of 50 mM, a pH of 7.4, and a NaCl content of 80 to 180 mmol / L.

[0008] Preferably, the silicon dots and Ru(bpy)2(dppx) in the buffer 2+The concentrations were 400 μg / mL and 20 μmol / L, respectively.

[0009] Another object of the present invention is to provide a method for preparing the above fluorescent probe, wherein Ru(bpy)2(dppx) 2+ The SiNDs were vortexed and mixed in the buffer.

[0010] Another object of the present invention is to provide the use of the above fluorescent probe in DNA detection.

[0011] Preferably, when DNA detection is performed again, DNA is added to the fluorescent probe, and its fluorescence spectrum is measured. The excitation wavelength used is 359 nm, the excitation and emission slits are 10 nm and 5 nm respectively, and the signal ratio I is calculated based on the fluorescence intensity at wavelengths of 601 nm and 448 nm. 601 / I 448 .

[0012] The present invention simply mixes SiNDs and Ru(bpy)2(dppx) 2+ , a method for ratiometric fluorescence detection of DNA in aqueous phase was established. Ru(bpy)2(dppx) 2+ It can specifically and efficiently recognize DNA, and the interaction between the two does not affect Ru(bpy)2(dppx) 2+ Based on this, SiNDs-Ru(bpy)2(dppx) was constructed for ratiometric fluorescence detection of DNA. 2+ Probe. This probe has the advantages of high sensitivity, good selectivity, and strong anti-interference ability, and can realize the visual detection of low-concentration DNA and the quantitative detection of target DNA in human serum. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 For this application SiNDs-Ru(bpy)2(dppx) 2+ Schematic diagram of probes used for ratiometric fluorescence detection of DNA;

[0014] Figure 2 Ru(bpy)2(dppx) 2+ 、SiNDs and SiNDs-Ru(bpy)2(dppx) 2+ Spectrum of Ru(bpy)2(dppx) from left to right in (A) 2+ (B) is the fluorescence spectrum of SiNDs (400 μg / mL) along with the concentration of Ru(bpy)2(dppx) 2+The concentration (0, 7.5, 15, 20, 30, 37.5, 56.2, 75, 93.7, 112.5 μM, from top to bottom) changes. The inset shows the fluorescence intensity of SiNDs at 448 nm as Ru(bpy)2(dppx) 2+ Changes in concentration;

[0015] Figure 3 Fluorescence spectra of raw materials, probes and sample solutions at different DNA concentrations, including (a) SiNDs, (b) SiNDs+Ru(bpy)2(dppx) 2+ ,(c)SiNDs+Ru(bpy)2(dppx) 2+ +65nM DNA, (d)SiNDs+Ru(bpy)2(dppx) 2+ +500nM DNA and (e)SiNDs+Ru(bpy)2(dppx) 2+ +1000nM DNA, SiNDs and Ru(bpy)2(dppx) 2+ The concentrations were 400 μg / mL and 20 μM;

[0016] Figure 4 Ru(bpy)2(dppx) in the presence of different concentrations of DNA under Xe lamp irradiation 2+ and SiNDs-Ru(bpy)2(dppx) 2+ Dark box plots with DNA concentrations ranging from 0–750 nM from left to right, SiNDs and Ru(bpy)2(dppx) 2+ The concentrations were 400 μg / mL and 20 μM, respectively.

[0017] Figure 5 SiNDs-Ru(bpy)2(dppx) 2+ Fluorescence spectra and fluorescence intensity ratios at different DNA concentrations. (A) is the fluorescence spectrum, where the curve of fluorescence intensity at 601 nm from strong to weak represents DNA concentrations of 0, 20, 50, 75, 100, 200, 400, 500, 750, 1000, 1250, 1500 nM, and (B) is the fluorescence intensity ratio I 601 / I 448 , SiNDs and Ru(bpy)2(dppx) 2+ The concentrations were 400 μg / mL and 20 μM, respectively. DETAILED DESCRIPTION

[0018] For a better understanding of the present invention, the following examples are provided to further illustrate the present invention, but the present invention is not limited to the following examples.

[0019] Ru(bpy)2(dppx) 2+ The synthesis method can be referred to the literature Ling LS, He ZK, Song GW, et al. Anovel method for determination of DNA by use of molecular 'Light Switch' complex of Ru(bipy)2(dppx) 2+ [J].Anal.Chim.Acta,2000,403:209-217.

[0020] The synthesis method of SiNDs can refer to the literature Feng YL, Liu YF, Su C, et al. New fluorescent pH sensor based on label-free silicon nanodots[J]. Sens. Actuators, B: Chem. 2014, 203: 795-801 and Zhang YN, Hou DJ, Yu X L. Facile preparation of FITC-modified silicon nanodots for ratiometric pH sensing and imaging[J]. Spectrochim. Acta A, 2020, 234: 118276.

[0021] HIV double-stranded DNA (5′-CGAGTTAAGAAGAAAAAAGATTGAGC-3′ / 5′-GCTCAATCTTTTTTCTTCTTAACTCG-3′) was purchased from Shanghai Biotechnology Co., Ltd., China.

[0022] Human serum was provided by Chengwen Immunochemistry Laboratory, Beijing, China.

[0023] Example 1

[0024] First, Ru(bpy)2(dppx) 2+ and SiNDs were vortexed in 50 mM PBS buffer (pH 7.4, 100 mM NaCl), where Ru(bpy)2(dppx) 2+ The concentration was 0-112.5 μM, the SiNDs concentration was 400 μg / mL, and SiNDs-Ru(bpy)2(dppx) was obtained. 2+ Then, the above SiNDs-Ru(bpy)2(dppx) 2+DNA of appropriate concentration and 0-10 μL of human serum were added to the solution, and the total volume of the solution was 1 mL. After 10 minutes, the obtained solution was used as a test sample to measure its fluorescence spectrum. The excitation wavelength used was 359 nm, and the excitation and emission slits were 10 nm and 5 nm, respectively. The signal ratio I was calculated based on the fluorescence intensity at 601 nm and 448 nm. 601 / I 448 All measurements were performed in triplicate at room temperature.

[0025] Figure 1 SiNDs-Ru(bpy)2(dppx) 2+ Schematic diagram of the probe used for ratiometric fluorescence detection of DNA. Ru(bpy)2(dppx) 2+ It is a molecular "light switch" that selectively recognizes double-stranded DNA. It has no fluorescence in aqueous solution because water molecules can quench its fluorescence through the proton transfer mechanism. When double-stranded DNA is present, its dppx ligand can be inserted into the double helix structure of DNA, thereby emitting red fluorescence. Based on the internal filter effect (IFE), Ru(bpy)2(dppx) 2+ It can effectively quench the blue fluorescence of SiNDs. When the target DNA is present, Ru(bpy)2(dppx) 2+ The red fluorescence is emitted, while the blue fluorescence signal of the quenched SiNDs remains unchanged, so the two can be used as the response signal and the reference signal respectively.

[0026] Figure 2 Ru(bpy)2(dppx) 2+ 、SiNDs and SiNDs-Ru(bpy)2(dppx) 2+ From the spectrum of Ru(bpy)2(dppx), we can see that 2+ The UV-visible absorption spectrum of Ru(bpy)2(dppx) overlaps with the excitation spectrum and emission spectrum of SiNDs, which indicates that 2+ The presence of IFE in SiNDs can effectively quench the fluorescence of SiNDs. We further investigated the 2+ The quenching effect of SiNDs was 97% at a relatively low Ru complex concentration. In order to facilitate the visualization of DNA, 20 μM Ru(bpy)2(dppx) was selected. 2+ Quenching SiNDs.

[0027] Figure 3Figure 2 is the fluorescence spectra of the raw material, probe and sample solution at different DNA concentrations. It can be seen that the fluorescence signal of SiNDs in the probe is significantly lower than that of the raw material. In the presence of different concentrations of DNA, the fluorescence signal of Ru(bpy)2(dppx) in the probe 2+ The fluorescence intensity at 601 nm increased with the increase of DNA concentration, while the fluorescence intensity of SiNDs at 448 nm remained unchanged. This indicates that DNA and Ru(bpy)2(dppx) 2+ Specific binding to Ru(bpy)2(dppx) 2+ The IFE of SiNDs was not affected and SiNDs could be used as a fluorescent reference signal in detecting DNA ratio probes.

[0028] Figure 4 Ru(bpy)2(dppx) in the presence of different concentrations of DNA under Xe lamp irradiation 2+ and SiNDs-Ru(bpy)2(dppx) 2+ The dark box diagram shows that as the DNA concentration increases, compared with Ru(bpy)2(dppx) 2+ (red), SiNDs-Ru(bpy)2(dppx) 2+ The probe showed a series of color changes (from blue to red), and in the presence of 20 nM DNA, it could be clearly distinguished from the blank control, which showed that the probe could be well applied to the visual detection of low-concentration DNA.

[0029] Figure 5 SiNDs-Ru(bpy)2(dppx) 2+ Fluorescence spectra and fluorescence intensity ratios at different DNA concentrations show that the Ru(bpy)2(dppx) 2+ The fluorescence intensity of increases with the increase of DNA concentration, while the fluorescence intensity of SiNDs remains unchanged; in the range of 20-1500nM DNA, there is a good linear relationship between the signal intensity ratio of the two and the DNA concentration.

[0030] The above is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and changes can be made without departing from the principle of the present invention, and these improvements and changes are also regarded as the protection scope of the present invention.

Claims

1. A fluorescent probe, characterized in that Includes SiNDs and Ru(bpy)2(dppx) uniformly mixed in buffer 2+ , wherein bpy = 2,2'-bipyridine, dppx = 7,8-dimethylpyridine [3,2-a: 2',3'c] phenazine.

2. The fluorescent probe according to claim 1, characterized in that Silicon dots and Ru(bpy)2(dppx) in the buffer 2+ The ratio is 400μg / mL:7.5~112.5μmol / L.

3. The fluorescent probe according to claim 1, characterized in that The buffer is a PBS buffer with a phosphate concentration of 50 mM, a pH of 7.4, and a NaCl content of 80 to 180 mmol / L.

4. The fluorescent probe according to claim 1, characterized in that Silicon dots and Ru(bpy)2(dppx) in the buffer 2+ The concentrations were 400 μg / mL and 20 μmol / L, respectively.

5. The method for preparing the fluorescent probe according to any one of claims 1 to 4, characterized in that: Change Ru(bpy)2(dppx) 2+ The SiNDs were vortexed and mixed in the buffer.

6. Use of the fluorescent probe according to any one of claims 1 to 4 in DNA detection.

7. The use according to claim 6, characterized in that: DNA was added to the fluorescent probe and its fluorescence spectrum was measured. The excitation wavelength used was 359 nm, the excitation and emission slits were 10 nm and 5 nm, respectively, and the signal ratio I was calculated based on the fluorescence intensity at 601 nm and 448 nm. 601 / I 448 .

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