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Preparation method and application of rare earth up-conversion fluorescence probe for detecting DNA damage marker

A rare earth up-conversion and DNA damage technology, applied in the field of fluorescent nanoprobe detection, can solve the problems of long analysis time, complicated pretreatment, poor repeatability, etc., and achieves low background interference, low probe toxicity and wide linear detection range Effect

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  • Abstract
  • Description
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Problems solved by technology

[0003] Currently reported 8-OHdG detection methods mainly include high-performance liquid chromatography-tandem mass spectrometry (HPLC / MS), electrophoresis, enzyme-linked immunosorbent assay (ELISA), electrochemical methods, etc. , long analysis time, poor repeatability and other reasons show certain limitations, we need a simple and fast method for the detection of 8-OHdG

Method used

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  • Preparation method and application of rare earth up-conversion fluorescence probe for detecting DNA damage marker
  • Preparation method and application of rare earth up-conversion fluorescence probe for detecting DNA damage marker
  • Preparation method and application of rare earth up-conversion fluorescence probe for detecting DNA damage marker

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Embodiment 1

[0027] In the present invention, the reported solvothermal method is used to prepare the micro-nano up-conversion material NaYF4:Er,Yb. Add YCl3, YbCl3, ErCl3 solids to 5mL deionized water, add sodium citrate and 10mL deionized water and mix well. Then add the mixed liquid of NaCl, NH4F, oleic acid and ethylene glycol, stir for 30 minutes, put it into the reaction kettle, and react at 180° C. for 6 hours. The lower precipitate was collected by centrifugation, washed three times with ethanol, and finally dried to obtain the micronano up-conversion material NaYF4:Er,Yb powder.

[0028] In the present invention, the following steps are used to modify the surface of NaYF4:Er,Yb by amination. Disperse the NaYF4:Er,Yb powder of the micro-nano up-conversion material obtained above in isopropanol, add polyvinylpyrrolidone, mix and sonicate for 30 minutes, add distilled water and ammonia water in turn, seal at 35°C and stir rapidly with magnetic force for 15 minutes, A mixed solution...

Embodiment 2

[0030] Establishment of Standard Curve for 8-OHdG Detection Using Fluorescent Probes

[0031] Use an acidic buffer solution to prepare a certain concentration of 8-OHdG solution and mix it with acid fuchsin solution, then add NH2-UCNPs, take the same acidic buffer solution and add it so that the total volume is 3mL to obtain a detection solution. The fluorescence spectrum data at 545nm is recorded under 980nm excitation, and the quantitative detection of 8-OHdG is realized according to the difference in fluorescence intensity difference (F-F0) between the detection solution of different concentrations of 8-OHdG and the sample blank.

[0032] The scanning parameters of the fluorescence spectrum are as follows: the excitation wavelength is 980 nm, the scanning speed is 1200 nm / min, the excitation and emission slit widths are both 5 nm, and the PMT voltage is 700 V.

[0033] figure 2 A is the fluorescence response curve of the up-conversion detection system to different concent...

Embodiment 3

[0035] To study the influence of the adding order of each reagent in the detection system on the sensitivity of the system

[0036] Take 1mL of 4mg / L acid fuchsin solution and mix it with 3×10-8mol / L8-OHdG solution, add 1mL of 1mg / mL aminated NaYF4:Er,Yb solution, shake and mix well, add the same buffer solution to make the total volume 3mL, Obtain solution 1 to be tested; mix 1mL acid fuchsin solution with 1mL 1mg / mL aminated NaYF4:Er,Yb solution, add 3×10-8mol / L8-OHdG solution, shake and mix well, take the same acidic buffer solution and add Make the total volume 3 mL to obtain the test solution 2. Under the excitation light of 980 nm, use a fluorescence spectrophotometer to measure the fluorescence intensity of the test solution 1 and test solution 2 at 545 nm. The effect of the order of addition on the sensitivity of the system is as follows: image 3 As shown, the results show that the higher system sensitivity can be obtained when the addition sequence is acid fuchsin—8...

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Abstract

The invention discloses a preparation method and application of a rare earth up-conversion fluorescence probe for detecting a DNA damage marker. The preparation method comprises the following specificsteps: premixing acid fuchsin and 8-OHdG dissolved in an acid buffer solution, incubating for a period of time, then adding an up-conversion nano material NaYF4: Er, Yb(NH2-UCNPs) subjected to surface amination modification, performing full mixing, and detecting under the irradiation of 980 nm excitating light to find that the fluorescence intensity difference (F-F0) of the system at 545 nm and the blank fluorescence intensity of a sample are in direct proportion to the negative logarithm of the addition concentration of 8-OHdG in order to achieve the quantitative detection of 8-OHdG. The constructed probe has strong background interference resistance and low toxicity, and can be used for detecting the fluorescence intensity of the sample under the irradiation of 980nm exciting light. Theconstructed detection method is high in sensitivity, wide in linear range, high in selectivity, stable in system, easy and convenient to operate, free of aptamer modification materials and low in cost.

Description

technical field [0001] The invention belongs to the field of fluorescent nanometer probe detection, and specifically relates to a preparation method and application of a rare earth up-conversion fluorescent probe for detecting DNA damage markers. Background technique [0002] Reactive oxygen species (ROS) are continuously formed in living cells of aerobic organisms as part of physiological processes, metabolism, and other biochemical reactions, and due to their reactivity and a series of exogenous factors, can damage cell membrane lipids, proteins, and DNA Causes oxidative damage that leads to aging, malignancy and other degenerative diseases. Therefore, the development of a rapid and sensitive method for detecting DNA damage is of great significance for evaluating the impact of various environmental pollution problems on human health and early prediction of diseases. The biomarker 8-hydroxydeoxyguanosine (8-OHdG) was first reported by Kasai and Nishimura in 1984. It can be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/85C09K11/02G01N21/64
CPCC09K11/7773C09K11/025G01N21/6428G01N2021/6432G01N2021/6417
Inventor 于慧郭会琴颜流水刘小明李可心田凌溪
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