CN111562244A

CN111562244A - Rare earth time-resolved fluorescent probe and kit for detecting daunorubicin

Info

Publication number: CN111562244A
Application number: CN202010448523.8A
Authority: CN
Inventors: 吴勇权; 陈小勇; 周慧; 李勋; 范小林
Original assignee: Gannan Normal University
Current assignee: Gannan Normal University
Priority date: 2020-05-25
Filing date: 2020-05-25
Publication date: 2020-08-21
Anticipated expiration: 2040-05-25
Also published as: CN111562244B

Abstract

The invention provides a rare earth time-resolved fluorescence probe and a kit for detecting daunorubicin, belonging to the technical field of drug detection and aiming at combining a 22AG sequence and Tb sequence³⁺Incubating the ions at 20-30 ℃ for 8-12 min to obtain a fluorescent probe; the 22AG sequence has a nucleotide sequence shown as SEQ ID No. 1. The fluorescent probe provided by the invention has the detection concentration range of 0-1 mu M for daunorubicin, the detection limit of 42nM, and the fluorescent probe has the advantages of low background, simplicity in operation, short response time, no mark, low price and the like.

Description

Rare earth time-resolved fluorescent probe and kit for detecting daunorubicin

Technical Field

The invention belongs to the technical field of drug detection, and particularly relates to a rare earth time-resolved fluorescence probe and a kit for detecting daunorubicin.

Background

Daunorubicin (Daunorubicin) is a typical anthracycline anticancer drug, is mainly used for acute lymphocytic or granulocytic leukemia resistant to common antitumor drugs, and has been applied to clinical treatment of various cancers. The anti-tumor activity of daunorubicin can be attributed to good DNA sequence selectivity, and anthracene rings in the structure can be inserted into a DNA chain in cells, so that the conformation of the DNA is changed. However, clinical use of daunorubicin also shows serious dose-limiting side effects, and overdose can cause myocardial damage, arrhythmia, electrocardiographic abnormality, and other adverse reactions. Therefore, the development of a medical clinical daunorubicin detection method has practical value.

The fluorescence detection has the advantages of simple and rapid realization, strong real-time property, simple operation, good reproducibility and the like, and is an effective detection tool. However, most fluorescence assays suffer from the problem that the fluorescence background signal is difficult to eliminate, which is detrimental to the improvement of detection sensitivity. To effectively overcome this drawback, time-resolved luminescence (TRL) detection techniques can use the time domain to distinguish between long-lived and short-lived background fluorescence, which is an ideal tool for completely eliminating background luminescence. Furthermore, due to the unique spectral characteristics of the luminescent probe based on the DNA sensitized rare earth ions, such as long luminescent life, narrow emission band and the like, the luminescent probe based on the DNA sensitized rare earth ions is very suitable for time resolved fluorescence (TRL) detection, and can realize sensing application with low background fluorescence interference to the maximum extent. At present, the designed fluorescent probe for nucleic acid sensitized rare earth ions has the defects of more or less complex probe design, poor sensitizing effect and the like.

Disclosure of Invention

In view of the above, the present invention aims to provide a rare earth time-resolved fluorescence probe and a kit for detecting daunorubicin, and the fluorescence probe provided by the present invention can eliminate background fluorescence interference in buffer solution and serum, thereby realizing rapid, sensitive, label-free and cheap fluorescence detection of daunorubicin.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a rare earth time-resolved fluorescence probe for detecting daunorubicin, which is prepared by combining a 22AG sequence and Tb³⁺Incubating the ions at 20-30 ℃ for 8-12 min to obtain a fluorescent probe;

the 22AG sequence has a nucleotide sequence shown as SEQ ID No. 1.

Preferably, the incubation is performed in Tris-HAc buffer.

Preferably, the Tris-HAc buffer has a pH value of 7.9 and a concentration of 10 mM.

Preferably, the concentration of the 22AG sequence in the Tris-HAc buffer solution during the incubation is 1-5 mu M.

Preferably, during the incubation, Tb in the Tris-HAc buffer solution³⁺The concentration of the ions is 1 to 10 mu M.

Preferably, the incubation time is 10 min.

The invention also provides a kit for detecting daunorubicin, which comprises the fluorescent probe in the technical scheme.

The invention provides a rare earth time-resolved fluorescence probe for detecting daunorubicin, which is prepared by combining a 22AG sequence and Tb³⁺Incubating the ions at 20-30 ℃ for 8-12 min to obtain a fluorescent probe; the 22AG sequence has a nucleotide sequence shown as SEQ ID No. 1.

Tb³⁺The ion can be sensitized and luminesced by 22AG rich in G base in aqueous solution, when daunorubicin is added into a solution system, because 22AG contains a plurality of AG bases, daunorubicin can be inserted into 22AG in a detection system to cause the change of DNA conformation, and when the 22AG conformation is changed, the daunorubicin can react with Tb³⁺The sensitization of the ions will decrease rapidly leading to a decrease in fluorescence. Therefore, the daunorubicin in the solution can be rapidly detected by the on-off mode of fluorescence of the detection system before and after the reaction. The fluorescent probe provided by the invention has the advantages of low background, simple operation, short response time, no mark, low price and the like.

The detection concentration range of the fluorescent probe provided by the invention for daunorubicin is 0-1 mu M, and the detection limit is 42 nM.

Drawings

FIG. 1 is a time-resolved fluorescence spectrum of a fluorescent probe added with 0-10 μ M daunorubicin;

FIG. 2 is a linear fitting graph of fluorescent probe to daunorubicin detection, wherein the fluorescent probe 547nm in a is added with the change of fluorescent intensity of solutions with different concentrations of daunorubicin; and b, linearly fitting the concentration of the daunorubicin to the fluorescence intensity of the fluorescent probe, wherein the concentration of the daunorubicin is 0-1 mu M linearly.

Detailed Description

In the invention, the 22AG sequence has a nucleotide sequence shown as SEQ ID No.1, and specifically comprises the following steps:

5’-AGGGTTAGGGTTAGGGTTAGGG-3’。

in the present invention, the incubation is preferably carried out in Tris-HAc buffer, the pH value of the Tris-HAc buffer is preferably 7.9, and the concentration of the Tris-HAc buffer is preferably 10 mM. In the invention, the incubation time is preferably 10min, and the incubation temperature is preferably 23-27 ℃. In the present invention, the concentration of 22AG sequence in the Tris-HAc buffer solution is preferably 1-5 μ M, and Tb in the Tris-HAc buffer solution³⁺The concentration of the ions is preferably 1 to 10 μ M.

In the present invention, the method of using the fluorescent probe preferably includes: and mixing the solution to be detected with the fluorescent probe, incubating for 10min at 37 ℃, and detecting the time-resolved fluorescence spectrum by using a multifunctional enzyme-linked immunosorbent assay to obtain a result. In the invention, the multifunctional microplate reader tests the time-resolved luminescence spectrum (TRL) of the probe, and the test conditions are as follows: the excitation wavelength is 290nm, the delay time is 50 mus, the collection time is 2ms, and the collection spectrum range is 450 nm-725 nm. In the present invention, the fluorescent probe is preferably incubated in the form of a fluorescent probe solution, and the preparation of the fluorescent probe solution comprises: 22AG sequence and Tb were added to Tris-HAc buffer pH 7.910 mM³⁺And (4) incubating the mixture for 10min at room temperature to obtain a fluorescent probe solution. In the invention, the concentration of the 22AG sequence in the mixture is preferably 1-5 mu M, and Tb in the mixture³⁺The concentration of the ions is preferably 1 to 10 μ M. In the present invention, the volume ratio of the solution to be measured to the solution containing the fluorescent probe is preferably 1: 7.5.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Preparation of fluorescent probe solution: 22AG sequence and Tb were added to Tris-HAc buffer pH 7.910 mM³⁺Ion, incubating the mixture at room temperature for 10min to obtain a solution containing the fluorescent probe, wherein the concentration of the 22AG sequence in the mixture is 1 mu M, and Tb in the mixture³⁺The concentration of the ions was 1. mu.M.

Example 2

Preparation of fluorescent probe solution: 22AG sequence and Tb were added to Tris-HAc buffer pH 7.910 mM³⁺Ion, incubating the mixture at room temperature for 10min to obtain a solution containing the fluorescent probe, wherein the concentration of the 22AG sequence in the mixture is 5 mu M, and Tb in the mixture³⁺The concentration of the ions was 10. mu.M.

Example 3

Preparation of fluorescent probe solution: 22AG sequence and Tb were added to Tris-HAc buffer pH 7.910 mM³⁺Ion, incubating the mixture at room temperature for 10min to obtain a solution containing the fluorescent probe, wherein the concentration of the 22AG sequence in the mixture is 3 mu M, and Tb in the mixture³⁺The concentration of the ions was 2. mu.M.

Example 4

Preparing daunorubicin aqueous solutions (the concentration is 0-10 mu M) with different concentrations by using ultrapure water, then respectively adding the daunorubicin aqueous solutions into the solutions containing the fluorescent probes prepared in the example 1 (the volume ratio of the erythromycin solution to the solution containing the fluorescent probes is 1:7.5), incubating for 10min in an environment at 37 ℃, and measuring the fluorescence spectra of the fluorescent probes under different conditions by using a multifunctional microplate reader. The time resolution luminescence spectrum (TRL) of the probe is tested by adopting a multifunctional microplate reader, and the test conditions are as follows: the excitation wavelength was 290nm, the delay time was 50 μ s, the collection time was 2ms, and the collection spectral range was 547 nm. The results are shown in FIGS. 1 and 2.

From FIG. 1, it can be concluded that the luminescence intensity of the fluorescent probe decreases with the addition of daunorubicin; as can be seen from FIG. 2, the detection concentration range of daunorubicin is 0-1 μ M, and the detection limit is 42nM according to the formula 3 σ/k.

Example 5

Detection of daunorubicin in serum:

tb is to be³⁺Mixing/22 Ag (2. mu.M and 3. mu.M, respectively) with 4-fold reaction buffer (100mM Tris, pH 7.9), and adding daunorubicin 0-300 nM to Tb containing 2.5% serum³⁺In the/22 Ag probe solution, the reaction system is 150. mu.L. Immediately thereafter, the fluorescence intensity was measured by setting the excitation wavelength to 290nm, the delay time to 50. mu.s and the collection time to 2ms, and the emission intensity value at 547nm was recorded. The values obtained were all the mean of triplicate determinations plus the standard deviation. The results are shown in Table 1.

And (3) probe construction: DNA 22AG (3. mu.M) and Tb³⁺(2. mu.M) solution.

TABLE 1 spiked recovery of daunorubicin from actual serum samples

From table 1, the recovery rate of the serum labeling experiment of daunorubicin is 93% -103%, which indicates that the detection method of the fluorescent probe for daunorubicin in the actual biological sample (serum) is feasible and has good capability of resisting fluorescence background interference.

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 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> university of Jiangxian teachers

<120> rare earth time-resolved fluorescence probe and kit for detecting daunorubicin

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

agggttaggg ttagggttag gg 22

Claims

1. A rare earth time-resolved fluorescence probe for detecting daunorubicin is characterized in that 22AG sequence and Tb sequence are combined³⁺Incubating the ions at 20-30 ℃ for 8-12 min to obtain a fluorescent probe;

the 22AG sequence has a nucleotide sequence shown as SEQ ID No. 1.

2. The rare earth time-resolved fluorescence probe according to claim 1, wherein the incubation is performed in Tris-HAc buffer.

3. The rare earth time-resolved fluorescence probe of claim 2, wherein the Tris-HAc buffer has a pH of 7.9 and a concentration of 10 mM.

4. The rare earth time-resolved fluorescence probe of claim 2 or 3, wherein the concentration of the 22AG sequence in the Tris-HAc buffer solution is 1-5 μ M during the incubation.

5. The rare earth time-resolved fluorescence probe of claim 2 or 3, wherein the incubation is performed with Tb in the Tris-HAc buffer³⁺The concentration of the ions is 1 to 10 mu M.

6. The rare earth time-resolved fluorescence probe of claim 1, wherein the incubation time is 10 min.

7. A kit for detecting daunorubicin, characterized by comprising the rare earth time-resolved fluorescence probe of any one of claims 1 to 6.