CN111562244B - A rare earth time-resolved fluorescent probe and kit for detecting daunorubicin - Google Patents

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 sequence ³⁺ 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 and the detection limit of 42nM, and has the advantages of low background, simplicity in operation, short response time, no mark, low price and the like.

Description

A rare earth time-resolved fluorescent probe and kit for detecting daunorubicin

technical field

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

Background technique

Daunorubicin is a typical anthracycline anticancer drug, which is mainly used for acute lymphocytic or myeloid leukemia that is resistant to commonly used antineoplastic drugs, and has been used in the clinical treatment of various cancers. The antitumor activity of daunorubicin can be attributed to its good DNA sequence selectivity, and the anthracycline in the structure can be embedded in the DNA chain in the cell, so that the conformation of the DNA changes. However, the clinical application of daunorubicin also shows serious dose-limiting side effects, and excessive use can cause adverse reactions such as myocardial damage, arrhythmia, and abnormal electrocardiogram. Therefore, the development of medical clinical daunorubicin detection method has practical value.

Fluorescence detection has the advantages of simple implementation, rapidity, strong real-time performance, simple operation, and good reproducibility, and is an effective detection tool. However, most fluorescence analysis will encounter the problem that the fluorescence background signal is difficult to eliminate, which is not conducive to the improvement of detection sensitivity. To effectively overcome this shortcoming, time-resolved luminescence (TRL) detection technology can utilize the time domain to distinguish long-lived luminescence from short-lived background fluorescence, which is an ideal tool for completely eliminating background luminescence. Further, DNA-sensitized rare-earth ion-based luminescent probes are very suitable for time-resolved fluorescence ( TRL) detection, which can realize sensing applications with maximum low background fluorescence interference. At present, the fluorescent probes designed for nucleic acid sensitization to rare earth ions have more or less shortcomings such as complex probe design and poor sensitization effect.

Contents of the invention

In view of this, the object of the present invention is to provide a kind of rare earth time-resolved fluorescent probe and kit for detecting daunorubicin, adopting the fluorescent probe provided by the present invention can eliminate background fluorescence interference in buffer solution and serum, thereby realizing Rapid, sensitive, label-free and inexpensive fluorescent detection of daunorubicin.

In order to realize the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides a rare earth time-resolved fluorescent probe for detecting daunorubicin. The fluorescent probe is obtained by incubating 22AG sequence and Tb ³⁺ ions at 20-30°C for 8-12 minutes;

The 22AG sequence has the nucleotide sequence shown in SEQ ID No.1.

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

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

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

Preferably, during the incubation, the concentration of Tb ³⁺ ions in the Tris-HAc buffer is 1-10 μM.

Preferably, the incubation time is 10 min.

The present invention also provides a kit for detecting daunorubicin, including the fluorescent probe described in the above technical solution.

The invention provides a rare earth time-resolved fluorescent probe for detecting daunorubicin. The 22AG sequence is incubated with Tb ³⁺ ions at 20-30°C for 8-12 minutes to obtain the fluorescent probe; the 22AG sequence has a SEQ ID Nucleotide sequence shown in No.1.

Tb ³⁺ ions in aqueous solution can be sensitized by 22AG rich in G bases to emit light. When daunorubicin is added to the solution system, since 22AG contains multiple AG bases, daunorubicin may be inserted into the detection system The 22AG in the DNA causes the conformational change of DNA. When the conformational change of 22AG occurs, its sensitization to Tb ³⁺ ions will decrease rapidly, resulting in the weakening of fluorescence. Therefore, through the "on-off" mode of the fluorescence of the detection system before and after the reaction, daunorubicin in the solution can be quickly detected. The fluorescent probe provided by the invention has the advantages of low background, simple operation, short response time, label-free and cheap.

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

Description of drawings

Fig. 1 is the time-resolved fluorescence spectrum of the fluorescent probe that adds 0～10 μ M daunorubicin;

Fig. 2 is the linear fitting figure that fluorescent probe detects to daunorubicin, wherein a fluorescent probe 547nm place corresponds to the solution fluorescence intensity change of adding different concentrations of daunorubicin; b daunorubicin concentration is to the effect of fluorescent probe Fluorescence intensity was linearly fitted, and the concentration of daunorubicin was linear in the range of 0-1 μM.

Detailed ways

The invention provides a rare earth time-resolved fluorescent probe for detecting daunorubicin. The 22AG sequence is incubated with Tb ³⁺ ions at 20-30°C for 8-12 minutes to obtain the fluorescent probe; the 22AG sequence has a SEQ ID Nucleotide sequence shown in No.1.

In the present invention, the 22AG sequence has the nucleotide sequence shown in SEQ ID No.1, specifically as follows:

5'-AGGGTTAGGGTTAGGGTTAGGG-3'.

In the present invention, the incubation is preferably performed in Tris-HAc buffer, the pH of the Tris-HAc buffer is preferably 7.9, and the concentration of the Tris-HAc buffer is preferably 10 mM. In the present invention, the incubation time is preferably 10 min, and the incubation temperature is preferably 23-27°C. In the present invention, during the incubation, the concentration of 22AG sequence in the Tris-HAc buffer is preferably 1-5 μM, and the concentration of Tb ³⁺ ions in the Tris-HAc buffer is preferably 1-10 μM.

In the present invention, the method of using the fluorescent probe preferably includes: mixing the solution to be tested with the fluorescent probe and incubating at 37°C for 10 minutes, and then detecting the time-resolved fluorescence spectrum with a multifunctional microplate reader to obtain the result . In the present invention, the time-resolved luminescence spectrum (TRL) of the probe is tested by the multifunctional microplate reader, and the test conditions are: excitation wavelength 290nm, delay time 50μs, collection time 2ms, collection spectrum range 450nm～725nm. 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 includes: adding 22AG sequences and Tb ³⁺ ions in pH 7.9 10mM Tris-HAc buffer solution, and the mixture Incubate at room temperature for 10 min to obtain a fluorescent probe solution. In the present invention, the concentration of 22AG sequence in the mixture is preferably 1-5 μM, and the concentration of Tb ³⁺ ions in the mixture is preferably 1-10 μM. In the present invention, the volume ratio of the solution to be tested to the solution containing the fluorescent probe is preferably 1:7.5.

The present invention also provides a kit for detecting daunorubicin, including the fluorescent probe described in the above technical solution.

The technical solutions provided by the present invention will be described in detail below in conjunction with the examples, but they should not be interpreted as limiting the protection scope of the present invention.

Example 1

Preparation of fluorescent probe solution: 22AG sequence and Tb ³⁺ ions were added to pH 7.9 10mM Tris-HAc buffer, and the mixture was incubated at room temperature for 10 min to obtain a solution containing fluorescent probe. The concentration of 22AG sequence in the mixture was 1 μM, The concentration of Tb ³⁺ ions in the mixture was 1 μM.

Example 2

Preparation of fluorescent probe solution: 22AG sequence and Tb ³⁺ ions were added to pH 7.9 10mM Tris-HAc buffer, and the mixture was incubated at room temperature for 10min to obtain a solution containing fluorescent probe. The concentration of 22AG sequence in the mixture was 5 μM, The concentration of Tb ³⁺ ions in the mixture was 10 μM.

Example 3

Preparation of fluorescent probe solution: 22AG sequence and Tb ³⁺ ions were added to pH 7.9 10mM Tris-HAc buffer, and the mixture was incubated at room temperature for 10min to obtain a solution containing fluorescent probe. The concentration of 22AG sequence in the mixture was 3 μM, The concentration of Tb ³⁺ ions in the mixture was 2 μM.

Example 4

Prepare different concentrations of daunorubicin aqueous solution (concentration is 0-10 μ M) with ultrapure water, then add respectively in the solution containing fluorescent probe prepared in embodiment 1 (erythromycin solution and the solution containing fluorescent probe The volume ratio is 1:7.5), incubated at 37°C for 10 min, and measured the fluorescence spectra of the fluorescent probes under different conditions with a multi-functional microplate reader. The time-resolved luminescence spectrum (TRL) of the probe was tested by a multifunctional microplate reader, and the test conditions were: the excitation wavelength was 290 nm, the delay time was 50 μs, the collection time was 2 ms, and the collection spectrum range was 547 nm. The results are shown in Figures 1 and 2.

It can be drawn from Fig. 1 that the luminescent intensity of the fluorescent probe decreases with the addition of daunorubicin; it can be drawn from Fig. 2 that the detection concentration range of daunorubicin is 0～1 μ M, according to the formula 3σ/k , resulting in a detection limit of 42 nM.

Example 5

Detection of daunorubicin in serum:

Mix Tb ³⁺ /22Ag (2μM and 3μM, respectively) with 4x reaction buffer (100mM Tris, pH 7.9), and add different concentrations of daunorubicin 0-300nM to Tb ³ containing 2.5% serum ⁺ /22Ag probe solution, the reaction volume is 150 μL. Immediately afterwards, the fluorescence intensity was measured, the excitation wavelength was set to 290nm, the delay time was 50μs, and the collection time was 2ms, and the emission intensity value at 547nm was recorded. The values obtained are the mean plus standard deviation of triplicate determinations. The results are shown in Table 1.

Probe construction: DNA 22AG (3 μM) and Tb ³⁺ (2 μM) solution.

The spiked recovery of daunorubicin in table 1 actual serum samples

Can obtain from table 1, the rate of recovery of the serum spiked experiment of daunorubicin is 93%～103%, illustrate that the detection method of daunorubicin in the actual biological sample (serum) of fluorescent probe is feasible, and has good Anti-fluorescence background interference ability.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

sequence listing

<110> Gannan Normal University

<120> A Rare Earth Time-Resolved Fluorescent Probe and Kit for Detecting Daunorubicin

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 22

<212>DNA

<213> Artificial Sequence

<400> 1

agggttaggg ttagggttag gg 22

Claims (3)

1. A rare earth time-resolved fluorescence probe for detecting daunorubicin is characterized in that 22AG sequences are usedColumn and Tb ³⁺ 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 incubation is carried out in Tris-HAc buffer;

the pH value of the Tris-HAc buffer solution is 7.9, and the concentration of the Tris-HAc buffer solution is 10mM;

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

during the incubation, tb in the Tris-HAc buffer solution ³⁺ The concentration of the ions is 1-10 mu M;

the fluorescence probe adopts a microplate reader to test the time-resolved luminescence spectrum of the probe, and the test conditions are that the excitation wavelength is 290nm, the delay time is 50 mu s, the collection time is 2ms, and the collection spectrum range is 547nm.

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

3. A kit for detecting daunorubicin, comprising the rare earth time-resolved fluorescent probe of claim 1 or 2.

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