CN111562244B - Rare earth time-resolved fluorescent probe and kit for detecting daunorubicin - Google Patents
Rare earth time-resolved fluorescent probe and kit for detecting daunorubicin Download PDFInfo
- Publication number
- CN111562244B CN111562244B CN202010448523.8A CN202010448523A CN111562244B CN 111562244 B CN111562244 B CN 111562244B CN 202010448523 A CN202010448523 A CN 202010448523A CN 111562244 B CN111562244 B CN 111562244B
- Authority
- CN
- China
- Prior art keywords
- daunorubicin
- time
- fluorescent probe
- sequence
- rare earth
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
Landscapes
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Optics & Photonics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
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
Technical Field
The invention belongs to the technical field of drug detection, and particularly relates to a rare earth time-resolved fluorescent 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 used for 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 disadvantageous for improving the 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 the advantages of 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 the sensing application of 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 this, 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 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.
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 10mM.
Preferably, the concentration of the 22AG sequence in the Tris-HAc buffer solution during the incubation is 1-5 mu M.
Preferably, tb during the incubation is contained in the Tris-HAc buffer solution 3+ The concentration of the ions is 1 to 10 mu M.
Preferably, the incubation time is 10min.
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 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.
Tb 3+ 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 Tb 3+ 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 42nM.
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 a fluorescent probe 547nm is added with corresponding change in fluorescence intensity of solutions of daunorubicin of different concentrations; b, linearly fitting the concentration of the daunorubicin to the fluorescence intensity of the fluorescent probe, wherein the concentration of the daunorubicin is linearly 0-1 mu M.
Detailed Description
The invention provides a rare earth time-resolved fluorescence probe for detecting daunorubicin, which is prepared by combining a 22AG sequence 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.
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 carried outThe incubation is preferably carried out in Tris-HAc buffer, preferably at pH 7.9, preferably at a concentration of 10mM. In the present invention, the incubation time is preferably 10min, and the incubation temperature is preferably 23 to 27 ℃. In the present invention, the concentration of 22AG sequence in the Tris-HAc buffer solution is preferably 1 to 5. Mu.M, and the concentration of Tb in the Tris-HAc buffer solution is preferably 1 to 5. Mu.M during the incubation 3+ The concentration of the ions is preferably 1 to 10. Mu.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 (ELISA) instrument 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 a pH 7.9 10mM Tris-HAc buffer solution 3+ And (4) incubating the mixture for 10min at room temperature to obtain a fluorescent probe solution. In the present invention, the concentration of the 22AG sequence in the mixture is preferably 1 to 5. Mu.M, tb in the mixture 3+ The concentration of the ions is preferably 1 to 10. Mu.M. In the present invention, the volume ratio of the solution to be tested to the solution containing the fluorescent probe is preferably 1.
The invention also provides a kit for detecting daunorubicin, which comprises the fluorescent probe in the technical scheme.
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 the pH 7.9 10mM Tris-HAc buffer 3+ 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 mixture 3+ The concentration of the ions was 1. Mu.M.
Example 2
Preparation of fluorescent probe solution: 22AG sequence and Tb were added to a pH 7.9 10mM Tris-HAc buffer solution 3+ 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 mixture 3+ The concentration of the ions was 10. Mu.M.
Example 3
Preparation of fluorescent probe solution: 22AG sequence and Tb were added to a pH 7.9 10mM Tris-HAc buffer solution 3+ 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 μ M, and Tb in the mixture 3+ 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 1.5), incubating for 10min in an environment of 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: excitation wavelength 290nm, delay time 50 μ s, collection time 2ms, collection spectral range 547nm. 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. Mu.M, and the detection limit is 42nM according to the formula 3. Sigma./k.
Example 5
Detection of daunorubicin in serum:
tb is to be 3+ Per 22Ag (2. Mu.M and 3. Mu.M, respectively) was mixed with 4-fold reaction buffer (100mM Tris, pH 7.9), and then daunorubicin was added at 0-300 nM to Tb containing 2.5% serum at different concentrations 3+ In the/22 Ag probe solution, the reaction system is 150. Mu.L. The fluorescence intensity was then measured immediately, setting the excitation wavelength at 290nm, the delay time at 50. Mu.s, the collection time at 2ms, and the emission at 547nm was recordedA radiation intensity value. 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 Tb 3+ (2. Mu.M) solution.
TABLE 1 spiked recovery of daunorubicin from actual serum samples
As can be obtained from Table 1, the recovery rate of the serum labeling experiment of daunorubicin is 93% -103%, which indicates that the fluorescent probe is feasible in the detection method of daunorubicin in the actual biological sample (serum), and has good anti-fluorescence background interference capability.
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 (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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010448523.8A CN111562244B (en) | 2020-05-25 | 2020-05-25 | Rare earth time-resolved fluorescent probe and kit for detecting daunorubicin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010448523.8A CN111562244B (en) | 2020-05-25 | 2020-05-25 | Rare earth time-resolved fluorescent probe and kit for detecting daunorubicin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111562244A CN111562244A (en) | 2020-08-21 |
| CN111562244B true CN111562244B (en) | 2023-02-07 |
Family
ID=72074989
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010448523.8A Active CN111562244B (en) | 2020-05-25 | 2020-05-25 | Rare earth time-resolved fluorescent probe and kit for detecting daunorubicin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111562244B (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1988002784A1 (en) * | 1986-10-17 | 1988-04-21 | Wallac Oy | Hybridization assay and means to be used in the assay |
| CN1723288A (en) * | 2002-10-10 | 2006-01-18 | 英国国防部 | Detection system |
| CN101003525A (en) * | 2005-12-02 | 2007-07-25 | 北卡罗来纳大学查珀尔希尔分校 | Dicationic compounds which selectively recognize G-quadruplex DNA |
| CN101240338A (en) * | 2007-02-09 | 2008-08-13 | 株式会社东芝 | Method of detecting gene mutation |
| CN101275166A (en) * | 2007-03-28 | 2008-10-01 | 株式会社东芝 | Nucleotide primer set and nucleotide probe for detecting genotype of n-acetyltransferase 2 (NAT2) |
| CN101935689A (en) * | 2009-06-29 | 2011-01-05 | 中国科学院化学研究所 | Method for identifying telomere DNA fragments and c-kit gene promoter DNA sub-fragments |
| CN105806831A (en) * | 2016-03-04 | 2016-07-27 | 北京农业质量标准与检测技术研究中心 | Method for detecting chlorophenol pollutants by utilizing chemiluminescent method |
| CN106714846A (en) * | 2014-08-14 | 2017-05-24 | 泰克年研究发展基金会公司 | Compositions and methods for therapeutics prescreening |
| CN111051521A (en) * | 2017-07-06 | 2020-04-21 | 条形码诊断公司 | Method for prescreening therapeutic agents in body fluids |
-
2020
- 2020-05-25 CN CN202010448523.8A patent/CN111562244B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1988002784A1 (en) * | 1986-10-17 | 1988-04-21 | Wallac Oy | Hybridization assay and means to be used in the assay |
| CN1723288A (en) * | 2002-10-10 | 2006-01-18 | 英国国防部 | Detection system |
| CN101003525A (en) * | 2005-12-02 | 2007-07-25 | 北卡罗来纳大学查珀尔希尔分校 | Dicationic compounds which selectively recognize G-quadruplex DNA |
| CN101240338A (en) * | 2007-02-09 | 2008-08-13 | 株式会社东芝 | Method of detecting gene mutation |
| CN101275166A (en) * | 2007-03-28 | 2008-10-01 | 株式会社东芝 | Nucleotide primer set and nucleotide probe for detecting genotype of n-acetyltransferase 2 (NAT2) |
| CN101935689A (en) * | 2009-06-29 | 2011-01-05 | 中国科学院化学研究所 | Method for identifying telomere DNA fragments and c-kit gene promoter DNA sub-fragments |
| CN106714846A (en) * | 2014-08-14 | 2017-05-24 | 泰克年研究发展基金会公司 | Compositions and methods for therapeutics prescreening |
| CN105806831A (en) * | 2016-03-04 | 2016-07-27 | 北京农业质量标准与检测技术研究中心 | Method for detecting chlorophenol pollutants by utilizing chemiluminescent method |
| CN111051521A (en) * | 2017-07-06 | 2020-04-21 | 条形码诊断公司 | Method for prescreening therapeutic agents in body fluids |
Non-Patent Citations (1)
| Title |
|---|
| 《Affinity of the anthracycline antitumor drugs Doxorubicin and Sabarubicin for human telomeric G-quadruplex structures》;Ilse Manet,etal;《Physical Chemistry Chemical Physics》;20111231;第13卷;第541页右栏-542页左栏 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111562244A (en) | 2020-08-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0755458B1 (en) | Electrogenerated chemiluminescence labels for analysis and/or referencing | |
| Li et al. | Imaging of formaldehyde in plants with a ratiometric fluorescent probe | |
| CN113075269B (en) | Electrochemical luminescence aptamer sensor for specifically detecting chloramphenicol and preparation method and application thereof | |
| CN104819970B (en) | A kind of method that super molecular complex fluorescence probe determines carbendazim in water | |
| CN105132524A (en) | Application of dual application reaction of Exo (exonuclease) III-assisted cycle and DNAzyme cycle to Hg<2+> detection | |
| CN111239118A (en) | Analysis method for detecting organophosphorus pesticide based on alkaline phosphatase triggered fluorescence and colorimetric dual-reading sensor | |
| Ge et al. | A novel label-free fluorescent molecular beacon for the detection of 3′–5′ exonuclease enzymatic activity using DNA-templated copper nanoclusters | |
| CN111471743A (en) | Urease-based microRNA detection kit | |
| CN108398406B (en) | Biosensor for detecting uracil glycosylase (UDG) and application thereof | |
| CN112575067B (en) | Method for detecting structure-specific nuclease FEN1 by using DNA ligation reaction and rolling circle amplification combined biosensor | |
| Kang et al. | Recent progress and developments of iridium-based compounds as probes for environmental analytes | |
| CN111562244B (en) | Rare earth time-resolved fluorescent probe and kit for detecting daunorubicin | |
| CN103940797A (en) | Method for detecting copper by using copper ion specificity DNA and SYBR Green I fluorescence method | |
| CN109233812B (en) | Chair type copper iodine cluster and selective sensing application of nitrophenol thereof | |
| Guo et al. | Label-free Detection of Zn2+ Based on G-quadruplex | |
| Gong et al. | A fluorescence enhancement-based sensor using glycosylated metalloporphyrin as a recognition element for levamisole assay | |
| CN108956991B (en) | Fluorescence resonance energy transfer biosensor and application thereof | |
| CN116179654B (en) | Rolling circle amplification detection system for detecting cadmium ions in water and application thereof | |
| CN113913432B (en) | Tetracycline bivalent aptamer sequence and non-enzyme label-free detection method | |
| CN116642868A (en) | Application of rare earth complex fluorescent probe in detection of 2, 6-pyridine dicarboxylic acid | |
| CN108997255A (en) | A kind of vinyl ethers Hg2+Fluorescence probe and its preparation method and application | |
| Lever | Rapid fluorometric or spectrophotometric determination of isoniazid | |
| CN110698390B (en) | Fluorescent probe for identifying bisulfite and preparation method and detection method thereof | |
| Xiao et al. | Selective recognition of luteolin and quercetin based on the specific interaction of ortho-dihydroxy substituents with a zinc (ii) complex | |
| CN112578107A (en) | Working solution for high-throughput determination of double-stranded DNA concentration and determination method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |