CN109781692B - Fluorescence sensor for detecting quencher and detection method - Google Patents
Fluorescence sensor for detecting quencher and detection method Download PDFInfo
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Abstract
The invention discloses a fluorescence sensor for detecting a quencher and a detection method, and relates to the technical field of fluorescence sensors. The fluorescence sensor consists of an aptamer chain segment of a quencher and a fluorophore; wherein the fluorophore is modified at the 3 'end or the 5' end of the aptamer segment. The fluorescence sensor provided by the invention can be used for detecting the concentration of the quencher in an unknown solution, has high sensitivity and selectivity, is convenient and quick to operate, has good biocompatibility, and has a wide application prospect in the fields of biological detection, medical diagnosis and the like.
Description
Technical Field
The invention relates to the technical field of fluorescence sensors. And more particularly, to a fluorescence sensor for quencher detection and a detection method.
Background
The aptamer serving as a novel biological material has the characteristics of strong specificity, good biocompatibility, easiness in modification and the like, and is widely applied to the aspects of biosensing, medical research and the like. Aptamers are used in the sensing field, primarily using signal changes caused by changes in the conformation of aptamer segments caused by analytes. Common aptamer sensors contain three moieties, a fluorophore, an aptamer chain, and a quenching group, with little consideration given to the nature of the detection species itself. And in the detection object, metal ions such as Cu2+Biological small molecules such as ATP have certain fluorescence quenching capacity.
At present, most of the construction of organic molecular fluorescent probes is realized by modifying recognition groups on the existing fluorophores. The recognition group can be selectively and highly sensitively combined with an analyte, thereby realizing the detection of the analyte. The sensitivity and selectivity of the detection depends on the recognition group. In this respect, the aptamer functions in accordance with the organic recognition group, and its performance is even superior to that of the organic recognition group. We envision that detection of a quencher-type detector can be achieved by quenching the detector itself without the aid of a quencher. In 2003, Guonan Chen et al reported the use of Hg for the first time2+As a quenching agent, the method realizes the reaction to Hg under the condition of no additional auxiliary quenching agent2+Designing a DNA probe for detection. The report isThe assumption that we utilized the detector as a quencher was verified. However, this method utilizes another DNA helper strand, increasing the complexity of the system and increasing the stabilization time for the assay.
Disclosure of Invention
The invention aims to provide a fluorescent sensor for detecting a quencher based on an aptamer chain segment marked by a fluorescent molecule, and provides a new idea for the design of the sensor.
Another object of the present invention is to provide a method for detecting the concentration of a quencher in an unknown solution using the fluorescence sensor as described above.
In order to achieve the first purpose, the invention adopts the following technical scheme:
a fluorescent sensor for the detection of a quencher, the fluorescent sensor consisting of a quencher-specific aptamer segment and a fluorophore; wherein the fluorophore is modified at the 3 'end or the 5' end of the aptamer segment.
Preferably, in the fluorescent sensor, when the aptamer segment is an Ag aptamer segment, an Hg aptamer segment, an ATP aptamer segment, or a dopamine aptamer segment, the fluorophore is 6-FAM;
or in the fluorescent sensor, when the aptamer segment is a Cu aptamer segment, the fluorophore is 6-FAM, TAMRA, or ROX.
Preferably, the sequence of the Cu aptamer chain segment is shown as SEQ ID No. 1; the sequence of the Ag aptamer chain segment is shown in SEQ ID No. 2; the sequence of the Hg aptamer chain segment is shown as SEQ ID No. 3; the sequence of the ATP aptamer chain segment is shown as SEQ ID No. 4; the sequence of the segment of the dopamine aptamer is shown as SEQ ID No. 5.
Preferably, when the aptamer segment is a Cu aptamer segment, an Ag aptamer segment, an Hg aptamer segment, or an ATP aptamer segment, the fluorophore is modified at the 5' end of the aptamer segment;
when the aptamer segment is a dopamine aptamer segment, the fluorophore is modified at the 3' end of the aptamer segment.
The invention also provides a method as defined in any one of the aboveThe fluorescence sensor is used for detecting a quencher Cu2+、Ag+、Hg2+ATP or dopamine.
In order to achieve the second purpose, the invention adopts the following technical scheme:
a detection method for detecting a quencher using the fluorescence sensor as described in any one of the above, comprising the steps of:
selecting a proper concentration of the fluorescence sensor and a proper detection solution system, selecting a specific excitation wavelength under a fluorescence spectrometer test system, and drawing a standard curve with the maximum fluorescence emission peak intensity as a vertical coordinate and the quencher concentration as a horizontal coordinate;
selecting an analogue which has a similar structure with the quencher and can generate interference for carrying out the selective test of the fluorescence sensor;
and (3) diluting the solution to be detected by a detection solution system by multiple times, detecting the fluorescence intensity value at the moment, comparing the fluorescence intensity value with a standard curve, and calculating the concentration of the quencher in the solution to be detected.
Preferably, when a standard curve is drawn, the concentration of the fluorescence sensors adopted by the fluorescence sensors Cu-ADFs, Cu-TA-ADFs, Cu-RO-ADFs, Ag-ADFs, Hg-ADFs and dopamine-ADFs is 300 nM; fluorescence sensor ATP-ADFs used a fluorescence sensor concentration of 500 nM.
Preferably, when the standard curve is plotted,
the detection solution system of Cu-ADFs is 25mM HEPES, 100mM NaCl, 1mM MgCl2A buffer solution system (PH 7.0);
the detection solution system of Cu-TA-ADFs and Cu-RO-ADFs is 100mM NaCl, 1mM MgCl2Ultra pure aqueous systems.
The detection solution system of Ag-ADFs and Hg-ADFs is a 25mM HEPES buffer solution system (PH 7.0);
the detection solution system of ATP-ADFs and dopamine-ADFs is a 1xPBS buffer solution system (PH is 7.4).
Preferably, under a fluorescence spectrometer test system,
the excitation wavelength selected by the fluorescence sensors Cu-ADFs, Ag-ADFs, Hg-ADFs, ATP-ADFs and dopamine-ADFs is 485 nm;
the excitation wavelength selected by the fluorescence sensor Cu-TA-ADFs is 560 nm; the excitation wavelength selected for the fluorescence sensor Cu-RO-ADFs was 585 nm.
The invention has the following beneficial effects:
the invention selects the aptamer chain segment with strong selectivity and high sensitivity as the recognition group, and the fluorophore modified at the tail end as the detection unit, constructs a new method for detecting the quencher based on the aptamer chain segment modified by the fluorophore, and provides a new idea for the design of the fluorescence sensor and the application and popularization of the aptamer. Compared with the prior art, the invention has the obvious advantages that:
(1) compared with the traditional organic fluorescent molecular sensor, the aptamer chain segment has sensitivity and selectivity superior to those of an organic molecular recognition group as the recognition group; meanwhile, the sensor related by the invention does not need complex synthesis, and has simple structure and convenient use.
(2) Compared with the traditional aptamer type fluorescent sensor, the sensor designed by the invention belongs to an aptamer sensor without assistance of quenching groups, and is more economical and practical compared with the aptamer type sensor with double-end modification and middle modification; meanwhile, redundant quenching agents are not introduced, a pretreatment process is avoided, and the operation is quick and convenient.
(3) The sensor designed by the invention is more beneficial to the detection of a biological system, and DNA is taken as an important genetic material, has very excellent biocompatibility and has great application prospect in the fields of biological detection, medical diagnosis and the like.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows a schematic diagram of the mechanism of quencher detection using the fluorescence sensor provided by the present invention in examples 1, 2, 3, 4, and 5 of the present invention.
FIG. 2 shows Cu modification of TAMRA with Cu aptamer modification in example 1 of the present invention2+Detection and Cu by TAMRA directly2+Titration curves for detection.
Fig. 3 shows the selectivity of the TAMRA fluorescence sensor with Cu aptamer modification in example 1 of the present invention.
FIG. 4 shows the Cu in the sewage system with TAMRA fluorescence sensor modified by Cu aptamer in example 1 of the present invention2+And detecting a conclusion graph.
FIG. 5 shows Ag for 6-FAM fluorescence sensor modified with Ag aptamer in example 2 of the present invention+Detection and direct utilization of 6-FAM for Ag+Titration curves for detection.
FIG. 6 shows Hg with respect to a 6-FAM fluorescence sensor modified with Hg aptamer in example 3 of the present invention2+Detection and direct utilization of 6-FAM for Hg2+Titration curves for detection.
FIG. 7 shows the titration curves for ATP detection using ATP aptamer-modified 6-FAM fluorescence sensor and ATP detection directly using 6-FAM in example 4 of the present invention.
FIG. 8 shows titration curves for dopamine detection using dopamine aptamer-modified 6-FAM fluorescence sensor and direct dopamine detection using 6-FAM in example 5 of the present invention.
Detailed Description
In order to make the technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.
It should be noted that 6-FAM, TAMRA and ROX are common fluorescent molecules in the present invention; wherein "FAM" refers to carboxyfluorescein, which is one of fluorescein derivatives; the term "TAMRA" is entirely called carboxytetramethylrhodamine, and is a rhodamine-based fluorescein derivative.
The 6-FAM, TAMRA and ROX fluorescence sensors modified by Cu aptamer chain segments are respectively marked as Cu-ADFs, Cu-TA-ADFs and Cu-RO-ADFs; the 6-FAM fluorescence sensor modified by the Ag aptamer chain segment, the Hg aptamer chain segment, the ATP aptamer chain segment and the dopamine aptamer chain segment is respectively marked as Ag-ADFs, Hg-ADFs, ATP-ADFs and dopamine-ADFs.
In one aspect, the invention provides a fluorescent sensor for the detection of a quencher, the fluorescent sensor consisting of a quencher-specific aptamer segment and a fluorophore; wherein the fluorophore is modified at the 3 'end or the 5' end of the aptamer segment. Compared with the traditional organic fluorescent molecular sensor, the fluorescent sensor has the advantages that the aptamer chain segment serving as the recognition group has higher sensitivity and selectivity than the organic molecular recognition group, and the sensor does not need complex synthesis, has a simple structure and is convenient to use; compared with the traditional aptamer-type fluorescent sensor, the fluorescent sensor belongs to an aptamer-type sensor without assistance of quenching groups, is more economical and practical compared with the aptamer-type sensor with double-end modification and middle modification, does not introduce redundant quenching agents, has no pretreatment process, and is quick and convenient to operate.
The analyte of the present invention requires some quenching of the fluorescence of the fluorophore being modified, e.g., Cu2+，Ag+，Hg2+ATP, dopamine on 6-FAM, Cu2+For TAMRA and ROX; the aptamer may be an ionic aptamer, such as Cu2+， Ag+，Hg2+Or a small biological molecule, such as ATP, dopamine. The specific listing of several classes of fluorophores and analyte quenchers in the present invention is merely a further description of the present invention. Those skilled in the art can select other suitable combinations of fluorophores and aptamer segments to prepare other fluorescence sensors based on the design concept provided by the present invention, which is not further illustrated in the present invention.
In a preferred embodiment of the invention, seven aptamer-based fluorescence sensors are constructed in total, based on the relationship between the analyte and the fluorophore, including Cu-ADFs, Cu-TA-ADFs, Cu-RO-ADFs, Ag-ADFs, Hg-ADFs, ATP-ADFs, dopamine-ADFs. Specifically, in the fluorescence sensor, when the aptamer segment is an Ag aptamer segment, an Hg aptamer segment, an ATP aptamer segment, or a dopamine aptamer segment, the fluorophore is 6-FAM; when the aptamer segment is a Cu aptamer segment, the fluorophore is 6-FAM, TAMRA, or ROX.
The sequence of the five aptamer chain segments is shown in SEQ ID No. 1; the sequence of the Ag aptamer chain segment is shown in SEQ ID No. 2; the sequence of the Hg aptamer chain segment is shown as SEQ ID No. 3; the sequence of the ATP aptamer chain segment is shown as SEQ ID No. 4; the sequence of the segment of the dopamine aptamer is shown as SEQ ID No. 5.
TABLE 1 adapter segment sequence Listing
The position of the fluorophore modification on the aptamer segment needs to be selected on a case-by-case basis. After the aptamer chain segment is combined with an analyte, a specific secondary structure is formed, the embedding position of the fluorophore on the aptamer chain segment is adjusted, the distance between the fluorophore and the quencher can be changed, and different quenching characteristics are generated. In particular embodiments of the invention, when the aptamer segment is a Cu aptamer segment, an Ag aptamer segment, an Hg aptamer segment, or an ATP aptamer segment, the fluorophore is modified at the 5' end of the aptamer segment; when the aptamer segment is a dopamine aptamer segment, the fluorophore is modified at the 3' end of the aptamer segment. In other embodiments of the invention, the fluorophore may also be selectively modified at other suitable positions in the aptamer segment, as the case may be.
The invention also provides a fluorescence sensor for detecting the quencher Cu2+、Ag+、Hg2+ATP or dopamine. It is understood that, based on the design concept of the present invention, one skilled in the art can design other kinds of fluorescence sensors directly using fluorescent molecules to label the aptamer segment for detecting other kinds of quenchers.
In another aspect, the present invention also provides a method for detecting a quencher using the fluorescence sensor as described in any one of the above, comprising the steps of:
(1) selecting a proper concentration of the fluorescence sensor and a proper detection solution system, selecting a proper excitation wavelength under a fluorescence spectrometer test system, and drawing a standard curve with the maximum fluorescence emission peak intensity as a vertical coordinate and the quencher concentration as a horizontal coordinate;
(2) selecting an analogue which has a similar structure with the quencher and can generate interference for carrying out the selective test of the fluorescence sensor;
(3) and (3) diluting the solution to be detected by a detection solution system by multiple times, detecting the fluorescence intensity value at the moment, comparing the fluorescence intensity value with a standard curve, and calculating the concentration of the quencher in the solution to be detected.
The method for drawing the standard curve of the fluorescence sensor comprises the following steps:
accurate configuration of 5mM Cu2+，0.05mM Ag+，0.5mM Hg2+50mM ATP, 100mM dopamine standard solution, was titrated as a quencher. Accurately prepare 25mM HEPES, 100mM NaCl, 1mM MgCl2Buffer solution system (PH 7.0) as the detection solution system for Cu-ADFs. Accurate preparation of 100mM NaCl, 1mM MgCl2The solution system is used as a detection solution system of the Cu-TA-ADFs and Cu-RO-ADFs fluorescence sensor. A25 mM HEPES buffer solution system (pH 7.0) is accurately prepared to be used as a detection solution system of Ag-ADFs and Hg-ADFs. A1 xPBS buffer solution system is accurately configured to be used as a detection solution system of ATP-ADFs and dopamine-ADFs.
And drawing standard curves of Cu-ADFs, Cu-TA-ADFs, Cu-RO-ADFs, Ag-ADFs, Hg-ADFs and dopamine-ADFs, and adopting the concentration of the sensor of 300 nM. 3uL 100uM of the above fluorescence sensor was removed and added to 1mL of the corresponding detection solution system. For the ATP-ADFs standard curve, using 500nM sensor concentration, 5uL of 100uM ATP-ADFs were removed and added to 1mL of 1xPBS buffer.
FL-4600 fluorescence spectrometer (Hitachi) is used as a detection platform. The excitation wavelength was chosen to be 485nm for Cu-ADFs, Ag-ADFs, Hg-ADFs, ATP-ADFs, dopamine-ADFs, and 560nm and 585nm for Cu-TA-ADFs, Cu-RO-ADFs, respectively. Respectively dripping standard solution of quenching agent into the detection solution system containing the aptamer fluorescence sensor, recording the fluorescence intensity of the corresponding maximum emission peak at different concentrations of the quenching agent, and normalizingAnd (6) processing. The normalization treatment is carried out by labeling the detection solution system without the quencher to have a fluorescence intensity F at the maximum emission peak0The fluorescence intensity at the maximum emission peak of the detection solution system labeled with different concentrations of quencher (labeled as x) is Fx as Fx/F0The values were taken as relative fluorescence intensities. A standard curve of relative fluorescence intensity versus quencher concentration was plotted.
The method comprises the following experimental steps of selective test of the sensor:
accurate preparation of 5mM Cd2+，Ni2+，Zn2+，Cr3+，Co2+，Pb2+，Hg2+，Ca2+，Mg2+And (4) standard solution. 300nM of Cu-TA-ADFs was added to a solution containing 100mM NaCl, 1mM MgCl2The ultrapure water detection solution of (1). Respectively adding 50uM of the interference ions into the system, and testing the fluorescence intensity value at the maximum emission wavelength in the presence of the interference ions.
The fluorescence sensor is applied to the test of the concentration of a quencher in an unknown solution system, taking the Cu-TA-ADFs fluorescence sensor for detecting the sewage soaked with a copper pipe in the industry as an example, the method comprises the following steps:
the adopted solution to be detected is sewage soaked with copper pipes in industry, obvious impurities in a solution system to be detected are removed, and a plurality of supernatant solutions are taken. Using 100mM NaCl, 1mM MgCl2Diluting a sewage sample to be detected by 10 times by using the ultrapure water solution, controlling the total volume of the detection solution to be 1mL, adding 300nM Cu-TA-ADFs into the system, recording the fluorescence intensity at the maximum emission wavelength, and carrying out normalization treatment to obtain a relative fluorescence intensity value. The measurement was repeated three times, and the average value was taken as the test result. Substituting into a nonlinear equation of standard curve fitting, and calculating to obtain Cu2+The concentration was 61.95uM, and there was only a 2.026% deviation in the fluorescence assay compared to 60.72uM for atomic absorption.
The following is a detailed description of specific embodiments.
Example 1
1) Designing and preparing Cu aptamer modified 6-FAM, TAMRA and ROX fluorescence sensors which are respectively marked as Cu-ADFs, Cu-TA-ADFs and Cu-RO-ADFs, and the basic structures of the sensors are respectively as follows: 6-FAM-ATCGCGATATTTTCTGTAGCGATTCTTGTTTGAGCGCTCGGTACGAACAGA; TAMRA-ATCGCGATATTTTCTGTAGCGATTCTTGTTTGAGCGCTCGGTACGAACAGA; ROX-ATCGCGATATTTTCTGTAGCGATTCTTGTTTGAGCGCTCGGTACGAACAGA.
2) Accurately preparing Cu with standard concentration of 5mM2+Solution, used for calibration of the standard curve. The 25mM HEPES, 100mM NaCl, 1mM MgCl was prepared2The buffer solution system (PH 7.0) was used as a detection solution system for Cu-ADFs. The preparation of 100mM NaCl, 1mM MgCl2The ultra-pure water solution system is used as a detection solution system of Cu-TA-ADFs and Cu-RO-ADFs. Selecting a FL-4600 fluorescence spectrometer (Hitachi) as a detection instrument, selecting 485nm as the excitation wavelength of Cu-ADFs, selecting 560nm as the excitation wavelength of Cu-TA-ADFs, selecting 585nm as the excitation wavelength of Cu-RO-ADFs, collecting the fluorescence intensity at the maximum emission peak, and carrying out normalization treatment. The normalization treatment is carried out such that the fluorescence intensity of the maximum emission peak of the system labeled with the detection solution not containing the quencher is F0The maximum emission peak fluorescence intensity of the detection solution system labeled with different quencher concentrations (labeled as x) is Fx as Fx/F0The values were taken as relative fluorescence intensities.
3) For Cu-ADFs, the analyte Cu was added dropwise at 2uM intervals2+Simultaneous recording of different Cu2+The relative fluorescence intensity values at concentration were sampled up to 50uM and repeated three times. For Cu-TA-ADFs and Cu-RO-ADFs, the analyte Cu was added dropwise at 5uM intervals2+Simultaneous recording of different Cu2+The relative fluorescence intensity values at concentration were sampled up to 50uM and repeated three times. A standard curve of relative fluorescence intensity values versus quencher concentration was plotted. As can be seen from FIG. 2, Cu follows the system2+The fluorescence intensity of Cu-TA-ADFs gradually decreases with the increase of the concentration. In contrast, the fluorescence intensity of the free TAMRA solution hardly changes at the same ion concentration, indicating that the aptamer plays a very important role in enhancing fluorescence quenching.
4) 3uL of 100uM Cu-TA-ADFs was added to 1mL of 100mM NaCl, 1mM MgCl2In the ultra-pure aqueous solution system of (1). Accurate configuration 5mM Cd2+，Ni2+，Zn2+，Cr3+，Co2+，Pb2+，Hg2+，Ca2+，Mg2+And (4) standard solution. Respectively adding 50uM of the interference ions into the system, and testing the fluorescence intensity value at the maximum emission wavelength in the presence of the interference ions. Fluorescence intensity F in the absence of interfering ionsintialThe fluorescence intensity of the label added with 50uM interfering ions is FendWith Fintial/FendAs reference value, wherein Fintial/FendA larger number indicates a more severe interference, Fintial/FendSmaller values indicate less interference. As can be seen from FIG. 3, when the concentration of the interfering substance is 50uM, Cr is excluded3+Besides certain interference, other metal ions hardly interfere the Cu-TA-ADFs, which shows that the Cu-TA-ADFs has excellent selectivity.
5) 100uL of the wastewater to be tested was added to 900uL of 100mM NaCl, 1mM MgCl2In the ultra-pure aqueous solution system of (1). And adding 3uL 100uM Cu-TA-ADFs into the system, testing the fluorescence intensity at the moment, repeating the steps for three times, taking the average value as a test result, and performing normalization treatment to obtain an average relative fluorescence intensity value. Substituting the relative fluorescence intensity values into the standard curve nonlinear equation, as can be seen from FIG. 4, Cu is obtained by calculation2+Is 61.95uM, there is only a 2.026% deviation compared to the data obtained from atomic absorption measurements.
Example 2
1) The Ag aptamer modified 6-FAM fluorescence sensor is designed and prepared, is marked as Ag-ADFs, and has the following basic structure: 6-FAM-CTCTCTTCTCTTCATTTTTCAACACAACACAC.
2) Accurately preparing Ag with standard concentration of 0.05mM+Solution, used for calibration of the standard curve. A25 mM HEPES buffer solution system (pH 7.0) was prepared and used as a detection solution system for Ag-ADFs. Selecting a FL-4600 fluorescence spectrometer (Hitachi) as a detection instrument, selecting 485nm as the excitation wavelength of Ag-ADFs, collecting the fluorescence intensity at the maximum emission peak, and carrying out normalization processing. The normalization treatment is carried out by labeling the detection solution without the quencherThe maximum emission peak fluorescence intensity of the system is F0The maximum emission peak fluorescence intensity of the detection solution system labeled with different quencher concentrations (labeled as x) is Fx as Fx/F0The values were taken as relative fluorescence intensities.
3) For Ag-ADFs, the analyte Ag was added dropwise at 0.2uM intervals+Simultaneously recording different Ag+The relative fluorescence intensity at concentration was sampled up to 3.6uM and repeated three times. A standard curve of relative fluorescence intensity values versus quencher concentration was plotted. As can be seen from FIG. 5, Ag follows the system+The fluorescence intensity of Ag-ADFs gradually decreases with the increase of the concentration. Compared with a free 6-FAM solution, the fluorescence intensity hardly changes under the same ion concentration, which indicates that the aptamer plays a very important role in enhancing fluorescence quenching.
Example 3
1) The Hg aptamer modified 6-FAM fluorescence sensor is designed and prepared, is marked as Hg-ADFs, and has the following basic structure: 6-FAM-TTCTTTCTTCCCCTTGTTTGTT.
2) Accurately preparing Hg with standard concentration of 0.5mM2+Solution, used for calibration of the standard curve. 25mM HEPES was prepared and used as a detection solution system for Hg-ADFs. Selecting a FL-4600 fluorescence spectrometer (Hitachi) as a detection instrument, selecting 485nm as the excitation wavelength of Hg-ADFs, collecting the fluorescence intensity at the maximum emission peak, and carrying out normalization processing. The normalization treatment is carried out such that the fluorescence intensity of the maximum emission peak of the system labeled with the detection solution not containing the quencher is F0The maximum emission peak fluorescence intensity of the detection solution system labeled with different quencher concentrations (labeled as x) is Fx as Fx/F0The values were taken as relative fluorescence intensities.
3) For Hg-ADFs, the analyte Hg was added dropwise at 1uM intervals2+Simultaneous recording of different Hg+The relative fluorescence intensity values at concentration were sampled up to 12uM and repeated three times. A standard curve of relative fluorescence intensity values versus quencher concentration was plotted. As can be seen from FIG. 6, as the system Hg2+The fluorescence intensity of Hg-ADFs gradually decreases with increasing concentration. While the free 6-FAM solution was compared in the same wayThe fluorescence intensity hardly changes at the ion concentration of (a), which indicates that the aptamer plays a very important role in enhancing fluorescence quenching.
Example 4
1) Designing and preparing an ATP aptamer modified 6-FAM fluorescence sensor which is marked as ATP-ADFs, wherein the basic structure is as follows: 6-FAM-ACCTGGGGAGTATTGCGGAGGAAGGT.
2) ATP solution with standard concentration of 50mM is prepared accurately and used for calibration of the standard curve. 1xPBS buffer solution is prepared and used as a detection solution system of ATP-ADFs. Selecting FL-4600 fluorescence spectrometer (Hitachi) as a detection instrument, selecting 485nm as the excitation wavelength of ATP-ADFs, collecting the fluorescence intensity at the maximum emission peak, and carrying out normalization processing. The normalization treatment is carried out such that the fluorescence intensity of the maximum emission peak of the system labeled with the detection solution not containing the quencher is F0The maximum emission peak fluorescence intensity of the detection solution system labeled with different quencher concentrations (labeled as x) is Fx as Fx/F0The values were taken as relative fluorescence intensities.
3) For ATP-ADFs, the analyte ATP was added dropwise at 0.2mM intervals, while the relative fluorescence intensity values at different ATP concentrations were recorded, sampled up to 5mM, and repeated three times. A standard curve of relative fluorescence intensity values versus quencher concentration was plotted. As can be seen from FIG. 7, the fluorescence intensity of ATP-ADFs gradually decreased as the ATP concentration of the system increased. Compared with a free 6-FAM solution, under the same ATP concentration, although the fluorescence intensity is reduced to a certain degree, the fluorescence quenching phenomenon of ATP-ADFs is more obvious, and the fact that the aptamer plays a very important role in the process of enhancing fluorescence quenching is shown.
Example 5
1) Designing and preparing a dopamine aptamer modified 6-FAM fluorescence sensor which is marked as dopamine-ADFs, wherein the basic structure of the sensor is as follows: GTCTCTGTGTGCGCCAGAGACACTGGGGCAGATATGGGCCAGCACAGAATGAGGCCC-6-FAM.
2) A solution of the dopamine with a standard concentration of 100mM is prepared accurately and used for calibration of a standard curve. 1xPBS buffer solution is prepared and used as a detection solution system of dopamine-ADFs. Selecting FL-4600 fluorescent lightAnd (3) taking a spectrometer (Hitachi) as a detection instrument, selecting 485nm as the excitation wavelength of the dopamine-ADFs, collecting the fluorescence intensity at the maximum emission peak, and carrying out normalization treatment. The normalization treatment is carried out such that the fluorescence intensity of the maximum emission peak of the system labeled with the detection solution not containing the quencher is F0The maximum emission peak fluorescence intensity of the detection solution system labeled with different quencher concentrations (labeled as x) is Fx as Fx/F0The values were taken as relative fluorescence intensities.
3) For the dopamine-ADFs, the analyte dopamine was added dropwise at 0.5mM intervals, while the relative fluorescence intensity values at different dopamine concentrations were recorded, sampled to 10mM, and repeated three times. A standard curve of relative fluorescence intensity values versus quencher concentration was plotted. As can be seen from FIG. 8, the fluorescence intensity of the dopamine-ADFs gradually decreased with increasing concentration of the dopamine in the system. Compared with a free 6-FAM solution, under the same dopamine concentration, although the fluorescence intensity is reduced to a certain extent, the fluorescence quenching phenomenon of the dopamine-ADFs is more obvious, and the fact that the aptamer plays a very important role in the process of enhancing fluorescence quenching is demonstrated.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.
Sequence listing
<110> research institute of physical and chemical technology of Chinese academy of sciences
University OF CHINESE ACADEMY OF SCIENCES
<120> fluorescent sensor for detecting quencher and detection method
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Claims (7)
1. A fluorescent sensor for detection of a quencher, wherein the fluorescent sensor is comprised of a quencher-specific aptamer segment and a fluorophore; wherein the fluorophore is modified at the 3 'end or the 5' end of the aptamer segment;
in the fluorescent sensor, when the aptamer segment is an Ag aptamer segment, an Hg aptamer segment, an ATP aptamer segment, or a dopamine aptamer segment, the fluorophore is 6-FAM;
or in the fluorescent sensor, when the aptamer segment is a Cu aptamer segment, the fluorophore is 6-FAM, TAMRA, or ROX;
the sequence of the Cu aptamer chain segment is shown as SEQ ID No. 1; the sequence of the Ag aptamer chain segment is shown in SEQ ID No. 2; the sequence of the Hg aptamer chain segment is shown as SEQ ID No. 3; the sequence of the ATP aptamer chain segment is shown as SEQ ID No. 4; the sequence of the segment of the dopamine aptamer is shown as SEQ ID No. 5.
2. The fluorescence sensor of claim 1, wherein when the aptamer segment is a Cu aptamer segment, an Ag aptamer segment, an Hg aptamer segment, or an ATP aptamer segment, the fluorophore is modified at the 5' end of the aptamer segment;
when the aptamer segment is a dopamine aptamer segment, the fluorophore is modified at the 3' end of the aptamer segment.
3. Use of a fluorescence sensor according to any of claims 1-2 in the detection of the quencher Cu2+、Ag+、Hg2+ATP or dopamine.
4. A method for detecting a quencher using the fluorescence sensor according to any one of claims 1-2, comprising the steps of:
selecting a proper concentration of the fluorescence sensor and a proper detection solution system, selecting a specific excitation wavelength under a fluorescence spectrometer test system, and drawing a standard curve with the maximum fluorescence emission peak intensity as a vertical coordinate and the quencher concentration as a horizontal coordinate;
selecting an analogue which has a similar structure with the quencher and can generate interference for carrying out the selective test of the fluorescence sensor;
and (3) diluting the solution to be detected by a detection solution system by multiple times, detecting the fluorescence intensity value at the moment, comparing the fluorescence intensity value with a standard curve, and calculating the concentration of the quencher in the solution to be detected.
5. The assay of claim 4, wherein the fluorescence sensors Cu-ADFs, Cu-TA-ADFs, Cu-RO-ADFs, Ag-ADFs, Hg-ADFs and dopamine-ADFs are used at a concentration of 300nM when plotting the standard curve; fluorescence sensor ATP-ADFs used a fluorescence sensor concentration of 500 nM.
6. The detection method according to claim 4, wherein when a standard curve is plotted,
the detection solution system of Cu-ADFs is 25mM HEPES, 100mM NaCl, 1mM MgCl2The pH of the buffer solution system is 7.0;
the detection solution system of Cu-TA-ADFs and Cu-RO-ADFs is 100mM NaCl, 1mM MgCl2An ultra-pure aqueous solution system;
the detection solution system of Ag-ADFs and Hg-ADFs is a 25mM HEPES buffer solution system, and the pH value is 7.0;
the detection solution system of ATP-ADFs and dopamine-ADFs is a 1xPBS buffer solution system, and the PH is 7.4.
7. The detection method according to claim 4, wherein, under a fluorescence spectrometer test system,
the excitation wavelength selected by the fluorescence sensors Cu-ADFs, Ag-ADFs, Hg-ADFs, ATP-ADFs and dopamine-ADFs is 485 nm;
the excitation wavelength selected by the fluorescence sensor Cu-TA-ADFs is 560 nm; the excitation wavelength selected for the fluorescence sensor Cu-RO-ADFs was 585 nm.
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