CN114438091A - DNA fluorescent probe and method for detecting cadmium ions by using same - Google Patents
DNA fluorescent probe and method for detecting cadmium ions by using same Download PDFInfo
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- CN114438091A CN114438091A CN202210119587.2A CN202210119587A CN114438091A CN 114438091 A CN114438091 A CN 114438091A CN 202210119587 A CN202210119587 A CN 202210119587A CN 114438091 A CN114438091 A CN 114438091A
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- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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Abstract
The disclosure provides a DNA fluorescent probe and a method for detecting cadmium ions by adopting the DNA fluorescent probe, wherein the 3 'end and the 5' end of the DNA fluorescent probe are simultaneously provided with pyrene molecular markers. The DNA fluorescent probe can be specifically combined with cadmium ions, when the cadmium ions exist, a pyrene molecular marker fluorescent signal is enhanced, and the detection of the cadmium ions can be realized according to the change of the fluorescent signal. The fluorescent probe analysis method has the advantages of quick response, high sensitivity, high stability and good selectivity, and can be used for detecting cadmium ions in a complex sample matrix.
Description
Technical Field
The disclosure relates to the technical field of detection, in particular to a DNA fluorescent probe and a method for detecting cadmium ions by adopting the DNA fluorescent probe.
Background
The heavy metal cadmium is widely distributed in nature and widely used in industrial and agricultural production, such as metallurgy, pigment, battery, semiconductor material, fertilizer, plastics and other fields. With the development of mineral resources and the production and use of cadmium-containing products, cadmium is also released into the environment through various ways, causing pollution of water, soil, air, etc. In addition, cadmium can enter a human body through drinking water, food chains and the like and is enriched in the human body, cadmium ions can react with biomolecules such as protein, nucleic acid and the like to influence the normal functions of the corresponding biomolecules, thus causing harm to the health of people and causing serious diseases such as pain diseases (itai-itai disease), renal function injury, immune system lesion, nerve function injury, cancer and the like. The world organization for cancer ranks cadmium as a grade 1 carcinogen. The content of cadmium in water and food is strictly limited in all countries.
The existing analysis method commonly used for detecting the content of cadmium and cadmium ions mainly comprises atomic absorption spectroscopy, inductively coupled plasma emission spectroscopy, inductively coupled plasma mass spectrometry, atomic fluorescence spectroscopy and the like. However, these methods also suffer from limitations, such as expensive equipment, complicated operation, requirement of specialized personnel, etc., and are not suitable for performing rapid on-site testing.
Disclosure of Invention
In view of the above, the present disclosure provides a DNA fluorescent probe and a method for detecting cadmium ions using the DNA fluorescent probe, so as to at least partially solve the above technical problems.
One aspect of the present disclosure provides a DNA fluorescent probe, including: any one DNA sequence of the first sequence to the fifth sequence, wherein the DNA sequence comprises an aptamer which is marked with pyrene molecules at the 3 'end and the 5' end respectively:
sequence one: 5'-GGG TTC ACA GTC C-3', respectively;
and (2) sequence II: 5'-CGG GTT CAC AGT CCG-3', respectively;
and (3) sequence III: 5'-ACG GGT TCACAG TCC GT-3', respectively;
and (4) sequence IV: 5'-GAC GGG TTC ACA GTC CGT T-3', respectively;
and a fifth sequence: 5'-CGA CGG GTT CAC AGT CCG TTG-3' are provided.
Another aspect of the present disclosure provides a method for detecting cadmium ions by using the DNA fluorescent probe, comprising:
detecting a reaction solution obtained by reacting the DNA fluorescent probe with cadmium ion standard solutions with different concentrations at a second preset temperature by a fluorescence detection method at a first preset temperature, and drawing a calibration curve within a preset emission wavelength range;
at a second preset temperature, incubating the DNA fluorescent probe and a sample solution containing cadmium ions in a preset buffer solution;
recording the fluorescence intensity of a fluorescence response signal of a sample solution containing cadmium ions in a preset emission wavelength range by a fluorescence detection method at a first preset temperature;
and determining the concentration of cadmium ions in the sample solution containing cadmium ions according to the fluorescence intensity of the fluorescence response signal and the calibration curve.
According to the embodiment of the disclosure, the preset emission wavelength range is 365-600 nm.
According to an embodiment of the present disclosure, the preset buffer solution includes a Tris-HCl buffer solution containing sodium ions and magnesium ions.
According to the embodiment of the disclosure, the pH value of the preset buffer solution is 6-9.
According to the embodiment of the disclosure, the concentration of sodium ions in the predetermined buffer solution is 0-100 mM, and the concentration of magnesium ions is 0-10 mM.
According to an embodiment of the present disclosure, the first preset temperature includes 4-50 ℃.
According to an embodiment of the present disclosure, the second preset temperature includes 4-25 ℃.
According to an embodiment of the present disclosure, the reaction time includes 0 to 60 minutes.
Another aspect of the present disclosure provides a kit for detecting cadmium ions, including the above DNA fluorescent probe, a predetermined buffer solution, and a standard of cadmium ions.
According to the embodiment of the disclosure, the DNA fluorescent probe is obtained by adopting the nucleic acid aptamer with reasonable design and respectively labeling pyrene molecules at the 5 'end and the 3' end of the nucleic acid aptamer, and can be applied to the rapid detection of cadmium ions based on the change of the fluorescence intensity of the fluorescence response signal before and after the DNA fluorescent probe is combined with cadmium ions.
Drawings
FIG. 1 schematically shows changes in fluorescence intensity before and after binding of a DNA fluorescent probe with cadmium ions according to an embodiment of the present disclosure;
FIG. 2 schematically shows fluorescence intensities of fluorescence response signals of DNA fluorescent probes of different sequences at an emission wavelength of 485nm according to an embodiment of the present disclosure;
FIG. 3 is a graph schematically showing fluorescence spectra after different concentrations of cadmium ions were bound to DNA fluorescent probes in example 2;
FIG. 4 is a graph schematically showing the relationship between fluorescence intensity and cadmium ion concentration according to the 485nm emission wavelength of the reaction solution in which the DNA fluorescent probe of example 2 is combined with cadmium ions of different concentrations;
FIG. 5 is a graph schematically showing the relationship between fluorescence intensity and cadmium ion concentration according to the 485nm emission wavelength of the reaction solution in which the DNA fluorescent probe of example 3 is combined with cadmium ions of different concentrations;
FIG. 6 is a graph schematically showing the fluorescence intensity of the fluorescence response signal of the reaction solution in which the DNA fluorescent probe in example 4 is bound to different metal ions;
FIG. 7 is a graph schematically showing the fluorescence intensity of a fluorescence response signal when the DNA fluorescent probe in example 5 detects cadmium ions in a diluted water sample;
FIG. 8 is a schematic diagram showing the change in fluorescence intensity of the fluorescence response signal at different detection temperatures when the DNA fluorescent probe in example 6 is bound to the same concentration of cadmium ions.
Detailed Description
For the purpose of promoting a better understanding of the objects, aspects and advantages of the present disclosure, reference is made to the following detailed description taken in conjunction with the accompanying drawings.
A first aspect of the present disclosure provides a DNA fluorescent probe comprising: any one DNA sequence of the first sequence to the fifth sequence, wherein the DNA sequence comprises an aptamer which is marked with pyrene molecules at the 3 'end and the 5' end respectively:
sequence one: 5'-GGG TTC ACA GTC C-3', respectively;
and (2) sequence II: 5'-CGG GTT CAC AGT CCG-3', respectively;
and (3) sequence III: 5'-ACG GGT TCACAG TCC GT-3', respectively;
and (4) sequence IV: 5'-GAC GGG TTC ACA GTC CGT T-3', respectively;
and a fifth sequence: 5'-CGA CGG GTT CAC AGT CCG TTG-3' are provided.
According to the embodiment of the disclosure, the DNA fluorescent probe is obtained by adopting the nucleic acid aptamer with reasonable design and respectively labeling pyrene molecules at the 5 'end and the 3' end of the nucleic acid aptamer, and can be applied to the rapid detection of cadmium ions based on the obvious change of the fluorescence intensity of the fluorescence response signal before and after the DNA fluorescent probe is combined with the cadmium ions.
The second aspect of the present disclosure provides a method for detecting cadmium ions by using the DNA fluorescent probe, comprising:
detecting a reaction solution obtained by reacting the DNA fluorescent probe with cadmium ion standard solutions with different concentrations at a second preset temperature by a fluorescence detection method at a first preset temperature, and drawing a calibration curve within a preset emission wavelength range;
at a second preset temperature, incubating the DNA fluorescent probe and a sample solution containing cadmium ions in a preset buffer solution;
recording the fluorescence intensity of a fluorescence response signal of a sample solution containing cadmium ions in a preset emission wavelength range by a fluorescence detection method at a first preset temperature;
and determining the concentration of cadmium ions in the sample solution containing cadmium ions according to the fluorescence intensity of the fluorescence response signal and the calibration curve.
According to the embodiment of the disclosure, the principle of detecting cadmium ions by using a DNA fluorescent probe comprises the following steps: when the DNA fluorescent probe is not combined with cadmium ions, the structure of the aptamer in the DNA fluorescent probe is loose, the pyrene molecules at the 5 'end and the 3' end are far away from each other, when the DNA fluorescent probe is combined with the cadmium ions, the structure of the DNA fluorescent probe is changed, and the pyrene molecules at the 5 'end and the 3' end are close to each other, so that the fluorescent signal is changed.
According to the embodiment of the present disclosure, the predetermined emission wavelength range is 365-600 nm, for example: 365nm, 450nm, 500nm, 550nm and 600 nm.
Fig. 1 schematically shows changes in fluorescence intensity before and after binding of a DNA fluorescent probe to cadmium ions according to an embodiment of the present disclosure.
As shown in FIG. 1, when the emission wavelength is less than 365nm, or more than 600nm, the fluorescence intensity curves of the blank sample and the sample solution containing cadmium ions are very close, which means that when the emission wavelength is less than 365nm, or more than 600nm, the change of the fluorescence signal before and after the DNA fluorescent probe is combined with cadmium ions becomes weak, which is not favorable for detection. Therefore, the preset emission wavelength range is 365-600 nm, and the change of the fluorescence signal before and after the DNA fluorescent probe is combined with the cadmium ions becomes strong in the emission wavelength range, so that the detection method can be used for detecting the cadmium ions.
According to an embodiment of the present disclosure, the preset buffer solution includes a Tris-HCl buffer solution containing sodium ions and magnesium ions.
According to an embodiment of the present disclosure, the concentration of sodium ions in the predetermined buffer solution includes 0 to 100mM, for example: 10mM, 20mM, 50mM, 100 mM. The concentration of magnesium ions includes 0 to 10mM, for example: 1mM, 2mM, 5mM, 10 mM.
According to an embodiment of the present disclosure, the sodium ions and the magnesium ions in the buffer solution may be provided by sodium chloride and magnesium chloride.
According to an embodiment of the present disclosure, the pH value of the predetermined buffer solution includes 6 to 9, for example: 6.0, 6.5, 7.0, 7.5, 7.8, 8.0, 8.5, 9.0.
According to an embodiment of the present disclosure, the first preset temperature includes 4 to 50 ℃, for example: 4 ℃, 10 ℃, 20 ℃, 25 ℃ and 50 ℃.
According to an embodiment of the present disclosure, the second preset temperature includes 4 to 25 ℃, for example: 4 ℃, 10 ℃, 15 ℃, 20 ℃ and 25 ℃.
According to an embodiment of the present disclosure, the second preset temperature is a temperature at which the DNA fluorescent probe is incubated with cadmium ions. The first preset temperature is a fluorescence detection temperature.
According to an embodiment of the present disclosure, the reaction time includes 0 to 60 minutes.
Another aspect of the disclosure provides a kit for detecting cadmium ions, which includes the DNA fluorescent probe, a predetermined buffer solution and a standard of cadmium ions.
The experimental procedures in the following examples are all conventional ones unless otherwise specified. The test materials and reagents used in the following examples were purchased from conventional reagents companies unless otherwise specified.
The DNA sequence used was synthesized, prepared and purified by Biotechnology engineering (Shanghai) GmbH.
Example 1
DNA fluorescent probes corresponding to the first to fifth sequences labeled with 50nM pyrene molecules were incubated with cadmium ions in a reaction buffer solution (20mM Tris-HCl (pH 7.5) +20mM NaCl) at 25 ℃ and then the change of fluorescent signal in the blank sample containing no cadmium ions and the sample solution containing 1000nM cadmium ions was measured by fluorescence detection at 25 ℃.
Fig. 1 schematically shows changes in fluorescence intensity before and after binding of a DNA fluorescent probe to cadmium ions according to an embodiment of the present disclosure.
As shown in FIG. 1, when the fluorescence spectra of the blank sample of the DNA fluorescent probe corresponding to sequence two and the reaction solution containing 1000nM cadmium ions combined with the DNA fluorescent probe corresponding to sequence two are compared, it can be seen that the fluorescence intensities of the two are clearly distinguished at any emission wavelength in the wavelength range of 365-600 nM.
As shown in FIG. 2, for example, with an emission wavelength of 485nm, the fluorescence intensity of the DNA fluorescent probe corresponding to sequence one before binding with cadmium ions (blank sample) is 96. After binding with cadmium ion (Cd)2+) The fluorescence intensity was 357. The DNA fluorescent probe corresponding to sequence two has a fluorescence intensity of 158 before binding with cadmium ions (blank sample). After binding with cadmium ion (Cd)2 +) The fluorescence intensity was 945. The fluorescent DNA probe corresponding to SEQ ID NO, before binding to cadmium ions (blank sample), has a fluorescence intensity of 224. After binding with cadmium ions (Cd)2+) The fluorescence intensity was 576. The DNA fluorescent probe corresponding to sequence four had a fluorescence intensity of 13 before binding to cadmium ions (blank sample). After binding with cadmium ion (Cd)+) The fluorescence intensity was 86. The DNA fluorescent probe corresponding to the fifth sequence had a fluorescence intensity of 66 before binding to cadmium ions (blank sample). After binding with cadmium ion (Cd)+) The fluorescence intensity was 158. Therefore, the fluorescence signal intensity of the five DNA fluorescent probes provided by the embodiment of the disclosure is obviously changed before and after the five DNA fluorescent probes are combined with cadmium ions.
Example 2
And detecting cadmium ions by using a DNA fluorescent probe corresponding to the second sequence. A pyrene molecule labeled DNA fluorescent probe corresponding to 50nM sequence II and cadmium ions with different concentrations are incubated in a reaction buffer solution (20mM Tris-HCl (pH 7.5) +20mM NaCl) for 10 minutes at 25 ℃, and then fluorescence detection is carried out at 10 ℃ to obtain a calibration curve, for example, the calibration curve is the calibration curve of the reaction solution of fluorescence intensity corresponding to 485nM emission wavelength and cadmium ions with different concentrations.
And then, carrying out incubation reaction on the DNA fluorescent probe corresponding to the second sequence and cadmium ions with unknown concentration under the same condition, carrying out fluorescence detection on the reaction solution to obtain the fluorescence intensity of the reaction solution with unknown cadmium ion concentration, and substituting the fluorescence intensity of the reaction solution with unknown cadmium ion concentration into a calibration curve to obtain the concentration of the cadmium ions in the unknown solution.
FIG. 3 schematically shows fluorescence spectrum curves after different concentrations of cadmium ions were bound to DNA fluorescent probes in example 2.
As shown in FIG. 3, the fluorescence spectra curve obtained by performing fluorescence detection on the DNA fluorescent probe corresponding to the second sequence and the sample solution with cadmium ion concentration of 0, 7.8nM, 15.6nM, 31.3nM, 62.5nM, 125nM, 250nM, 500nM, 1000nM, 2000nM, 4000nM and 8000nM respectively from bottom to top in FIG. 3 is shown. As is clear from FIG. 3, the higher the separation degree between the fluorescence spectrum curve and the fluorescence spectrum curve of the blank sample (cadmium ion concentration: 0) as the cadmium ion concentration increases, the more significant the change in fluorescence intensity before and after the binding of the DNA fluorescent probe to the cadmium ion.
FIG. 4 is a schematic diagram showing the relationship between fluorescence intensity and cadmium ion concentration according to the 485nm emission wavelength of the reaction solution in which the DNA fluorescent probe of example 2 is combined with cadmium ions of different concentrations.
As shown in FIG. 4, at an emission wavelength of 485nm, the fluorescence intensity increases with increasing concentration of cadmium ions. Aiming at the detection of cadmium ions, according to the fact that the difference value between the fluorescence intensity value of the sample solution and the fluorescence intensity value of the blank sample solution is more than 3 times of the deviation of the fluorescence intensity value of the blank solution, the detection limit of the method can be determined to be 7.8nlM cadmium ions.
Example 3
The procedure was the same as in example 2 except that the DNA fluorescent probe corresponding to sequence two in example 2 was replaced with the DNA fluorescent probe corresponding to sequence three.
FIG. 5 is a schematic diagram showing the relationship between fluorescence intensity and cadmium ion concentration according to the 485nm emission wavelength of the reaction solution in which the DNA fluorescent probe in example 3 is combined with cadmium ions of different concentrations.
As shown in FIG. 5, at an emission wavelength of 485nm, the fluorescence intensity increases with increasing concentration of cadmium ions. Aiming at the detection of cadmium ions, according to the fact that the difference value between the fluorescence intensity value of the sample solution and the fluorescence intensity value of the blank sample solution is more than 3 times of the deviation of the fluorescence intensity value of the blank sample solution, the detection limit of the method can be determined to be 1.95nM cadmium ions.
Example 4
By using the experimental conditions and the DNA fluorescent probe in example 2, the cadmium ion solutions with different concentrations were replaced with copper ion solution, nickel ion solution, lead ion solution, zinc ion solution, mercury ion solution, manganese ion solution, magnesium ion solution, and calcium ion solution, all of which had a concentration of 1000 nM.
FIG. 6 is a graph schematically showing the fluorescence intensity of the fluorescence response signal of the reaction solution in which the DNA fluorescent probe in example 4 is bound to different metal ions.
As shown in fig. 6, the fluorescence intensity of the blank sample was 300, and the fluorescence intensity of the sample solution containing copper ions, the fluorescence intensity of the sample solution containing nickel ions, the fluorescence intensity of the sample solution containing lead ions, the fluorescence intensity of the sample solution containing zinc ions, the fluorescence intensity of the sample solution containing mercury ions, the fluorescence intensity of the sample solution containing manganese ions, the fluorescence intensity of the sample solution containing magnesium ions, and the fluorescence intensity of the sample solution containing calcium ions were all around 300, and the difference from the fluorescence intensity of the blank sample was small, and it was found that the detected ions did not produce a significant change in fluorescence signal (fluorescence intensity at 485 nM) in the presence of these ions as compared with the blank sample solution, while the fluorescence signal significantly increased in the presence of cadmium ions (1000nM) as a control as compared with the blank sample.
Example 5
And (3) detecting cadmium ions in the diluted tap water by using the fluorescent probe corresponding to the sequence 2 in the example 2. The tap water sample was diluted 20 times with the reaction buffer. In diluted water samples containing cadmium ions with different concentrations, the detection is carried out under the same detection conditions in example 2. The results of the experiment are shown in FIG. 7.
FIG. 7 schematically shows the fluorescence intensity of the fluorescence response signal of the DNA fluorescent probe in example 5 in a diluted cadmium ion solution.
The fluorescence signal at 485nm gradually increased with increasing concentration of cadmium ions. Aiming at the detection of cadmium ions, the fluorescence intensity of the cadmium ion diluted water sample with different concentrations is similar to that of the cadmium ion sample solution with corresponding concentration shown in fig. 4, and it can be seen that the method of the embodiment of the disclosure can combine the DNA fluorescent probe and the cadmium ions by using the buffer solution with lower concentration, and detect the cadmium ions by using the change of the fluorescence signal before and after the DNA fluorescent probe is combined with the cadmium ions.
Example 6
Adopting a DNA fluorescent probe corresponding to the second sequence, and carrying out fluorescence detection at different temperatures after incubating a pyrene molecular marker DNA fluorescent probe corresponding to the second sequence of 50nM and cadmium ions with 1000nM concentration in a reaction buffer solution (20mM Tris-HCl (pH 7.5) +20mM NaCl) for 10 minutes at 25 ℃.
FIG. 8 is a schematic diagram showing the change in fluorescence intensity of the fluorescence response signal at different detection temperatures when the DNA fluorescent probe in example 6 is bound to the same concentration of cadmium ions.
As can be seen from FIG. 8, the fluorescence intensity of the reaction solution slowly increased with the increase in temperature between 0 ℃ and 10 ℃, and gradually decreased with the increase in temperature when the fluorescence intensity was more than 10 ℃. Therefore, the detection temperature range with proper fluorescence intensity is 4-50 ℃ selected in the embodiment of the disclosure.
In summary, the embodiment of the present disclosure provides a fluorescent molecular method capable of rapidly and sensitively detecting cadmium ions under optimized experimental conditions by reasonably designing an aptamer sequence and using an aptamer with a pyrene molecule labeled at the end of the sequence. The method has the advantages of simple operation, high sensitivity, rapid detection, good stability, high selectivity, low cost and the like. The fluorescence signal rises along with the increase of the concentration of the cadmium ions, the signal change is obvious, the detection limit of the cadmium ions can be lower than 10nM, and the method can be used for sensitively detecting the cadmium ions in the actual water sample.
The above-described embodiments, objects, technical solutions and advantages of the present disclosure are further described in detail, it should be understood that the above-described embodiments are only examples of the present disclosure, and should not be construed as limiting the present disclosure, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.
Claims (10)
1. A DNA fluorescent probe comprising: any one DNA sequence of the first sequence to the fifth sequence, wherein the DNA sequence comprises an aptamer, the 3 'end and the 5' end of which are respectively marked with pyrene molecules:
sequence one: 5'-GGG TTC ACA GTC C-3', respectively;
and (2) sequence II: 5'-CGG GTT CAC AGT CCG-3', respectively;
and (3) sequence III: 5'-ACG GGT TCACAG TCC GT-3', respectively;
and (4) sequence IV: 5'-GAC GGG TTC ACA GTC CGTT-3', respectively;
and a fifth sequence: 5'-CGA CGG GTT CAC AGT CCG TTG-3' are provided.
2. A method for detecting cadmium ions by using the DNA fluorescent probe of claim 1, comprising:
detecting a reaction solution obtained after the DNA fluorescent probe and a cadmium ion standard solution containing different concentrations react at a second preset temperature through a fluorescence detection method at a first preset temperature, and drawing a calibration curve in a preset emission wavelength range;
incubating the DNA fluorescent probe and a sample solution containing cadmium ions in a preset buffer solution at the second preset temperature;
recording the fluorescence intensity of the fluorescence response signal of the sample solution containing cadmium ions in the preset emission wavelength range by a fluorescence detection method at the first preset temperature;
and determining the concentration of cadmium ions in the sample solution containing cadmium ions according to the fluorescence intensity of the fluorescence response signal and the calibration curve.
3. The method of claim 2, wherein the predetermined emission wavelength range is 365-600 nm.
4. The method of claim 2, wherein the predetermined buffer solution comprises a Tris-HCl buffer solution containing sodium ions and magnesium ions.
5. The method according to claim 4, wherein the pH value of the predetermined buffer solution comprises 6 to 9.
6. The method according to claim 5, wherein the predetermined buffer solution contains 0-100 mM of sodium ions and 0-10 mM of magnesium ions.
7. The method of claim 2, wherein the first preset temperature comprises 4-50 ℃.
8. The method of claim 2, wherein the second preset temperature comprises 4-25 ℃.
9. The method of claim 2, wherein the reaction time comprises 0 to 60 minutes.
10. A kit for detecting cadmium ions, comprising: the DNA fluorescent probe of claim 1, a predetermined buffer solution, and a standard containing cadmium ions.
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