CN109799197B - Application of cyanine dye in lead ion detection, lead ion detection kit and method - Google Patents
Application of cyanine dye in lead ion detection, lead ion detection kit and method Download PDFInfo
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Abstract
The invention provides an application of cyanine dye in detection of lead ion concentration, a lead ion detection kit and a method. The method comprises the following steps: (1) mixing the sample with cyanine dye and aptamer; (2) performing spectrum detection on the mixture obtained in the step (1); (3) and determining whether the lead ions exist in the sample or not and the lead ion concentration based on the spectrum detection result. The detection specificity is strong, the sensitivity is high, and the detection limit of the detection on lead ions is 200 nM.
Description
Technical Field
The invention relates to the field of detection, in particular to application of cyanine dye in detection of lead ions, a lead ion detection kit and a lead ion detection method.
Background
Lead is a highly toxic metal pollutant, is a serious hazard to the human body, has an effect on all systems of the body, and particularly has a greater effect on children. Its main toxic effect is anemia, nerve function disturbance, kidney injury, and genital system injury, and the lead content in blood is up to 100mg L-1Can cause a series of problems of nervous system, reproduction, cardiovascular and development disorders and the like. The American environmental protection agency specifies that the maximum allowable amount of lead ions in drinking water is 72 nM. Conventional inspection of trace lead ions in the current environment mainly comprises an atomic absorption method, a fluorescence spectroscopy method, inductive coupling and the like; plasma emission spectroscopy, electrochemical methods, and the like. These methods are cumbersome to operate, require cumbersome pre-treatments, specialized analytical technicians, and expensive instruments, and are not conducive to rapid on-site analysis and detection.
Thus, the detection of lead ions remains to be developed and improved.
Disclosure of Invention
The present application is based on the discovery and recognition by the inventors of the following facts and problems:
the inventor finds that the aptamer (TBA) exists in a G-quadruplex form under the condition of a potassium/sodium ion buffer system, the G-quadruplex can induce cyanine dye to form J-polymer to generate J-polymer signal, and lead ions can form more stable TBA G-quadruplex with the aptamer (TBA), so that the J-polymer is depolymerized, the J-polymer signal is lost, and the concentration of the lead ions can be detected according to the detection of the signal change of the J-polymer. Based on the discovery of the problems, the inventor proposes the use of cyanine dye in quantitative detection of lead ions, proposes the use of cyanine dye in preparation of a kit for detecting the concentration of lead ions, and proposes a method for detecting the concentration of lead ions in a sample. The detection has the advantages of high sensitivity, high specificity, easily obtained reagents, quick detection, no need of special instruments, low detection cost and convenient popularization and application in the industry.
In a first aspect of the invention, the invention proposes the use of a cyanine dye for detecting the concentration of lead ions. According to an embodiment of the invention, the use is the use of cyanine dyes for detecting lead ion concentration. The inventors found that the J-aggregates formed by cyanine dyes are disaggregated by lead ions, and the concentration of lead ions is reflected by detecting the signal change of the J-aggregates. According to the embodiment of the invention, the detection limit of the lead ion concentration reaches 200nmol/L, the reagent is easy to obtain, the detection is rapid, no special instrument is needed, the detection cost is low, and the method is convenient to popularize and apply in the industry.
According to an embodiment of the present invention, the use of the cyanine dye for detecting the lead ion concentration may further include at least one of the following additional technical features:
according to an embodiment of the invention, the use further comprises a nucleic acid aptamer. The inventor finds that the aptamer (TBA) exists in a G-quadruplex form under the condition of a potassium/sodium ion buffer system, can further induce the cyanine dye to form J-aggregates to generate a J-aggregate signal, and when lead ions are added, the lead ions are combined with the aptamer (TBA) to form more stable TBA G-quadruplex, so that the J-aggregates are gradually depolymerized, and further, the lead ions in a sample can be detected with higher sensitivity and higher specificity by detecting the change of the J-aggregate signal.
According to an embodiment of the present invention, the cyanine dye is a compound represented by formula I,
formula I
Wherein: r1Is C1～6Alkyl, phenyl optionally substituted by alkyl, R2、R3、R4And R5Each independently selected from H or C1～6Or R is2And R3Together with the carbon atom to which they are attached form a 5-to 7-membered ring structure, or R4And R5Together with the carbon atom to which they are attached form a 5-to 7-membered ring structure, R6And R7Each independently is C optionally substituted by a sulfonic acid group1～6Alkyl radical, X1And X2Each independently selected from C, O, S, Se or Te; y is a counterion based on R6And R7Charged and selected. According to the embodiment of the invention, the cyanine dye can be more effectively used for detecting the concentration of the lead ions, has specificity on the detection of the concentration of the lead ions, and further improves the detection sensitivity of the concentration of the lead ions.
According to an embodiment of the invention, said C1-C6The alkyl group of (a) is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, n-hexyl or isohexyl.
According to an embodiment of the invention, when R6And R7When alkyl, Y is a halide anion; when R is6And R7At least one of them carries a sulfonate radical and does not contain Y; when R is6And R7When the compounds carry sulfonic acid groups, Y is triethylamine cation.
According to an embodiment of the present invention, the cyanine dye is at least one selected from the group consisting of compounds represented by formula II and formula III below:
in the formula II, the compound is shown in the specification,
formula III.
The inventor finds that the cyanine dyes of the compounds shown in the formulas II and III can be more effectively used for detecting the concentration of lead ions, have specificity on the detection of the concentration of the lead ions, and further improve the detection sensitivity of the concentration of the lead ions.
According to the embodiment of the invention, the mole ratio of the cyanine dye to the aptamer is 1 (0.05-0.1). Through a large number of research experiments, the inventor finds that when the mole ratio of the cyanine dye to the aptamer is not in the range of 1 (0.05-0.1), no J-aggregate signal is generated.
According to an embodiment of the present invention, the nucleic acid aptamer has the sequence of SEQ ID NO: 1.
GGTTGGTGTGGTTGG(SEQ ID NO：1)。
The inventor finds that the aptamer (TBA) exists in a G-quadruplex form under the condition of a potassium/sodium ion buffer system, the cyanine dye can be further induced to form J-aggregates to generate a J-aggregate signal, when lead ions are added, the lead ions are combined with the aptamer (TBA) to form more stable TBA G-quadruplex, the J-aggregates are gradually depolymerized, and the lead ions in a sample can be detected with higher sensitivity and higher specificity by detecting the change of the J-aggregate signal.
In a second aspect of the invention, the invention provides the use of a cyanine dye in the preparation of a kit for detecting the lead ion concentration. The kit provided by the embodiment of the invention has the advantages of high detection sensitivity and strong specificity on lead ions, is easy to obtain reagents, is quick to detect, does not need special instruments, is low in detection cost, and is convenient to popularize and apply in the industry.
According to an embodiment of the present invention, the above-mentioned use may further comprise at least one of the following additional technical features:
according to an embodiment of the present invention, the kit further comprises a nucleic acid aptamer. The inventor finds that the aptamer (TBA) exists in a G-quadruplex form under the condition of a potassium/sodium ion buffer system, can further induce the cyanine dye to form J-aggregates to generate a J-aggregate signal, and when lead ions are added, the lead ions are combined with the aptamer (TBA) to form more stable TBA G-quadruplex, so that the J-aggregates are gradually depolymerized, and further, the lead ions in a sample can be detected with higher sensitivity and higher specificity by detecting the change of the J-aggregate signal.
According to an embodiment of the present invention, the nucleic acid aptamer has the sequence of SEQ ID NO: 1.
According to an embodiment of the present invention, the cyanine dye is a compound represented by formula I,
formula I
Wherein: r1Is C1～6Alkyl, phenyl optionally substituted by alkyl, R2、R3、R4And R5Each independently selected from H or C1～6Or R is2And R3Together with the carbon atom to which they are attached form a 5-to 7-membered ring structure, or R4And R5Together with the carbon atom to which they are attached form a 5-to 7-membered ring structure, R6And R7Each independently is C optionally substituted by a sulfonic acid group1～6Alkyl radical, X1And X2Each independently selected from C, O, S, Se or Te; y is a counterion based on R6And R7Charged and selected. According to the embodiment of the invention, the cyanine dye can be more effectively used for detecting the concentration of the lead ions, has specificity on the detection of the concentration of the lead ions, and further improves the detection sensitivity of the concentration of the lead ions.
According to an embodiment of the invention, said C1-C6The alkyl group of (a) is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, n-hexyl or isohexyl.
According to an embodiment of the invention, when R6And R7When alkyl, Y is a halide anion; when R is6And R7At least one of them carries a sulfonate radical and does not contain Y; when R is6And R7When all carry a sulfonate group, YIs a triethylamine cation.
According to an embodiment of the present invention, the cyanine dye is at least one selected from the group consisting of compounds represented by formula II and formula III below:
in the formula II, the compound is shown in the specification,
formula III.
The inventor finds that the cyanine dye in the kit is a compound shown in a formula II or a formula III, and the kit can be more effectively used for detecting the concentration of lead ions, has higher detection sensitivity and further enhances specificity.
According to an embodiment of the invention, the kit further comprises a buffer.
According to an embodiment of the invention, the buffer is Tris-HCl-145mM Na+-5mM K+. The inventor finds that the buffer solution can maintain the biological activity of the aptamer to the maximum extent, so that lead ions can effectively induce the depolymerization of the J-aggregate of the cyanine dye to cause the signal change of the J-aggregate. Therefore, the buffer solution enables the kit to be used for detecting the lead ion concentration, and the specificity and the sensitivity of the kit are further improved.
According to the embodiment of the invention, the pH value of the buffer solution is 5.0-8.2. The inventors have found that the above pH maximizes the biological activity of the aptamer, and thus lead ions are effective in inducing disaggregation of the cyanine dye J-aggregates, resulting in a change in J-aggregate signal. Thus, the buffer at the pH allows the kit to be used for detecting lead ion concentrations, with further improvements in specificity and sensitivity.
In a third aspect of the present invention, there is provided a method for detecting lead ions in a sample, comprising: (1) mixing the sample with cyanine dye and aptamer; (2) performing spectrum detection on the mixture obtained in the step (1); (3) determining whether lead ions are present in the sample and a lead ion concentration based on a result of the spectroscopic detection. The method provided by the embodiment of the invention has the characteristics of strong specificity and high sensitivity for the detection of lead ions, is easy to obtain a reagent, is quick to detect, does not need special instruments, is low in detection cost, and is convenient to popularize and apply in the industry.
According to an embodiment of the present invention, the method may further include at least one of the following additional features:
according to an embodiment of the present invention, the spectrum detection comprises at least one selected from circular dichroism, ultraviolet spectroscopy, and fluorescence spectroscopy. The inventors have found that the detection of the signal of the J-aggregate by the above method has high sensitivity and enables the specific detection of lead ions.
According to an embodiment of the invention, the spectroscopic detection is circular dichroism detection. The inventor finds that the circular dichroism chromatogram has higher sensitivity for detecting the signal of the J-aggregate and can specifically detect the lead ions.
According to an embodiment of the invention, the wavelength range of the circular dichroism method is 200nm to 700 nm. The inventor finds that when the circular dichroism method is used for detection, the detection of lead ions in a sample with higher specificity and higher sensitivity can be realized by adopting the wavelength of 200 nm-700 nm.
According to an embodiment of the invention, in step (3), the presence of a 650nm peak in the circular dichroism detection result is an indication of the presence of lead ions in the sample. The inventors have surprisingly found that when lead ions are mixed with cyanine dyes and aptamers, and the mixture is subjected to circular dichroism detection, a specific peak can appear at 650nm, and therefore, the existence of a 650nm peak in the detection result of the circular dichroism detection of a sample to be detected is an indication of whether the lead ions exist in the sample.
According to an embodiment of the present invention, the concentration of lead ions in the sample is determined based on the area of the 650nm peak. The inventors found that the area of the specific peak 650nm corresponds to the content of lead ions in the sample.
According to an embodiment of the present invention, the amount of lead ions in the sample is determined by comparing the area of the 650nm peak with a standard curve. The inventors have found that by comparing the area of the 650nm peak with a standard curve, the amount of lead ions in the sample can be determined more accurately.
According to an embodiment of the invention, the standard curve is determined by performing parallel experiments using a plurality of samples containing known amounts of lead ions. The parallel experiment is an experiment performed under the condition that the detection conditions are completely the same. The inventor finds that the test conditions are completely consistent with the conditions of the method for detecting the lead ions, so that a peak area-lead ion concentration standard curve is drawn, and the lead ion content in the sample can be more accurately determined by comparing the area of the 650nm peak with the standard curve.
According to an embodiment of the invention, the pH of the sample is 5.0-8.2. The inventors found that Tris-HCl-145mM Na having a pH of 5.0 to 8.2+-5mM K+The buffer system further improves the sensitivity and specificity of the method for detecting lead ions.
According to an embodiment of the invention, the content of lead ions in the sample is at least 200 nM. The inventors have found that the detection is more accurate and sensitive when the lead ion content of the sample is at least 200 nM. According to the method of the embodiment of the invention, the minimum detection limit is 200 nM.
According to an embodiment of the present invention, the cyanine dye is a compound represented by formula I,
formula I
Wherein: r1Is C1～6Alkyl, phenyl optionally substituted by alkyl, R2、R3、R4And R5Each independently selected from H or C1～6Or R is2And R3Together with the carbon atom to which they are attached form a 5-to 7-membered ring structure, or R4And R5Together with the carbon atom to which they are attached form a 5-to 7-membered ring structure, R6And R7Each independently is C optionally substituted by a sulfonic acid group1～6Alkyl radical, X1And X2Each independently selected from C, O, S, Se or Te; y is a counterion based on R6And R7Charged and selected. According to the embodiment of the invention, the cyanine dye can be more effectively used for detecting the concentration of the lead ions, has specificity on the detection of the concentration of the lead ions, and further improves the detection sensitivity of the concentration of the lead ions.
According to an embodiment of the invention, said C1-C6The alkyl group of (a) is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, n-hexyl or isohexyl.
According to an embodiment of the present invention, the cyanine dye is at least one selected from the group consisting of compounds represented by formula II and formula III below:
in the formula II, the compound is shown in the specification,
formula III.
The inventor finds that the adopted cyanine dye is a compound shown in a formula II or a formula III, and the method has higher detection sensitivity and further enhanced specificity on the lead ion concentration.
According to the embodiment of the invention, the mole ratio of the cyanine dye to the aptamer is 1 (0.05-0.1). The inventor finds that when the mole ratio of the cyanine dye to the aptamer is 1 (0.05-0.1), the obtained circular dichroism detection peak pattern has a characteristic peak at 650nm, and if the mole ratio of the cyanine dye to the aptamer is not in the range of 1 (0.05-0.1), the circular dichroism detection peak pattern does not have a characteristic peak at 650 nm. The method provided by the embodiment of the invention can be used for detecting the lead ions in the sample with higher specificity and higher sensitivity.
According to an embodiment of the present invention, the nucleic acid aptamer has the sequence of SEQ ID NO: 1. The inventors found that a sample containing lead ions was mixed with a cyanine dye, a dye having the sequence of SEQ ID NO: 1, lead ions enable the aptamer to form a G-quadruplex structure, the G-quadruplex further induces cyanine dyes to form J-aggregates, a J-aggregate signal is generated, and the lead ion content in the sample can be determined by detecting the J-aggregate signal. The detection method provided by the embodiment of the invention has the advantages of strong specificity and high sensitivity, and is further improved.
According to the embodiment of the invention, the concentration of the cyanine dye is 4-16 micromoles/liter. The inventor finds that when the concentration of the cyanine dye is 4-16 micromoles/liter, the obtained circular dichroism chromatogram detection peak pattern has a characteristic peak at 650 nm. According to the method provided by the embodiment of the invention, the detection of the lead ions in the sample with high specificity and high sensitivity can be realized.
According to an embodiment of the present invention, the concentration of the cyanine dye is 12 μmol/l. The inventors found that the circular dichroism peak pattern is highest at 650nm when the concentration of the cyanine dye is 12 micromole/liter. The method provided by the embodiment of the invention can realize detection of lead ions in the sample with higher specificity and higher sensitivity.
According to the embodiment of the invention, the concentration of the aptamer is 0.6-2 micromoles/liter. The inventor finds that when the concentration of the aptamer is 0.6-2 micromoles/liter, the obtained circular dichroism chromatogram detection peak image has a characteristic peak at 650 nm. The method provided by the embodiment of the invention has high specificity and high sensitivity for detecting lead ions in the sample.
According to an embodiment of the present invention, the concentration of the nucleic acid aptamer is 1 micromole/liter. The inventors found that the circular dichroism peak pattern is highest at 650nm when the concentration of the aptamer is 1. mu. mol/l. The method provided by the embodiment of the invention has higher specificity and higher sensitivity for detecting lead ions in the sample.
Drawings
FIG. 1 is a statistical chart of the results of a specificity experiment according to an embodiment of the present invention;
FIG. 2 is a detection limit experimental circular dichroism chromatogram according to an embodiment of the invention;
FIG. 3 is a graph of the circular dichroism spectrum of an aptamer concentration screening assay according to an embodiment of the present invention;
FIG. 4 is a circular dichroism spectrum of cyanine dye concentration screening experiments according to embodiments of the present invention;
FIG. 5 is a graph of circular dichroism chromatogram detection of a screening experiment of the concentration ratio range of cyanine dyes and aptamers according to an embodiment of the present invention;
FIG. 6a is a circular dichroism spectrum of lead ion detection range experiment according to an embodiment of the present invention; and
fig. 6b is a detection spectrum of a lead ion linear relation experiment circular dichroism chromatogram according to the embodiment of the invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
In the following examples, the sample storage and detection temperatures were 4 ℃ to ensure stability and consistency of the detection results.
Example 1 specificity experiments
The cyanine dye used in this example has the structure shown in formula II, and the aptamer has the sequence shown in SEQ ID NO: 1, at a concentration of 1 micromolar.
This example is for 8 different metal ions (Li)+、NH4 +、Ca2+、Cu2+、Zn2+、Mg2+、Cd2+、Fe3+) The special-purpose research is carried out,
3 samples with different concentrations are prepared for each metal ion for verification, the concentration of each sample aptamer is 1 micromole/liter, and the concentration of the cyanine dye in methanol solution is 12 micromoles/liter.
The experimental procedure was as follows:
1)10mM Tris-HCl-5mM K+-145mM Na+The buffer solution dissolves inorganic salts (LiCl, NH)4Cl、CaCl2、CuCl2、ZnCl2、MgCl2、CdCl2、FeCl3) Obtaining mother liquor with the corresponding metal ion concentration of 10 micromoles/liter, and respectively and accurately measuring 50, 100 and 200 microliters of the metal ion mother liquor to be used as samples to be detected;
2) respectively adding 50 microliters of aptamer solution with the concentration of 20 micromoles per liter into the 3 metal ion samples to be detected; then, 60 microliters of methanol solution of cyanine dye with 200 micromoles/liter is sequentially added into the 3 samples;
3) respectively supplemented with Tris-HCl-5mM K+-145mM Na+840 microliter, 790 microliter and 690 microliter of buffer solution to obtain metal ions (Li) with the same total volume, the same concentrations of aptamer and cyanine dye+、NH4 +、Ca2+、Cu2+、Zn2+、Mg2+、Cd2+、Fe3+) 3 parts of samples to be detected with different concentrations;
4) detecting a solution to be detected by a circular dichroism method, selecting a wavelength of 200-700nm, analyzing the sample to be detected by a circular dichroism instrument, and storing a sample and performing all operations at 4 ℃;
5) and (4) analyzing results: FIG. 1 was prepared by using the absorption of the circular dichroism spectrum as the ordinate and the wavelength of the circular dichroism spectrum as the abscissa.
The results are shown in FIG. 1, the metal ion (Li)+、NH4 +、Ca2+、Cu2+、Zn2+、Mg2+、Cd2+、Fe3+) The concentration is 500, 1000 and 2000 nanomole/liter, and the characteristic peak signal (650nm) of the cyanine dye J-aggregate does not appear or appears in the detection system.
As can be seen from FIG. 1, the method provided by the invention can specifically detect lead ions and has strong specificity.
Example 2 detection Limit experiment
In this example, the cyanine dye used by the inventors has a structure represented by formula II.
The aptamer has the nucleotide sequence of SEQ ID NO: 1.
In this embodiment, 6 lead ion samples with different concentrations are verified, and the lead ion concentrations of each sample are respectively: 0. 10, 100, 200, 500, 1000 nanomolar.
The experimental procedure was as follows:
1)Tris-HCl-5mM K+-145mM Na+the buffer solution dissolves PbCl2To obtain PbCl with the concentration of 10 mu M2The solution was ready for use, and 10. mu.M of PbCl was added to each of 6 sample bottles2The solution is ready for use at 0, 1, 10, 20, 50, 100 microliter;
2) to the above 6 parts of PbCl2Respectively adding 10 microliters of aptamer 100 micromoles per liter and 60 microliters of cyanine dye methanol solution 200 micromoles per liter into a sample to be detected;
3) respectively supplemented with Tris-HCl-5mM K+-145mM Na+Buffer solution 930, 929, 920, 910, 880 and 830 microliters to prepare 6 parts of samples to be detected, which have the same total volume, the same aptamer and cyanine dye concentration and different lead ion concentrations;
4) and analyzing the sample to be detected by the circular dichroism spectrometer, wherein all operations are carried out at 4 ℃. The wavelength range of the circular dichroism is 200-700 nm.
5) The absorption of the circular dichroism spectrum is used as the ordinate, and the wavelength of the circular dichroism spectrum is used as the abscissa, thereby obtaining FIG. 2.
As can be seen from fig. 2, the detection limit of the method for detecting lead ions in a sample according to the present invention is 200 sodium moles.
Example 3 aptamer concentration screening
The cyanine dye used in this example has the structure shown in formula II, and the aptamer has the sequence shown in SEQ ID NO: 1, the lead ion concentration is 500 nM;
in this embodiment, 10 aptamer samples with different concentrations are verified, and the concentration of each aptamer sample is as follows: 0. 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0 micromoles/liter.
The experimental procedure was as follows:
1)Tris-HCl-5mM K+-145mM Na+buffer solution dilution of Pb (NO)3)2Mother liquor to obtain Pb at a concentration of 5. mu.M2+The solution to be measured is reserved, and Pb is respectively added into 10 sample bottles 2+100 microliter of solution to be detected;
2) to the above 10 parts of Pb (NO)3)2Respectively adding aptamer solutions with the concentration of 100 micromoles per liter into samples to be detected, wherein the volumes of the aptamer solutions are 0, 4, 6, 8, 10, 12, 14, 16, 18 and 20 microliters respectively; sequentially adding 60 microliters of cyanine dye methanol solution with the concentration of 200 micromoles/liter into the 10 samples;
3) respectively supplemented with Tris-HCl-5mM K+-145mM Na+Preparing 10 samples to be detected with the same total volume, the same lead ion and cyanine dye concentration and different aptamer concentrations by using buffer solutions 840, 836, 834, 832, 830, 828, 826, 824, 822 and 820 microliters;
4) detecting a solution to be detected by a circular dichroism method, selecting the wavelength of 200-700nm, analyzing the sample to be detected by a circular dichroism instrument, and performing all operations at 4 ℃;
5) and (4) analyzing results: FIG. 3 was prepared by using the absorption of the circular dichroism spectrum as the ordinate and the wavelength of the circular dichroism spectrum as the abscissa.
As can be seen from FIG. 3, when the aptamer concentration is 0.6-2 micromol/l, a J-aggregate characteristic peak signal (650nm) appears in the detection system, and when the aptamer concentration is 1 micromol/l, the circular dichroism signal (CD) intensity is strongest, so that the optimal aptamer concentration is determined to be 1 micromol/l.
Example 4 Cyanine dye concentration screening
The cyanine dye used in this example has the structure shown in formula II, and the aptamer has the sequence shown in SEQ ID NO: 1, the lead ion concentration is 500 nM;
in this example, 9 samples of cyanine dyes (methanol solutions) with different concentrations were tested, and the concentrations of the cyanine dyes in each sample were: 0.1, 2, 4, 8, 10, 12, 16, 20 micromoles/liter.
The experimental procedure was as follows:
1)Tris-HCl-5mM K+-145mM Na+buffer solution dilution of Pb (NO)3)2Mother liquor to obtain Pb at a concentration of 5. mu.M2+The solution to be measured is reserved, and Pb is respectively added into 10 sample bottles 2+100 microliter of solution to be detected;
2) adding 9 parts of Pb2+Respectively adding 10 microliters of aptamer solutions of 100 micromoles/liter into a sample to be detected; then adding 200 micromole/liter of methanol solution of cyanine dye into the 9 samples in turn, wherein the methanol solution of cyanine dye is 0, 5, 10, 20, 40, 50, 60, 80 and 100 microliters;
3) respectively supplementing Tris-HCl buffer solutions 890, 885, 880, 870, 850, 840, 830, 810 and 790 microliters to prepare 9 parts of samples to be detected with the same total volume, the same aptamer and lead ion concentration and different cyanine dye concentrations;
4) detecting a solution to be detected by a circular dichroism method, selecting the wavelength of 200-700nm, analyzing the sample to be detected by a circular dichroism spectrometer, and performing all operations at 4 ℃;
5) and (4) analyzing results: FIG. 4 was prepared by using the absorption of the circular dichroism spectrum as the ordinate and the wavelength of the circular dichroism spectrum as the abscissa.
As can be seen from FIG. 4, when the concentration of the cyanine dye is 4-16 micromoles/liter, a J-aggregate characteristic peak signal (650nm) appears in the detection system, and when the concentration of the cyanine dye is 12 micromoles/liter, the intensity of a circular dichroism (JASCO J-815) signal (CD) is strongest, so that the optimal concentration of the cyanine dye is determined to be 12 micromoles/liter.
Example 5 concentration ratio Range screening of cyanine dyes to aptamers
The cyanine dye used in this example has the structure shown in formula II, and the aptamer has the sequence shown in SEQ ID NO: 1, the lead ion concentration is 500nM, and the cyanine dye (methanol solution) concentration is 12 micromole/L.
In this embodiment, 10 samples (cyanine dye/aptamer) with different concentration ratios are verified, and the concentrations of the aptamer in each sample are: 0. 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2.0 micromoles/liter.
The experimental procedure was as follows:
1)Tris-HCl-5mM K+-145mM Na+buffer solution dilution of Pb (NO)3)2Mother liquor to obtain Pb at a concentration of 5. mu.M2+The solution to be measured is reserved, and Pb is respectively added into 10 sample bottles 2+100 microliter of solution to be detected;
2) adding 10 parts of Pb2+Respectively adding 60 microliters of methanol solution of cyanine dye with the concentration of 200 micromoles/liter into a sample to be detected; sequentially adding 100 micro mol/ L aptamer solutions 0, 4, 6, 8, 10, 12, 14, 16, 18 and 20 micro L into the 10 samples;
3) respectively supplemented with Tris-HCl-5mM K+-145mM Na+Preparing 10 parts of samples to be detected with the same total volume, the same cyanine dye and lead ion concentration and different aptamer concentrations by using buffer solutions 840, 836, 834, 832, 830, 828, 826, 824, 822 and 820 microliters;
4) detecting a solution to be detected by a circular dichroism method, selecting the wavelength of 200-700nm, analyzing the sample to be detected by a circular dichroism spectrometer, and performing all operations at 4 ℃;
5) and (4) analyzing results: FIG. 5 was prepared by using the absorption of the circular dichroism spectrum as the ordinate and the wavelength of the circular dichroism spectrum as the abscissa.
As can be seen from FIG. 5, the concentration ratio of cyanine dye to aptamer is in the range of 12. mu.M (0.6-2.0). mu.M (1 (0.05-0.1)), and the ratio is out of this range, and no J-aggregate signal is observed.
Example 6 test of detection Range and Linear relationship
In this example, the cyanine dye used by the inventors has a structure represented by formula II.
The aptamer has the nucleotide sequence of SEQ ID NO: 1.
In this embodiment, 10 lead ion samples with different concentrations are verified, and the lead ion concentrations of each sample are respectively: 0. 50, 100, 200, 500, 1000, 1500, 2000, 3000, 5000 nM.
The experimental procedure was as follows:
1)Tris-HCl-5mM K+-145mM Na+buffer solution dilution of Pb (NO)3)2Mother liquor to obtain Pb with a concentration of 10. mu.M2 +The solution to be measured is reserved, and Pb is respectively added into 10 sample bottles 2+0, 5, 10, 20, 50, 100, 150, 200, 300 and 500 microliter of solution to be detected;
5) adding 9 parts of Pb2+Respectively adding 10 microliters of aptamer 100 micromoles per liter and 60 microliters of cyanine dye methanol solution 200 micromoles per liter into a sample to be detected;
6) respectively supplementing Tris-HCl buffer solutions 930, 925, 920, 910, 880, 830, 780, 730, 630 and 430 microliters to prepare 9 parts of samples to be detected with the same total volume, the same aptamer and cyanine dye concentration and different lead ion concentrations;
7) and analyzing the sample to be detected by the circular dichroism spectrometer, wherein all operations are carried out at 4 ℃. The wavelength range of the circular dichroism is 200-700 nm.
5) FIG. 6a and FIG. 6b were prepared using the absorption of the circular dichroism spectrum as the ordinate and the wavelength of the circular dichroism spectrum as the abscissa.
As can be seen from FIGS. 6a and 6b, the linear range of the method for detecting lead ions proposed by the present invention is 200-2000 nanomolar.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> chemical research institute of Chinese academy of sciences
Application of <120> cyanine dye in lead ion detection, lead ion detection kit and method
<130> PIDC3175169
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 15
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence of nucleic acid aptamer
<400> 1
ggttggtgtg gttgg 15
Claims (8)
1. A method of detecting the concentration of lead ions in a sample, comprising:
(1) mixing the sample with cyanine dye and aptamer;
(2) performing spectrum detection on the mixture obtained in the step (1);
(3) determining whether lead ions exist in the sample and the lead ion concentration based on the result of spectrum detection;
wherein the sample is dissolved in Tris-HCl-5mM K+-145mM Na+In the buffer solution, the solution is added with a solvent,
the concentration of the aptamer is 0.6-2 micromoles/liter, and the aptamer has the nucleotide sequence shown in SEQ ID NO: 1, or a nucleotide sequence shown in the specification,
the concentration of the cyanine dye is 4-16 micromoles/liter, and the cyanine dye is at least one of compounds shown in the following formulas II and III:
the molar ratio of the cyanine dye to the aptamer is 1 (0.05-0.1);
the spectroscopic detection is a circular dichroism detection, and in the step (3), the existence of a 650nm peak in the detection result of the circular dichroism is an indication of the existence of lead ions in the sample.
2. The method of claim 1, wherein the wavelength range of the circular dichroism method is 200nm to 700 nm.
3. The method of claim 1, wherein the concentration of lead ions in the sample is determined based on the area of the 650nm peak.
4. The method of claim 1, wherein the amount of lead ions in the sample is determined by comparing the area of the 650nm peak to a standard curve,
the standard curve was determined by performing parallel experiments using a plurality of samples containing known amounts of lead ions.
5. The method of claim 1, wherein the sample has a pH of 5.0 to 8.2.
6. The method of claim 1, wherein the concentration of lead ions in the sample is at least 200 nM.
7. The method of claim 1, wherein the cyanine dye is at a concentration of 12 μm/l.
8. The method of claim 1, wherein the concentration of the aptamer is 1 micromole/liter.
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| CN110714011B (en) * | 2019-11-11 | 2023-12-08 | 暨南大学 | Lead ion nucleic acid aptamer, electrochemical sensor and preparation method |
| CN112345749B (en) * | 2020-10-16 | 2024-03-29 | 吉林烟草工业有限责任公司 | Nucleic acid molecule embedded organic semiconductor small molecule aggregate, preparation method and application thereof in heavy metal ion detection |
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