CN109839359B - Kit for detecting concentration of Pb2+ and detection method - Google Patents
Kit for detecting concentration of Pb2+ and detection method Download PDFInfo
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Abstract
The invention provides a method for detecting Pb2+The kit comprises the following components: cyanine dye, Pb2+Standard solution, DNA bisChain, acidic buffer solution and neutral buffer solution. The detection method comprises the following steps: drawing Pb under neutral and acidic conditions respectively2+Detecting a standard curve; mixing a sample to be detected, a DNA double strand and cyanine dye, incubating the mixed solution at 20-40 ℃ for 15-25min, measuring the absorbance at 448nm or the fluorescence intensity at 616nm, judging the acidity of the sample to be detected according to the detected absorbance or fluorescence intensity, and combining with Pb under neutral or acidic conditions2+Calculating Pb by using the detection standard curve2+And (4) concentration. The kit has the advantages of simple system, high detection method sensitivity, low detection limit and high accuracy, and can reflect the acidity condition of a sample to be detected, thereby providing a simple method for judging the migration rate of lead ions.
Description
Technical Field
The invention belongs to the technical field of environmental monitoring, and particularly relates to a method for detecting Pb2+A kit for detecting concentration and a detection method.
Background
The metal lead has excellent physical and chemical properties, and people find the characteristics of easy exploitation, softness and easy processing before thousands of years. At present, lead is widely used in the industries of alloys, building materials, batteries and the like. The toxicity of lead has been noted since the late 19 th century, mainly manifested as neurotoxicity, cardiovascular toxicity and reproductive toxicity, and is particularly serious in children. The toxicological mechanism is that the normal configuration of the ring-breaking enzyme leads the enzyme to lose the catalytic function; interfere the metabolism of metal ions in human body and further influence the normal physiological function of cells. As an important pollutant in environmental monitoring, the monitoring of lead in soil, wastewater and industrial waste has great significance for human health and ecological balance.
It has been shown that Pb is contained in soil2+The damage of (a) is not only dependent on its concentration but also on the migration rate of the lead ions. Lead in the soil can be absorbed through root systems in the growth process of agricultural products, and the migration rateThe larger the risk, the higher the lead enrichment in the plant. The pH value is one of important factors influencing the migration rate, and the analysis of the pH environment of the lead ions has practical significance when the lead ions in the soil are monitored.
The traditional instrument analysis methods, such as an Atomic Absorption Spectrometry (AAS), an electron coupled plasma method (ICP), an electron coupled plasma mass spectrometry (ICP-MS) and the like, have high selectivity and sensitivity for detecting lead ions. With the development of biotechnology, scientists have developed various convenient and fast biosensing devices suitable for on-site analysis, and many biosensing devices have been applied to the detection of heavy metal ions. The method has strong detection capability on the concentration of lead ions, but the existing instrument analysis method and the existing biosensor cannot well evaluate other pollution related information (such as pH value) of the lead ions in the sample, and certain limitations exist in the actual sample analysis.
In summary, it is one of the current trends in the field of analytical science to develop a detection method capable of measuring a target in a sample and analyzing other information of the sample.
Disclosure of Invention
In view of the above problems in the prior art, the present invention provides a method for detecting Pb2+The kit has the advantages of simple system, high sensitivity of the detection method, low detection limit and high accuracy, and can reflect the acidity condition of a sample to be detected.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
for detecting Pb2+A kit of concentrations comprising the following components: cyanine dye, Pb2+Standard solution, DNA double strand, acid buffer solution and neutral buffer solution;
the DNA double strand comprises a DNA single strand capable of forming a G-quadruplex structure and a DNA single strand which is complementary with the DNA single strand and capable of forming an i-motif structure;
the structural formula of the cyanine dye is as follows:
wherein R is1Is C1-C6Alkyl, phenyl, alkyl substituted phenyl of (a); r2、R3、R4And R5Independently selected from H or C1-C6Or R is2And R3Together with the carbon atom to which they are attached form a five-to seven-membered ring structure, or R4And R5Form a five-to seven-membered ring structure together with the carbon atoms to which they are attached; r6And R7Independently selected from C1-C6Alkyl groups of (a); y is halogen; x1，X2Independently selected from C, O, S, Se, Te.
Further, the structural formula of the cyanine dye is as follows:
further, the sequence of the DNA single strand capable of forming a G-quadruplex is: 5' -Ga1Yb1Ga2Yb2Ga3Yb3Ga4Yb4-3'; wherein a1-a4 represents the number of G and is an integer of more than 2, b1-b4 represents the number of Y and is an integer of 0-3, and Y represents A, T or C base.
Further, the sequence of the DNA single strand capable of forming a G-quadruplex is: 5'-GGTGGTGGTGGT-3', 5'-GGTGGTGGTGGTGTTGGTGGTGGTGGTTT-3', 5'-GGGTGGGTGGGTGGG-3', 5'-GGGATTGGGATTGGGATTGGGATT-3', 5'-GGTTGGTGTGGTTGG-3', or 5'-TGAGGGTGGGGAGGGTGGGGAA-3'.
Further, the sequence of the DNA single strand capable of forming i-motif is: 5' -Cn1Xm1Cn2Xm2Cn3Xm3Cn4Xm4-3'; whereinN1-n4 represent the number of C and are integers of 2 or more, m1-m4 represent the number of X and are integers of 0 to 3, and X represents A, T or G base.
Further, the sequence of the DNA single strand capable of forming i-motif is: 5'-CCACCACCACCACAACCACCACCAAA-3', 5'-CCACCACCACCACAACCACCACCACCAAA-3', 5'-CCCACCCACCCACCC-3', 5'-CCCTAACCCTAACCCTAACCCTAA-3', 5'-CCAACCACACCAACC-3', or 5'-ACTCCCACCCCTCCCACCCCTT-3'.
Further, Pb2+The standard solution is PbCl2A solution; the acid buffer solution is Tris-HAc buffer solution with the concentration of 10mM and the pH value of 4.0; the neutral buffer solution was Tris-HAc buffer solution with a concentration of 10mM and a pH of 7.0.
Pb detection by adopting kit2+A method of concentration comprising the steps of:
(1) mixing neutral buffer solution with Pb2+Mixing the standard solution, DNA double strand and cyanine dye, incubating the mixed solution at 20-40 deg.C for 15-25min, measuring its absorbance at 448nm, and plotting Pb under neutral condition2+Detecting a standard curve;
(2) mixing the acidic buffer solution with Pb2+Mixing the standard solution, DNA double strand and cyanine dye, incubating the mixed solution at 20-40 deg.C for 15-25min, measuring its fluorescence intensity at 616nm, and plotting Pb under acidic condition2+Detecting a standard curve;
(3) mixing a sample to be detected, a DNA double strand and cyanine dye, incubating the mixed solution at 20-40 ℃ for 15-25min, measuring the absorbance at 448nm or the fluorescence intensity at 616nm, if the absorbance is detected, indicating that the sample to be detected is neutral, and determining Pb under the neutral condition according to the combination of Pb and cyanine dye2+Detecting the standard curve, and calculating Pb2+Concentration; if stronger fluorescence intensity is detected, the sample to be detected is slightly acidic, and Pb is combined under the acidic condition2+Detecting the standard curve, and calculating Pb2+And (4) concentration.
The above-mentioned strong fluorescence intensity means 50X 10-9mol/L of PbCl2The standard solution was used as a control, and the measurement was carried out according to the above-mentioned detection methodThe fluorescence intensity at 616nm is higher than 20% of the fluorescence intensity, and is lower than 20% of the fluorescence intensity, so that the fluorescence intensity is very weak and can be ignored, namely, no fluorescence intensity at 616 nm.
When the absorbance value is less than 0.02, it is negligible, indicating no absorbance.
Further, the final concentration of cyanine dye in the detection process is 4 × 10-6mol/L。
Further, the concentration of DNA double strand during the detection process was 2X 10-6mol/L。
The invention provides a method for detecting Pb2+The concentration kit and the detection method have the following beneficial effects:
(1) the sensitivity is high, and the specific expression is that the lead ions and the hydrogen ions have obvious regulation and control functions on the secondary structure of the DNA, namely the secondary structure of the DNA can generate structural transformation under the condition of low-concentration lead ions or hydrogen ions, and meanwhile, the cyanine dye has good identification reaction capability on different secondary structures of the DNA and can convert the change of the structure of the DNA into an identifiable optical signal.
(2) The detection method of the invention is used for detecting Pb2+While the concentration is high, the analysis of other related information of the sample to be detected (such as the acidity condition of the sample to be detected) can be realized, the method is particularly embodied in that hydrogen ions are used for regulating and controlling a DNA secondary structure in the method to generate a detectable signal, and simultaneously, the hydrogen ions can also directly measure the acidity of the sample, namely, the mixed solution has relatively strong fluorescence intensity at 616nm, which indicates that the sample to be detected is slightly acidic, and if the mixed solution has relatively large light absorption value at 448nm, which indicates that the sample to be detected is slightly neutral, so that the Pb can be roughly judged2+The acidity of the environment, and further provides a simple method for judging the migration rate of the lead ions.
Drawings
FIG. 1 shows Pb produced under neutral conditions2+And (5) detecting a standard curve graph.
FIG. 2 shows Pb produced under acidic conditions2+And (5) detecting a standard curve graph.
Detailed Description
The invention provides a method for detecting Pb2+A kit of concentrations comprising the following components:
(1) the reagent I is cyanine dye solution for recognizing special DNA secondary structure in the system and converting it into detectable fluorescent signal with final concentration of 4 × 10-6mol/L。
The structural formula of the reagent I (cyanine dye) is as follows:
wherein R is1Is C1-C6Alkyl, phenyl, alkyl substituted phenyl of (a); r2、R3、R4And R5Independently selected from H or C1-C6Or R is2And R3Together with the carbon atom to which they are attached form a five-to seven-membered ring structure, or R4And R5Form a five-to seven-membered ring structure together with the carbon atoms to which they are attached; r6And R7Independently selected from C1-C6Alkyl groups of (a); y is halogen; x1，X2Independently selected from C, O, S, Se, Te.
(2) Reagent II was Tris-HAc buffer solution at a concentration of 10mM and pH 7.0, and was designated as buffer 1.
(3) Reagent III was Tris-HAc buffer at 10mM pH 4.0, and was designated buffer 2.
(4) The reagent IV is PbCl2Solution (Pb)2+Standard solution) for inducing the G-rich sequence to form a specific structure of G-quadruplex and reacting with cyanine dye, wherein the final concentration is 0-3 × 10-6mol/L。
(5) Reagent V is DNA single strand (Pb) rich in G base2+Response sequence) for the formation of a particular structure of G-quadruplexes, with a final concentration of 2X 10 during the detection-6mol/L。
The G-rich sequence has the general formula: 5' -Ga1Yb1Ga2Yb2Ga3Yb3Ga4Yb4-3′；
Wherein a1-a4 represents the number of G and is an integer of more than 2, b1-b4 represents the number of Y and is an integer of 0-3, and Y represents A, T or C base.
(6) Reagent VI is a DNA single strand rich in C base (H)+Response sequence) for forming an i-motif structure at a final concentration of 2X 10 during the detection-6mol/L。
The C-rich sequence has the general formula: 5' -Cn1Xm1Cn2Xm2Cn3Xm3Cn4Xm4-3′；
Wherein n1-n4 represent the number of C and are an integer of 2 or more, m1-m4 represent the number of X and are an integer of 0-3, and X represents A, T or G base.
(7) The reagent VII is a DNA complementary strand formed by a DNA single strand rich in G base and a DNA single strand rich in C base according to the volume ratio of 1:1, and the final concentration is 2x 10 in the detection process-6mol/L。
The detection method is concretely shown in the following examples:
example 1 Pb in neutral conditions2+Drawing of detection standard curve
The following treatments were performed on 9 EP tubes, numbered 1, 2, 3, 4,5, 6, 7, 8 and 9, respectively:
to EP tube No. 1, 940. mu.L of reagent II was added, followed by 20. mu.L of reagent II at a concentration of 100X 10-6mixing mol/L reagent VII, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6The volume of the solution is 1mL by using a mol/L reagent I to obtain a solution 1;
in EP tube No. 2, 937.5. mu.L of reagent II was added, followed by 20. mu.L of 100X 10-6mol/L of reagent VII and 2.5. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6The volume of the solution is 1mL by mol/L reagent I, and a solution 2 is obtained;
add 935. mu.L of reagent II to EP tube No. 3, followed by 20. mu.L of 100X 10-6mol/L of reagent VII and 5. mu.L of 100X 10-6The mol/L reagent IV is mixed evenly at 25 DEG CIncubating for 20min, and adding 40 μ L of 100 × 10-6A mol/L reagent I is used for ensuring that the volume of the solution is 1mL to obtain a solution 3;
in EP tube 4, 932.5. mu.L of reagent II was added, followed by 20. mu.L of 100X 10-6mol/L of reagent VII and 7.5. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6The volume of the solution is 1mL by mol/L reagent I, and a solution 4 is obtained;
to EP 5 tube 930. mu.L of reagent II was added followed by 20. mu.L of 100X 10-6mol/L of reagent VII and 10. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6The volume of the solution is 1mL by mol/L reagent I, and a solution 5 is obtained;
in EP 6 tube 927.5. mu.L of reagent II was added, followed by 20. mu.L of 100X 10-6mol/L of reagent VII and 12.5. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6The volume of the solution is 1mL by mol/L reagent I, and a solution 6 is obtained;
to EP tube 7, 925. mu.L of reagent II was added, followed by 20. mu.L of 100X 10-6mol/L of reagent VII and 15. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6The volume of the solution is 1mL by mol/L reagent I, and a solution 7 is obtained;
to EP 8 tube 922.5. mu.L of reagent II was added, followed by 20. mu.L of 100X 10-6mol/L of reagent VII and 17.5. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6The volume of the solution is 1mL by mol/L reagent I, so that a solution 8 is obtained;
add 920. mu.L of reagent II to EP 9. mu.L, and add 20. mu.L of reagent II to the tube at a concentration of 100X 10-6mol/L of reagent VII and 20. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6mol/L reagentI, enabling the volume of the solution to be 1mL to obtain a solution 9;
the solutions were mixed well and incubated at 25 ℃ for 20min, and the absorbance at 448nm of each solution was measured. With Pb in each solution2+The final concentration of (A) is plotted on the abscissa and the absorbance at each concentration is plotted on the ordinate as a standard curve, and the results are shown in FIG. 1 and are in the range of 0.5 to 1.75X 10-6A linear fit was performed over the concentration range of M.
Coefficient of linearity R20.99963, indicating that at Pb2+Within the concentration range, the detected absorbance and Pb2+There is a good linear relationship between concentrations.
By linear fitting, the regression equation is obtained as y-0.05363 x-0.01018. Wherein x is Pb in the system2+Y is corresponding to Pb2+Absorbance at concentration. Calculating corresponding Pb according to the absorbance obtained by detection2+And (4) concentration.
Example 2 Pb in acidic conditions2+Drawing of detection standard curve
Taking 11 EP tubes, which are respectively numbered as 1, 2, 3, 4,5, 6, 7, 8, 9, 10 and 11, respectively carrying out the following treatment:
to EP tube No. 1, 940. mu.L of reagent III was added, followed by 20. mu.L of reagent III at a concentration of 100X 10-6mixing mol/L reagent VII, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6The volume of the solution is 1mL by using a mol/L reagent I to obtain a solution 1;
to EP tube 2, 939.5. mu.L of reagent III was added, followed by 20. mu.L of 100X 10-6mol/L of reagent VII and 0.5. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6The volume of the solution is 1mL by mol/L reagent I, and a solution 2 is obtained;
to EP tube 3 was added 939. mu.L of reagent III, followed by 20. mu.L of 100X 10-6mol/L of reagent VII and 1. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6A mol/L reagent I is used for ensuring that the volume of the solution is 1mL to obtain a solution 3;
in EP tube 4, 937.5. mu.L of reagent III was added, followed by 20. mu.L of 100X 10-6mol/L of reagent VII and 2.5. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6The volume of the solution is 1mL by mol/L reagent I, and a solution 4 is obtained;
add 935. mu.L of reagent III to EP tube No. 5, followed by 20. mu.L of 100X 10-6mol/L of reagent VII and 5. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6The volume of the solution is 1mL by mol/L reagent I, and a solution 5 is obtained;
to EP tube 6, 932.5. mu.L of reagent III was added, followed by 20. mu.L of 100X 10-6mol/L of reagent VII and 7.5. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6The volume of the solution is 1mL by mol/L reagent I, and a solution 6 is obtained;
to EP 7 tube 930. mu.L of reagent III was added followed by 20. mu.L of 100X 10-6mol/L of reagent VII and 10. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6The volume of the solution is 1mL by mol/L reagent I, and a solution 7 is obtained;
to EP 8 tube 927.5. mu.L of reagent III was added, followed by 20. mu.L of 100X 10-6mol/L of reagent VII and 12.5. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6The volume of the solution is 1mL by mol/L reagent I, so that a solution 8 is obtained;
to EP 9 tube 925. mu.L of reagent III was added followed by 20. mu.L of 100X 10-6mol/L of reagent VII and 15. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6A mol/L reagent I, the volume of the solution is 1mL, and a solution 9 is obtained;
920 mul of reagent III was added to EP tube 10,then 20. mu.L of 100X 10-6mol/L of reagent VII and 20. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6The volume of the solution is 1mL by mol/L of reagent I, so that a solution 10 is obtained;
to EP tube 11, 910. mu.L of reagent III was added, followed by 20. mu.L of 100X 10-6mol/L of reagent VII and 30. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6The volume of the solution is 1mL by mol/L of reagent I, so that a solution 10 is obtained;
the solutions were mixed well and incubated at 25 ℃ for 20min, and the fluorescence intensity of each solution at 616nm was measured. With Pb in each solution2+The final concentration of (A) is plotted on the abscissa and the fluorescence intensity at each concentration is plotted on the ordinate as a standard curve, and the results are shown in FIG. 2 and are in the range of 0.5 to 1.5X 10-6A linear fit was performed over the concentration range of M.
Coefficient of linearity R20.99897, indicating that at Pb2+Within the concentration range, the obtained fluorescence intensity and Pb are detected2+There is a good linear relationship between concentrations.
By linear fitting, the regression equation is obtained as y-141.05012 x + 24.97509. Wherein x is Pb in the system2+Y is corresponding to Pb2+Fluorescence intensity at concentration. The corresponding Pb can be calculated according to the fluorescence intensity obtained by detection2+And (4) concentration.
Example 3
Pb under the conditions of example 12+Concentration determination, verification of Pb detection by the detection method2+The ability to be concentrated.
Taking 3 EP tubes, which are marked as 1, 2 and 3, and respectively carrying out the following treatment:
to EP tube 1, 932.5. mu.L of reagent II was added, followed by 20. mu.L of 100X 10-6mol/L of reagent VII and 7.5. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6The volume of the solution is 1mL by mol/L of reagent I to obtain a solutionLiquid 1;
to EP tube 2, 930. mu.L of reagent II was added, followed by 20. mu.L of 100X 10-6mol/L of reagent VII and 10. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6The volume of the solution is 1mL by mol/L reagent I, and a solution 2 is obtained;
to EP tube 3, 922.5. mu.L of reagent II was added, followed by 20. mu.L of 100X 10-6mol/L of reagent VII and 17.5. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6A mol/L reagent I is used for ensuring that the volume of the solution is 1mL to obtain a solution 3;
the solutions were mixed well and incubated at 25 ℃ for 20min, and the absorbance at 448nm of each solution was measured. Substituting each absorbance into Pb2+In the equation of the detection standard curve, the corresponding Pb under the absorbance is calculated according to the following formula2+Concentration:
y＝0.05363x-0.01018
the measurements were repeated 2 times and the mean concentration and Relative Standard Deviation (RSD) were further calculated, the results of which are shown in Table 1.
TABLE 1 neutral Condition Pb2+Statistical results of recovery test
As can be seen from the statistical calculation results, the method is used for Pb2+Has better detection capability.
Example 4
Pb under the conditions of example 22+Concentration determination, verification of Pb detection by the detection method2+The ability to be concentrated.
Taking 3 EP tubes, which are marked as 1, 2 and 3, and respectively carrying out the following treatment:
add 935. mu.L of reagent III to EP tube No. 1, then add 20. mu.L of 100X 10-6mol/L of reagent VII and 5. mu.L of 100X 10-6mixing the reagent IV with mol/L, incubating at 25 deg.C for 20min, and adding 40 μ LIs 100 x 10-6The volume of the solution is 1mL by using a mol/L reagent I to obtain a solution 1;
to EP tube 2, 932.5. mu.L of reagent III was added, followed by 20. mu.L of 100X 10-6mol/L of reagent VII and 7.5. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6The volume of the solution is 1mL by mol/L of reagent I to obtain a solution 2
To EP tube 3, 930. mu.L of reagent III was added, followed by 20. mu.L of 100X 10-6mol/L of reagent VII and 10. mu.L of 100X 10-6mixing with mol/L reagent IV, incubating at 25 deg.C for 20min, adding 40 μ L of 100 × 10-6A mol/L reagent I is used for ensuring that the volume of the solution is 1mL to obtain a solution 3;
the solutions were mixed well and incubated at 25 ℃ for 20min, and the fluorescence intensity of each solution at 616nm was measured. Substituting each fluorescence intensity into Pb2+In the equation of the detection standard curve, the corresponding Pb under the fluorescence intensity is calculated according to the following formula2+Concentration:
y＝141.05012x+24.97509
the measurements were repeated 2 times and the mean concentration and Relative Standard Deviation (RSD) were further calculated, the results of which are shown in Table 2.
TABLE 2 acidic Condition Pb2+Statistical results of recovery test
As can be seen from the statistical calculation results, the method is used for Pb2+Has better detection capability.
Wherein the cyanine dyes of reagent I used in examples 1 to 4 have the following structural formula:
the DNA double strand is prepared by mixing the DNA single strand rich in the C sequence and the DNA single strand rich in the G sequence in equal volume, heating at 90 ℃ for 2-15min, and naturally cooling to room temperature;
wherein the concentration of the single-stranded DNA rich in C sequence is 50X 10-6mol/L, the sequence is as follows: 5'-CCACCACCACCACAACCACCACCAAA-3', respectively;
the concentration of the G-rich sequence of the DNA single strand is 50X 10-6mol/L with the sequence 5'-GGTGGTGGTGGT-3'.
However, the present invention is not limited to the above cyanine dye structure and DNA sequence, and can also be cyanine dyes and DNA sequences of the following structures:
the cyanine dye structure:
DNA sequence:
5′-GGTGGTGGTGGTGTTGGTGGTGGTGGTTT-3′；
5′-CCACCACCACCACAACCACCACCACCAAA-3′；
5′-GGGTGGGTGGGTGGG-3′；
5′-CCCACCCACCCACCC-3′；
5′-CCCTAACCCTAACCCTAACCCTAA-3′；
5′-GGGATTGGGATTGGGATTGGGATT-3′；
5′-GGTTGGTGTGGTTGG-3′；
5′-CCAACCACACCAACC-3′；
5′-TGAGGGTGGGGAGGGTGGGGAA-3′；
5′-ACTCCCACCCCTCCCACCCCTT-3′。
EXAMPLE 5 Pb in aqueous samples2+Detection of concentration
Taking electroplating wastewater, filtering to remove impurities, using the electroplating wastewater as a sample to be detected, and respectively detecting Pb in the sample to be detected by the detection method, the traditional atomic absorption method and the ultraviolet-visible spectrophotometry2+，Pb2+The results of the concentration measurements are shown in Table 3:
TABLE 3 Pb2+Concentration detection result
| The detection method of the invention | Atomic absorption method | Ultraviolet-visible spectrophotometry | |
| Sample 1 | 0.73mg/L | 0.50mg/L | 0.42mg/L |
| Sample 2 | 0.70mg/L | 0.41mg/L | 0.28mg/L |
| Sample 3 | 0.74mg/L | 0.35mg/L | 0.32mg/L |
As can be seen from Table 3, the method of the present invention was used to detect Pb in electroplating wastewater2+The concentration is obviously higher than that of the traditional atomic absorption method and ultraviolet-visible spectrophotometry, and the difference between parallel samples is relatively small.
Further, Pb was measured2+When the concentration is higher than the preset value, the mixed solution has fluorescence intensity at 616nm, which indicates that the electroplating wastewater is acidic, and if the mixed solution has an absorbance value at 448nm, the electroplating wastewater is neutral. Meanwhile, the electroplating wastewater is determined by adopting pH test paper and the like, and the determination result is the same as the determination result of the pH test paper.
If Pb in the electroplating wastewater2+The concentration is very high, and the Pb of the electroplating wastewater can be accurately measured only by diluting the electroplating wastewater2+Concentration, when dilution times are increased in sequence, i.e. Pb in the waste water2+When the concentration is reduced in sequence, the traditional atomic absorption method and the ultraviolet-visible spectrophotometry are adopted to detect the Pb in the wastewater2+The detection method can still detect the Pb, and the wastewater is continuously diluted, and when the dilution multiple is increased by 10 times again, the detection method can still detect a small amount of Pb2 +Continuously diluting for 2 times and 4 times, and detecting no Pb when diluting for 4 times2+The sensitivity of the detection method is obviously higher than that of the traditional method, and the detection limit is low.
The detection method of the invention not only can detect Pb in electroplating wastewater2+The concentration of the solid can be detected by detecting other aqueous solution samples, such as urban domestic wastewater, other industrial wastewater, serum, urine and the like, and the Pb in the solid can be detected2+Concentration, such as soil, etc.
The kit and the method for detecting Pb in the sample to be detected2+At the concentration, Pb can be reflected2+The sample to be detected is acidic or neutral, and a simple method is provided for judging the migration rate of the lead ions.
Note: the cyanine dyes of the present invention have the following structural formula, molecular formula and nomenclature:
1、
structural formula (xvi):
the molecular formula is: c38H45N3O6S4；
Naming: 3,3 ' -bis (3-sulfo-propyl) -4,5,4 ', 5 ' -diphenyl-9-methyl-tricarbobenzothiazolocyanine dye triethylamine salt.
2、
Structural formula (xvi):
the molecular formula is: c39H57BrN2O2；
Naming: 3,3 ' -bis-methyl-4, 4 ' -bis-hexyl-5, 5 ' -bis-methyl-9-hexyl-tricarbobenzo
Bromine salt of an oxazolylcyanine dye.
3、
Structural formula (xvi):
the molecular formula is: c38H55IN2S2；
Naming: 3,3 '-bis-propyl-5, 5' -bis-hexyl-9-isopropyl-tricarbocyanine benzothiazole dye iodide salt.
4、
Structural formula (xvi):
the molecular formula is: c40H40ClN3；
Naming: 3,3 ' -bis-isopropyl-4, 5-bipyridyl-4 ', 5 ' -diphenyl-9-m-dimethylphenyl-tricarbobenzocyclo-cyanine dye chloride salt.
5、
Structural formula (xvi):
the molecular formula is: c34H39IN2OS；
Naming: 3-methyl-4, 5-diphenyl-benzo
Azole-3 ' -propyl-4 ', 5 ' -bicycloheptanyl benzothiazole-9-butyl-tricarbocyanine dye iodonium salt.
Claims (8)
1. For detecting Pb2+The kit of concentrations is characterized by comprising the following components: cyanine dye, Pb2+Standard solution, DNA double strand, acid buffer solution and neutral buffer solution;
the DNA double strand comprises a DNA single strand capable of forming a G-quadruplex structure and a DNA single strand which is complementary with the DNA single strand and capable of forming an i-motif structure;
the sequence of the DNA single strand capable of forming G-quadruplexes is: 5' -Ga1Yb1Ga2Yb2Ga3Yb3Ga4Yb4-3'; wherein a1-a4 represents the number of G and is an integer of more than 2, b1-b4 represents the number of Y and is an integer of 0-3, and Y represents A, T or C base;
the sequence of the DNA single strand capable of forming i-motif is as follows: 5' -Cn1Xm1Cn2Xm2Cn3Xm3Cn4Xm4-3'; wherein n1-n4 represent the number of C and are integers of more than 2, m1-m4 represent the number of X and are integers of 0-3, and X represents A, T or G base;
the structural formula of the cyanine dye is as follows:
wherein R is1Is C1-C6Alkyl, phenyl, alkyl substituted phenyl of (a); r2、R3、R4And R5Independently selected from H or C1-C6Or R is2And R3Together with the carbon atom to which they are attached form a five-to seven-membered ring structure, or R4And R5Form a five-to seven-membered ring structure together with the carbon atoms to which they are attached; r6And R7Independently selected from C1-C6Alkyl groups of (a); y is halogen; x1，X2Independently selected from C, O, S, Se, Te.
2. The method for detecting Pb according to claim 12+Concentration kit, characterized in thatThe structural formula of the cyanine dye is as follows:
3. the method for detecting Pb according to claim 12+A kit for concentration, characterized in that the sequence of a DNA single strand capable of forming a G-quadruplex is: 5'-GGTGGTGGTGGT-3', 5'-GGTGGTGGTGGTGTTGGTGGTGGTGGTTT-3', 5'-GGGTGGGTGGGTGGG-3', 5'-GGGATTGGGATTGGGATTGGGATT-3', 5'-GGTTGGTGTGGTTGG-3', or 5'-TGAGGGTGGGGAGGGTGGGGAA-3'.
4. The method for detecting Pb according to claim 12+A concentration kit is characterized in that the sequence of the DNA single strand capable of forming i-motif is as follows: 5'-CCACCACCACCACAACCACCACCAAA-3', 5'-CCACCACCACCACAACCACCACCACCAAA-3', 5'-CCCACCCACCCACCC-3', 5'-CCCTAACCCTAACCCTAACCCTAA-3', 5'-CCAACCACACCAACC-3', or 5'-ACTCCCACCCCTCCCACCCCTT-3'.
5. The method for detecting Pb according to claim 12+Kit of concentrations, characterized in that Pb2+The standard solution is PbCl2A solution; the acid buffer solution is Tris-HAc buffer solution with the concentration of 10mM and the pH value of 4.0; the neutral buffer solution was Tris-HAc buffer solution with a concentration of 10mM and a pH of 7.0.
6. Detection of Pb using the kit according to any one of claims 1 to 52+A method of concentration, comprising the steps of:
(1) mixing neutral buffer solution with Pb2+Mixing the standard solution, DNA double strand and cyanine dye, incubating the mixed solution at 20-40 deg.C for 15-25min, measuring its absorbance at 448nm, and plotting Pb under neutral condition2+Detecting a standard curve;
(2) mixing the acidic buffer solution with Pb2+Standard solution, DNA double strand and cyanineMixing dyes, incubating the mixed solution at 20-40 deg.C for 15-25min, measuring the fluorescence intensity at 616nm, and plotting Pb under acidic condition2+Detecting a standard curve;
(3) mixing a sample to be detected, a DNA double strand and cyanine dye, incubating the mixed solution at 20-40 ℃ for 15-25min, measuring the absorbance at 448nm or the fluorescence intensity at 616nm, if the absorbance is detected, indicating that the sample to be detected is neutral, and determining Pb under the neutral condition according to the combination of Pb and cyanine dye2+Detecting the standard curve, and calculating Pb2+Concentration; if stronger fluorescence intensity is detected, the sample to be detected is slightly acidic, and Pb is combined under the acidic condition2+Detecting the standard curve, and calculating Pb2+And (4) concentration.
7. Detecting Pb according to claim 62+A method of concentration, characterized in that the final concentration of cyanine dye during detection is 4X 10-6mol/L。
8. Detecting Pb according to claim 62+A method for detecting the concentration of a double strand of DNA, characterized in that the concentration of the double strand of DNA is 2X 10-6mol/L。
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