CN113155790A - Fluorescence detection of Pb in complex matrix based on DNA-Cu NMs2+Method (2) - Google Patents
Fluorescence detection of Pb in complex matrix based on DNA-Cu NMs2+Method (2) Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a fluorescence detection method for Pb in a complex matrix based on DNA-Cu NMs2+Belonging to the fields of analytical chemistry, materials science, nano biosensing and the like. The detection method adopts dendritic DNA macromolecules-Cu NMs as Pb2+The fluorescence detection probe is used for carrying out fluorescence detection on a sample to be detected; the dendritic DNA macromolecule-Cu NMs are prepared by taking dendritic DNA molecules with sticky ends as templates and ascorbic acid as a reducing agent. Fluorescent detection of Pb by dendritic DNA macromolecules-Cu NMs constructed by the invention2+In a concentration range of 2.0-100nM, F0F and Pb2+The concentration of (c) shows a good linear relationship. The detection limit was 0.75 nM; the response to other common metal ions is negligible. The method has the advantages of high sensitivity, high selectivity, rapid detection and the like.
Description
Technical Field
The invention relates to a fluorescence detection method for Pb in a complex matrix based on DNA-Cu NMs2+Belonging to the fields of analytical chemistry, materials science, nano biosensing and the like.
Background
Lead ion (Pb)2+) The environmental pollution caused by the pollution has attracted worldwide attention. Long-term intake of Pb2+Then, the blood will flow into the bodyThe fluid system and the nervous system cause serious damage, especially to the intelligence of children. At present, many methods for measuring trace Pb have been developed2+Such as atomic absorption spectrometry, inductively coupled plasma mass spectrometry, and resonance light scattering spectrometry. These methods all require expensive instruments and equipment, professional technical operators, complicated sample pretreatment and the like, and therefore a method with high detection sensitivity, simple operation and low detection cost is particularly required. Currently, fluorescence detection methods are of great interest because of the advantages of simplicity and high efficiency. Among them, metal nanoparticle-based fluorometry has received increasing attention because of its low manufacturing cost and the absence of complex instrumentation to detect heavy metals.
Chen et al developed highly selective and sensitive Pb using a copper nanomaterial (Cu NMs) prepared using DNA as a template as a fluorescent probe2+Detection methods (Chen, J.; Liu, J.; Fang, Z.; Zeng, L.random Dsdna-labeled Format of coater Nanoparticles as Novel Fluorescence Probes for Label-Free lines detection. chem. Commun.2012,48, 1057-. The principle is that Pb2+The ions can pass through 5d10(Pb2+)-3d10(Cu+)With Cu on the surface of DNA-Cu NMs+Reaction, resulting in quenching of DNA-Cu NMs fluorescence. Although the results are good, the DNA-copper nanomaterials (DNA-Cu NMs) prepared by this method can only be kept stable for a few hours and have not been implemented for use in biomatrix. At present, it remains a great challenge to prepare highly fluorescent DNA-Cu NMs with good stability in complex biological matrices (e.g. serum, cell lysates). Since single-and double-stranded DNA is easily degraded in biological matrices, the destruction of DNA templates will also reduce the stability and reproducibility of application of these DNA-Cu NMs. Therefore, the preparation of high-stability fluorescent DNA-Cu NMs is of great significance for sensing application in complex matrixes.
Disclosure of Invention
[ problem ] to
Single-stranded and double-stranded DNA is easily degraded in a biological matrix, so that the existing DNA-copper nano material has poor stability and poor reusability in a complex matrix.
[ solution ]
The invention provides a fluorescence detection method for Pb in a complex matrix based on DNA-Cu NMs2+The complex matrix comprises serum and cell lysate, and the detection principle is as follows: dendritic DNA macromolecules-Cu NMs are adopted as Pb2+Fluorescent detecting probe of (1), Pb2+The ions can pass through 5d10(Pb2+)-3d10(Cu+) With Cu on the surface of dendritic DNA macromolecules-Cu NMs+And (3) decomposing the dendritic DNA macromolecules-Cu NMs into DNA-Cu NMs with smaller sizes, and destroying the aggregation-induced fluorescence enhancement (AIEE) effect of the dendritic DNA macromolecules-Cu NMs. The dendritic DNA macromolecules-Cu NMs have good stability in serum, and can realize Pb in serum biological matrix2+And (4) carrying out quantitative detection.
In particular, the amount of the solvent to be used,
the invention provides dendritic DNA macromolecules-Cu NMs, which are prepared by the following steps:
(1) three equal oligonucleotide strands (designated Y0a, Y0b, Y0c, respectively) were placed in MOPS buffer (10mM, pH 7.5,150mM NaAc,1mM MgCl2) Mixing to obtain a mixed solution; then, the mixed solution was heated to 90 ℃, slowly cooled to 4 ℃, and the annealed sample was incubated at room temperature for 30min to obtain a Y-type DNA building block (named Y0), Y0 having a sticky end;
(2) another Y-type DNA building block (named Y1) was prepared by the procedure of (1) with three equal oligonucleotide strands (named Y1a, Y1b, Y1c, respectively), Y1 having three double arms with or without sticky ends;
(3) y0 and Y1 are named as Y0-SteX and Y1-SteX respectively, wherein X represents the number of bases, Y0-SteX and Y1-SteX can be subjected to base complementary pairing, and Y0-SteX and Y1-SteX are hybridized according to the concentration ratio of 1: 3 to obtain a dendritic DNA macromolecule (named as G1); then, the dendritic DNA macromolecule (G1, 500. mu.L, 500nM) was mixed with ascorbic acid (500. mu.L, 1.25mM), and Cu (Ac) was added immediately2(200. mu.L, 500nM) and incubation of the above solution for 20minAnd finishing the preparation of the dendritic DNA macromolecules-Cu NMs.
The invention also provides a method for detecting Pb by utilizing the fluorescence of the dendritic DNA macromolecules-Cu NMs2+The method comprises the following steps:
(1)Pb2+preparation of standard solutions:
Pb2+standard solutions were MOPS buffered (10mM, pH 7.5,150mM NaAc,1mM MgCl)2) Diluted to 2, 10, 20, 40, 60, 80, 100nM, respectively. With MOPS buffer (10mM, pH 7.5,150mM NaAc,1mM MgCl)2) The prepared DNA-Cu NMs were diluted to 62.5 nM.
(2)Pb2+Establishing a fluorescence detection standard curve:
mixing Pb in different concentrations2+Mixing and incubating the standard solution and the dendritic DNA macromolecule-Cu NMs solution for 20min, detecting by using a fluorescence spectrophotometer after mixing reaction, and establishing fluorescence quenching rate signals of the dendritic DNA macromolecule-Cu NMs and different Pb2+Standard curve (2.0-100nM) for the correlation between concentrations;
(3) calculating Pb in standard solution of sample to be measured by an external standard method2+The content of (a).
In one embodiment of the present invention, the buffer solution is a MOPS buffer. The concentration of the MOPS buffer chosen was 1-20mM MOPS, 15-300mM NaAc, 0.1-2mM MgCl2The pH value is 7.5-8.0.
In one embodiment of the invention, the fluorescence detection conditions are: the excitation broadband is 20nm, the slit width is 20nm, the excitation wavelength is 340nm, and the fluorescence value at the 590nm emission peak is detected.
In one embodiment of the invention, the concentration of the selected dendrimer-Cu NMs solution is in the range of 30 to 600 nM.
The invention also provides a method for detecting Pb in serum by utilizing the fluorescence of dendritic DNA macromolecules-Cu NMs2+The method comprises the steps of filtering serum, mixing and incubating with dendritic DNA macromolecules-Cu NMs, carrying out fluorescence test on the mixed solution and the like.
In one embodiment of the present invention, the recovery rate can be tested using a serum mimetic sample by the steps of:
(1) pretreatment of dendritic DNA macromolecule-Cu NMs solution: the prepared dendritic DNA macromolecules-Cu NMs were applied to MOPS buffer (10mM, pH 7.5,150mM NaAc,1mM MgCl)2) Diluting to appropriate concentration, storing at 4 deg.C, and performing next experiment in dark;
(2) sample pretreatment: adding Pb into serum at different concentrations2+(0.1, 0.4, 1.6mM), Pb was added2+Diluting the serum sample by 20 times, and filtering by a 0.22 mu m microporous filter membrane;
(3) pb in serum2+Determination of recovery rate of (1): 980. mu.L of dendritic DNA macromolecules-Cu NMs (62.5nM) with different concentrations of Pb than 20. mu.L2+The serum is mixed and incubated for 10min, and a fluorescence spectrophotometer is adopted for detection after the mixing reaction.
[ advantageous effects ]
(1) The invention prepares dendritic DNA macromolecules-Cu NMs with high fluorescence and high stability. Firstly, the viscous terminal branch-shaped DNA macromolecules are used as templates to prepare the Cu NMs which are used for Pb2+The detection reaction is sensitive (the detection limit is 0.75 nM), rapid (one-step reaction, 10min), and has good stability in complex biological matrixes such as serum.
(2) The detection method provided by the invention realizes the purpose of Pb2+High sensitivity fluorescence detection. In the concentration range of 2.0-100nM, F0F and Pb2+The concentration of (c) shows a good linear relationship. The linear range was 2.0-100nM and the detection limit was 0.75 nM. The limit of detection (LOD) is obviously lower than Pb in drinking water of the United states Environmental Protection Agency (EPA)2+The highest recommended concentration (72 nM).
(3) The fluorescent probe dendritic DNA macromolecules-Cu NMs synthesized by the method have high stability in a serum complex matrix. Research shows that the fluorescence intensity of dendritic DNA macromolecules-Cu NMs and serum is not changed greatly after the dendritic DNA macromolecules-Cu NMs and the serum are stored together for 6 days. However, dsDNA-templated Cu NMs remained stable for only 4h, with only 24% of fluorescence intensity remaining after 1 d.
(4) The fluorescence detection method for Pb based on dendritic DNA macromolecules-Cu NMs constructed by the invention2+The detection has high selectivity. The invention detects dendritic DNA macromolecule-Cu NMs pairsResponse of various heavy metal ions. At Pb2+Pb at an addition concentration of 0.3. mu.M and at an addition concentration of 3. mu.M of other common heavy metal ions2+The fluorescence quenching rate of the dendritic DNA macromolecules-Cu NMs is 80%, and the fluorescence quenching rates of other heavy metal ions are lower than 5%.
(5) Fluorescent detection Pb based on dendritic DNA macromolecules-Cu NMs constructed by the invention2+The method is expected to be further applied to aptamer biosensing (for example, the cohesive end of the dendritic DNA macromolecules-Cu NMs is designed into an aptamer sequence) and cell imaging (for example, the dendritic DNA macromolecules-Cu NMs are directly co-cultured with cells and are introduced into the cells for intracellular Pb2+Imaging of (a).
(6) The method has the advantages of high sensitivity, high selectivity, high stability, rapid detection and the like.
Drawings
FIG. 1 shows the preparation of DNA-Cu NMs and Pb2+Schematic diagram of detection.
FIG. 2 is a TEM image of DNA-Cu NMs having different numbers of bases at the cohesive ends; (A) G1-Ste0-Cu NASs, (B) G1-Ste6-Cu NASs, (C) G1-Ste13-Cu NASs, (D) G1-Ste20-Cu NASs, and (E) G1-Ste27-Cu NASs.
FIG. 3 is a graph showing the change of fluorescence spectra of viscous terminals of different lengths to DNA-Cu NMs.
FIG. 4 shows the viscous end versus Pb for different lengths2+Fluorescence spectrum change pattern of quenched DNA-Cu NMs.
FIG. 5 shows the addition of different concentrations of Pb2+The DNA-Cu NMs fluorescence curve and the standard curve of (1).
FIG. 6 is a graph comparing the stability of dendritic DNA-Cu NMs and dsDNA-CuNMs in serum.
FIG. 7 is a graph showing the selectivity of DNA-Cu NMs for detection of various heavy metal ions.
FIG. 8 is a schematic representation of the Y0 and Y1-SteX chains.
Detailed Description
The first embodiment is as follows: preparation method of dendritic DNA-Cu NMs
A method for synthesizing fluorescent Cu NMs by taking reference double-stranded DNA as a template prepares DNA dendrimers by utilizing a Y-shaped DNA structure (Y-DNA), and takes the DNA dendrimers as template molecules to prepare the fluorescent dendritic DNA-Cu NMs.
TABLE 1 preparation of nucleic acid template sequences for dendritic DNA macromolecules-Cu NMs
In particular, the method comprises the following steps of,
3 different oligonucleotide strands (Y0a, Y0b, Y0c) were mixed in equal amounts in MOPS buffer (10mM, pH 7.5,150mM NaAc,1mM MgCl2). The mixed solution was heated to 90 ℃ and slowly cooled to 4 ℃. The annealed samples were incubated at room temperature for 30min to obtain Y-type DNA building blocks (Y0). Another Y-type DNA building block (Y1-SteX) was prepared by a similar procedure using three different oligonucleotide strands (Y1a-SteX, Y1b-SteX, Y1c-SteX, X indicating the number of sticky end bases, 0, 6, 13, 20 or 27, respectively). The resulting Y0 and Y1-SteX chains are shown in FIG. 8.
Y0 and Y1-SteX can be hybridized at a concentration ratio of 1 to 3 by base complementary pairing to give a dendritic DNA macromolecule (designated G1-SteX). Then, the dendritic DNA macromolecule G1-SteX (500. mu.L, 500nM) was mixed with ascorbic acid (500. mu.L, 1.25mM), and Cu (Ac) was added immediately2(200. mu.L, 500 nM). The above solution was incubated for 20min to complete the preparation of DNA-Cu NMs and stored at 4 ℃ in the dark. Wherein the dendritic DNA macromolecule (G1-SteX) and ascorbic acid are both treated with MOPS buffer (10mM, pH 7.5,150mM NaAc,1mM MgCl)2) Dissolved and diluted to the desired concentration. Cu (Ac)2Dissolved and diluted to the desired concentration with ultrapure water (18.2 M.OMEGA.cm).
Example two: viscous ends of different lengths for Pb2+Effect of quenching DNA-Cu NMs fluorescence
DNA-Cu NMs with different length of the cohesive end dendritic DNA molecules (G1-SteX, DNA core) were prepared using the method of example oneThe acid single strand used was the sequence shown in Table 1) as a template, and the cohesive ends thereof were 0, 6, 13, 20, and 27 bases in length, respectively, under the same conditions as the others. DNA-Cu NMs (G1-SteX) for Pb2+A schematic diagram of fluorescence quenching detection is shown in FIG. 1.
1. Size of DNA-Cu NMs with different number of bases at cohesive end
DNA-Cu NMs were prepared using dendritic DNA macromolecules (G1-SteX) with different lengths at the sticky ends as templates, and their sizes were characterized by transmission electron microscopy, the results are shown in FIG. 2. The TEM image of fig. 2 shows that most of the smaller sized copper nanoclusters self-assemble into larger copper nano assemblies. As the number of cohesive end bases of G1 increased from 0 to 6, 13, 20, and 27, the average size of DNA-Cu NMs increased from 19nm to 131nm, 160nm, 220nm, and 264nm, respectively. Furthermore, Cu NMs showed a more dense morphology and ordered arrangement after introduction of G1 at the sticky end.
2. Fluorescence intensity of DNA-Cu NMs with different numbers of bases at cohesive ends
The fluorescence detection conditions are that the excitation broadband is 20nm, the slit width is 20nm, the excitation wavelength is 340nm, and the fluorescence value at the 590nm emission peak value is detected.
The fluorescence intensity results of DNA-Cu NMs prepared using DNA with different numbers of bases at the sticky end as a template are shown in FIG. 3. The fluorescence intensity of Cu NMs gradually increased with increasing length of the sticky end of G1. The fluorescence intensity of DNA-Cu NMs increases nearly 6-fold when increasing from 0 to 27 bases.
3. Viscous end pairs of different lengths to Pb2+Effect of quenching DNA-Cu NMs fluorescence
With MOPS buffer (10mM, pH 7.5,150mM NaAc,1mM MgCl)2) The prepared G1-Ste13-Cu NMs were diluted to 62.5 nM. 980 μ L of Cu NMs (62.5nM) and 20 μ L Pb2+(100nM) mixed and incubated at room temperature for 20 min.
Detecting by adopting a fluorescence photometer under the following detection conditions: the excitation wavelength was 340nm and the emission signal at 590nm was collected.
Applying viscous end pairs of different lengths to Pb2+The effect of quenching DNA-Cu NMs fluorescence is shown in FIG. 4. G1-Ste13-Cu NMs for Pb2+The detection is most sensitive, so that G1-Ste13-Cu NMs are selected as the optimal.
Example three: method for detecting Pb in serum by applying DNA-Cu NMs2+Method of content
Application example I prepared DNA-Cu NMs to detection of Pb in serum2+Content, preparation of DNA-Cu NMs and Pb2+The detection principle of (2) is shown in figure 1.
1、Pb2+Establishment of a Standard Curve
(1)Pb2+Preparation of standard solution:
Pb2+standard solutions were MOPS buffered (10mM, pH 7.5,150mM NaAc,1mM MgCl)2) Diluted to 2, 10, 20, 40, 60, 80, 100nM, respectively. With MOPS buffer (10mM, pH 7.5,150mM NaAc,1mM MgCl)2) The prepared DNA-Cu NMs were diluted to 62.5 nM. 980. mu.L of DNA-Cu NMs and 20. mu.L of Pb in different concentrations2+The standard solutions were mixed and incubated for 20min at room temperature. Fitting Stern-Volmer plots, F0/F＝1+Ksv[C]The quenching constant (K) in the above detection is estimatedsv) (ii) a In the formula, F, F0Are respectively provided with Pb2+And no Pb2+C is Pb2+Concentration in nM; k is a radical ofqIs the quenching rate constant, τ0The fluorescence lifetime of Cu NMs. Substituting the calculated fluorescence quenching rate signal into a standard curve, and calculating to obtain Pb in the detected sample2+The concentration of (c).
(2) And (3) fluorescence spectrum detection:
detecting by adopting a fluorescence photometer under the following detection conditions: the excitation wavelength was 340nm and the emission signal at 590nm was collected.
(3) Determination of linear relation and detection limit:
addition of Pb to DNA-Cu NMs2+The fluorescence intensity meter is F1DNA-Cu NMs without addition of Pb2+The fluorescence intensity meter of is F0The standard curve is plotted as in fig. 5. The standard curve is F0/F＝0.9905+0.01096C，R2＝0.989，KSV＝0.01096 nM-1. The linear range was 2.0-100nM and the detection limit was 0.75 nM.
2. Detecting bloodClearing away Pb2+Method of content
(1) Sample pretreatment: adding Pb into serum at different concentrations2+(0.1, 0.4, 1.6 mM). Addition of Pb2+The FBS sample was diluted 20 times and centrifuged at 10000rpm for 10min to obtain a supernatant. With MOPS buffer (10mM, pH 7.5,150mM NaAc,1mM MgCl)2) The prepared G1-Ste13-Cu NMs were diluted to 62.5 nM. 980. mu.L of Cu NMs and 20. mu.L of Pb containing different concentrations2+The supernatants were mixed and incubated for 20min at room temperature.
(2) And (3) fluorescence spectrum detection: detecting by adopting a fluorescence photometer under the following detection conditions: the excitation wavelength was 340nm and the emission signal at 590nm was collected.
3. Comparison of the stability of dendritic DNA-Cu NMs and dsDNA-Cu NMs in serum
The stability was compared in serum using G1-Ste13-Cu NMs and dsDNA-Cu NMs. Wherein, dsDNA-Cu NMs take DNA double strands without sticky ends (dsDNA-a and dsDNA-b are hybridized in the table I) as templates, the sequence of the templates is shown in the table 2, and ascorbic acid is used as a reducing agent; 500 μ L of the system contained 500nM dsDNA,1mM ascorbic acid, 100 μ M Cu2+MOPS (10mM, pH 7.5), NaAc (150 mM). The above solutions were mixed and incubated for 20min to complete the preparation of dsDNA-Cu NMs (Chen, J.; Liu, J.; Fan, Z.; Zeng, L.random Dsdna-structured Formation of coater Nanoparticles as Novel fluorescent Probes for Label-Free lines ion detection. chem. Commun.2012,48, 1057-. With MOPS buffer (10mM, pH 7.5,150mM NaAc,1mM MgCl)2) The prepared G1-Ste13-Cu NMs and dsDNA-Cu NMs were diluted to 500 nM. 100uL of G1-Ste13-Cu NMs or dsDNA-Cu NMs were added to 700uL of serum, incubated for several days, sampled periodically and observed for fluorescence change.
TABLE 2 preparation of nucleic acid template sequences for dsDNA-Cu NMs
Detection of Pb in serum by DNA-Cu NMs2+The stability of the contents is shown in FIG. 6. The fluorescence intensity of G1-Ste13-Cu NASs was not changed much after being stored with serum for 6 days. However, dsDNA-Cu NMs remained stable for only 4h, and retained only 24% of fluorescence intensity after 1d, which was unstable in complex matrices such as serum.
Example 4: accuracy and specificity of the method
1. Accuracy of the method
Adding standard Pb to serum sample2+The concentrations of the solutions were 5, 20 and 80nM, respectively, and then 3 times per sample were tested using G1-Ste13-Cu NMs as probes. Measured Pb2+The average concentrations are respectively 4.6, 19.3 and 72.88nM, the recovery rate is between 91.1% and 96.5%, and the Relative Standard Deviation (RSD) value is less than 4.0%, which shows that the method has better accuracy and precision.
2. Specificity of the method
The effect of common metal ions on the detection was examined, as shown in fig. 7. First, 980. mu.L of Cu NMs and 20. mu.L of each metal ion (Al)3+、Fe3+,Ca2+,Cd2+,Hg2+,Cu2+,Ni3+,Co2+,Mg2+,Mn2+,Zn2+,Pb2+) And (4) mixing. After incubation for 20min, fluorescence quenching results were analyzed. All measurements were performed in triplicate and the standard deviation was plotted as an error bar. The results show that they do not affect Pb2+Detection of (3).
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> method for fluorescence detection of Pb2+ in complex matrix based on DNA-Cu NMs
<130> BAA201524A
<160> 20
<170> PatentIn version 3.3
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Claims (10)
1. Fluorescence detection Pb2+The method is characterized in that dendritic DNA macromolecules-Cu NMs are used as Pb2+Fluorescent detecting probe of (1), Pb2+Through 5d10(Pb2+)-3d10(Cu+) With the metallophilic interaction of DNA macromolecules-Cu on the surface of Cu NMs+And (3) reacting to decompose the DNA macromolecules-Cu NMs into Cu NMs with smaller sizes, so that the aggregation-induced fluorescence enhancement effect of the DNA macromolecules-Cu NMs is destroyed.
2. The method of claim 1 for fluorescence detection of Pb2+The method of (1), characterized by detecting Pb in serum or cell lysate2+The content of (a).
3. A method of fluorescence detection of Pb according to claim 1 or 22+The method is characterized in that the preparation method of the dendritic DNA macromolecules-Cu NMs comprises the following steps:
(1) mixing three equal oligonucleotide chains Y0a, Y0b and Y0c in MOPS buffer solution to obtain mixed solution; then, the mixed solution was heated to 90 ℃, cooled to 4 ℃, and the annealed sample was incubated at room temperature to obtain Y-type DNA building block Y0;
(2) another Y-type DNA construct Y1 was prepared by referring to the three other equal oligonucleotide strands Y1a, Y1b, Y1c in step (1);
(3) y0 and Y1 were named Y0-SteX and Y1-SteX, respectively, wherein X represents the number of bases; hybridizing Y0-SteX and Y1-SteX according to the concentration ratio of 1: 3 by base complementary pairing of Y0-SteX and Y1-SteX to obtain a dendritic DNA macromolecule; then, the dendritic DNA macromolecules were mixed with ascorbic acid and Cu (Ac) was added immediately2The above solution was incubated to complete the preparation of dendritic DNA macromolecules-Cu NMs.
4. The method of claim 3 for fluorescence detection of Pb2+The method is characterized by comprising the following steps:
(1)Pb2+establishing a fluorescence detection standard curve:
mixing Pb in different concentrations2+Mixing and incubating the standard solution and the dendritic DNA macromolecule-Cu NMs solution, detecting by using a fluorescence spectrophotometer after mixing reaction, and establishing fluorescence quenching rate signals of the dendritic DNA macromolecule-Cu NMs and different Pb2+A standard curve of the correspondence between concentrations;
(2) calculating Pb in the sample to be detected by an external standard method2+The content of (a).
5. A method for fluorescence detection of Pb according to any one of claims 1 to 42+The method of (3), wherein the fluorescence detection conditions are: the excitation broadband is 20nm, the slit width is 20nm, the excitation wavelength is 340nm, and the fluorescence value at the 590nm emission peak is detected.
6. The method of claim 3 for fluorescence detection of Pb2+The method of (1) is characterized in that the concentration of the MOPS buffer solution is 1 to 20mM MOPS, 15 to 300mM NaAc, 0.1 to 2mM MgCl2The pH value is 7.5-8.0.
7. The method of claim 4 for fluorescence detection of Pb2+The method of (1), characterized in that the serum or cell lysate to be tested is filtered, mixed with dendritic DNA-Cu NMs for incubation, and the mixed solution is subjected to fluorescence detection.
8. The method of claim 4 for fluorescence detection of Pb2+The method of (1), wherein the concentration of the DNA macromolecule-Cu NMs solution is 30 to 600 nM.
9. A method for preparing dendritic DNA macromolecules-Cu NMs, which is characterized by comprising the following steps:
(1) mixing three equal oligonucleotide chains Y0a, Y0b and Y0c in MOPS buffer solution to obtain mixed solution; then, the mixed solution was heated to 90 ℃, cooled to 4 ℃, and the annealed sample was incubated at room temperature to obtain Y-type DNA building block Y0;
(2) another Y-type DNA construct Y1 was prepared by referring to the three other equal oligonucleotide strands Y1a, Y1b, Y1c in step (1);
(3) y0 and Y1 were named Y0-SteX and Y1-SteX, respectively, wherein X represents the number of bases; hybridizing Y0-SteX and Y1-SteX according to the concentration ratio of 1: 3 by base complementary pairing of Y0-SteX and Y1-SteX to obtain a dendritic DNA macromolecule; then, the dendritic DNA macromolecules were mixed with ascorbic acid and Cu (Ac) was added immediately2The above solution was incubated to complete the preparation of dendritic DNA macromolecules-Cu NMs.
10. Dendritic DNA macromolecules-Cu NMs prepared according to the method of claim 9 and their use in aptamer biosensors or cell imaging.
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