CN113155790B - Fluorescent detection of Pb in complex matrix based on DNA-Cu NMs 2+ Is a method of (2) - Google Patents

Abstract

The invention discloses a fluorescent detection method for Pb in a complex matrix based on DNA-Cu NMs ²⁺ Belongs to the fields of analytical chemistry, materialogy, nano biological sensing and the like. The detection method adopts dendritic DNA macromolecules-Cu NMs as Pb ²⁺ Performing fluorescence detection on a sample to be detected; the dendritic DNA macromolecules-Cu NMs are prepared by taking dendritic DNA molecules with sticky ends as templates and ascorbic acid as a reducing agent. Fluorescent detection Pb of dendritic DNA macromolecules-Cu NMs constructed by the invention ²⁺ In a concentration range of 2.0-100nM, F ₀ F and Pb ²⁺ Exhibits a good linear relationship with respect to the concentration of (a). The limit of detection was 0.75nM; 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

Fluorescent detection of Pb in complex matrix based on DNA-Cu NMs 2+ Is a method of (2)

Technical Field

The invention relates to a fluorescent detection method for Pb in complex matrix based on DNA-Cu NMs ²⁺ Belongs to the fields of analytical chemistry, materialogy, nano biological sensing and the like.

Background

Lead ion (Pb) ²⁺ ) The resulting environmental pollution has attracted worldwide attention. Long-term intake of Pb ²⁺ Later, serious damage to the blood system and nervous system of the body, especially serious mental damage to children, can occur. At present, many methods for determining trace Pb have been developed ²⁺ Such as atomic absorption spectrometry, inductively coupled plasma mass spectrometry, and resonance light scattering spectrometry. These methods all require expensive equipment, specialized technical operators, complicated sample pretreatment, etc., and thus a method with high detection sensitivity, simple operation, and low detection cost is particularly required. Currently, fluorescence detection methods are attracting great attention because of their advantages of simplicity and efficiency. Among them, metal nanoparticle-based fluorescence assays are receiving increasing attention because of their low manufacturing cost and the lack of complex instrumentation required to detect heavy metals.

Copper nanomaterials (Cu NMs) prepared by Chen et al using DNA as a template as a fluorescent probe developed Pb with high selectivity and high sensitivity ²⁺ Detection methods (Chen, J.; liu, J.; fang, Z.; zeng, L.random Dsdna-Templated Formation of Copper Nanoparticles as Novel Fluorescence Probes for Label-Free Lead ion detection. Chem. Commun. 20)12,48,1057-1059). The principle is that Pb ²⁺ The ions can pass through 5d ¹⁰ (Pb ²⁺ )-3d ¹⁰ (Cu ⁺⁾ Is associated with Cu on the surface of DNA-Cu NMs ⁺ The reaction, thus leading to fluorescence quenching of the DNA-Cu NMs. Although the results were good, the DNA-copper nanomaterial (DNA-Cu NMs) prepared by this method remained stable for only a few hours and was not implemented for use in biological matrices. Currently, preparing highly fluorescent DNA-Cu NMs with good stability in complex biological matrices (e.g. serum, cell lysates) remains a great challenge. Since single-and double-stranded DNA is easily degraded in biological matrices, disruption of the DNA template will also reduce the stability and reproducibility of the application of these DNA-Cu NMs. Thus, preparing fluorescent DNA-Cu NMs with high stability is of great importance for its sensing applications in complex matrices.

Disclosure of Invention

[ technical problem ]

Single-stranded and double-stranded DNA are easily degraded in biological matrices, resulting in poor stability and poor reusability of existing DNA-copper nanomaterials in complex matrices.

Technical scheme

The invention provides a fluorescent detection method for Pb in a complex matrix based on DNA-Cu NMs ²⁺ The complex matrix comprises serum and cell lysate, and the detection principle is as follows: dendritic DNA macromolecules-Cu NMs are adopted as Pb ²⁺ Fluorescent detection probe of Pb ²⁺ The ions can pass through 5d ¹⁰ (Pb ²⁺ )-3d ¹⁰ (Cu ⁺ ) Is associated with Cu on the surface of dendritic DNA macromolecules-Cu NMs ⁺ The reaction breaks down dendritic DNA macromolecules-Cu NMs into DNA-Cu NMs with smaller sizes, and breaks down the aggregation-induced fluorescence enhancement (AIEE) effect of the dendritic DNA macromolecules-Cu NMs. The dendritic DNA macromolecule-Cu NMs has good stability in serum, and can realize Pb in serum biological matrix ²⁺ Is a quantitative detection of (a).

In particular, the method comprises the steps of,

the invention provides dendritic DNA macromolecules-Cu NMs, which are prepared by the following steps:

(1) Three equal amounts of oligonucleotide strands (designated as Y0a, Y0b, Y0c, respectively) were incubated in MOPS buffer (10mM,pH 7.5,150mM NaAc,1mM MgCl) ₂ ) Mixing to obtain a mixed solution; then, the mixed solution was heated to 90 ℃ and cooled down slowly to 4 ℃, and the annealed sample was incubated at room temperature for 30min to obtain a Y-type DNA building block (designated as Y0), Y0 having a cohesive end;

(2) Another Y-type DNA construct (designated Y1) was prepared by passing three additional equal amounts of oligonucleotide strands (designated Y1a, Y1b, Y1c, respectively) through the step of (1), Y1 having three arms with or without sticky ends;

(3) Y0 and Y1 are respectively named as Y0-SteX and Y1-SteX, wherein X represents the number of bases, Y0-SteX and Y1-SteX can be subjected to base complementary pairing, and the Y0-SteX and Y1-SteX are hybridized according to the concentration ratio of 1 to 3 to obtain a dendritic DNA macromolecule (named as G1); then, dendritic DNA macromolecules (G1, 500. Mu.L, 500 nM) were mixed with ascorbic acid (500. Mu.L, 1.25, mM) and Cu (Ac) was immediately added ₂ (200. Mu.L, 500 nM) and incubating the above solution for 20min to complete the preparation of dendritic DNA macromolecules-Cu NMs.

The invention also provides the fluorescent detection of Pb by using dendritic DNA macromolecules-Cu NMs ²⁺ Comprising the steps of:

(1)Pb ²⁺ preparation of standard solution:

Pb ²⁺ MOPS buffer for Standard solution (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 62.5nM.

(2)Pb ²⁺ Establishment of a fluorescence detection standard curve:

pb at different concentrations ²⁺ Mixing and incubating the standard solution and the dendritic DNA macromolecule-Cu NMs solution for 20min, detecting by adopting a fluorescence spectrophotometer after the mixed reaction, and establishing fluorescence quenching rate signals of the dendritic DNA macromolecule-Cu NMs and different Pb ²⁺ A standard curve (2.0-100 nM) of correspondence between concentrations;

(3) Calculating to obtain a sample to be measured by an external standard methodPb in standard solution ²⁺ Is contained in the composition.

In one embodiment of the invention, the buffer solution is a MOPS buffer. The selected MOPS buffer concentration is 1-20mM MOPS,15-300mM NaAc,0.1-2mM MgCl ₂ The pH is 7.5-8.0.

In one embodiment of the present 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 dendritic DNA macromolecule-Cu NMs solution is 30-600 nM.

The invention also provides a fluorescent detection method for Pb in serum by using dendritic DNA macromolecules-Cu NMs ²⁺ 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 invention, a serum mimetic sample may be used to test recovery, the steps being:

(1) Pretreatment of dendritic DNA macromolecule-Cu NMs solution: the dendrimer-Cu NMs prepared were buffered with MOPS buffer (10mM,pH 7.5,150mM NaAc,1mM MgCl ₂ ) Diluted to the appropriate concentration, stored at 4 ℃, and subjected to the next experiment in the dark;

(2) Sample pretreatment: adding Pb with different concentration into serum ²⁺ (0.1, 0.4, 1.6 mM), pb was added ²⁺ Diluting the serum sample by 20 times, and filtering by a 0.22 mu m microporous filter membrane;

(3) Pb in serum ²⁺ Is measured by the recovery rate: 980. Mu.L dendritic DNA macromolecules-Cu NMs (62.5 nM) and 20. Mu.L Pb with different concentrations ²⁺ And (3) mixing and incubating for 10min, and detecting by adopting a fluorescence spectrophotometer after the mixing reaction.

[ advantageous effects ]

(1) The invention prepares dendritic DNA macromolecules-Cu NMs with high fluorescence and high stability. The Cu NMs are prepared by taking the cohesive end branch DNA macromolecules as templates for the first time, and the Cu NMs are used for Pb ²⁺ The detection reaction is sensitive (the detection limit is 0.75 nM) and quick (one-step reactionShould be 10 min), has good stability in complex biological matrix such as serum.

(2) The detection method provided by the invention realizes Pb ²⁺ Is a high-sensitivity fluorescent detection of (a). F in the concentration range of 2.0-100nM ₀ F and Pb ²⁺ Exhibits a good linear relationship with respect to the concentration of (a). The linear range was 2.0-100nM with a detection limit of 0.75nM. The detection Limit (LOD) is significantly lower than Pb in the United states Environmental Protection Agency (EPA) drinking water ²⁺ Is the highest recommended concentration (72 nM).

(3) The fluorescent probe dendritic DNA macromolecules-Cu NMs synthesized by the invention have high stability in a serum complex matrix. The study shows that the fluorescence intensity of the dendritic DNA macromolecules-Cu NMs is not greatly changed after the dendritic DNA macromolecules-Cu NMs are stored for 6 days together with serum. However, dsDNA templated Cu NMs remained stable for only 4h, only 24% of fluorescence intensity after 1 d.

(4) The fluorescence detection method based on dendritic DNA macromolecules-Cu NMs constructed by the invention is used for Pb ²⁺ The detection has high selectivity. The invention detects the response of dendritic DNA macromolecules-Cu NMs to various heavy metal ions. In Pb ²⁺ Pb at an addition concentration of 0.3. Mu.M and other common heavy metal ions at an addition concentration of 3. Mu.M ²⁺ The fluorescence quenching rate of the dendritic DNA macromolecules-Cu NMs is 80%, and the fluorescence quenching rate of other heavy metal ions is lower than 5%.

(5) Fluorescent detection Pb based on dendritic DNA macromolecules-Cu NMs constructed by the invention ²⁺ The method is expected to be further applied to aptamer biosensing (such as designing the cohesive end of the dendritic DNA macromolecule-Cu NMs as an aptamer sequence) and cell imaging (such as directly co-culturing the dendritic DNA macromolecule-Cu NMs with cells, introducing the cells and using the dendritic DNA macromolecule-Cu NMs in the cells for Pb in the cells) ²⁺ Is an imaging of (c).

(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 Pb ²⁺ Is a schematic diagram of detection.

FIG. 2 is a TEM image of DNA-Cu NMs with sticky ends containing different numbers of bases; (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 in fluorescence spectrum of cohesive ends of different lengths versus DNA-Cu NMs.

FIG. 4 is a graph of viscous ends of different lengths versus Pb ²⁺ Fluorescence spectrum change pattern of quenched DNA-Cu NMs.

FIG. 5 is a diagram of Pb addition at various concentrations ²⁺ DNA-Cu NMs fluorescence curve and standard curve.

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

Embodiment one: preparation method of dendritic DNA-Cu NMs

The fluorescent Cu NMs are prepared by taking a Y-shaped DNA structure (Y-DNA) as a template to prepare DNA dendrimers.

TABLE 1 preparation of nucleic acid template sequences for dendritic DNA macromolecules-Cu NMs

In particular, the method comprises the steps of,

equal amounts of 3 different oligonucleotide strands (Y0 a, Y0b, Y0 c) were mixed in MOPS buffer (10mM,pH 7.5,150mM NaAc,1mM MgCl) ₂ ). The mixed solution was heated to 90 ℃ and cooled slowly to 4 ℃. The annealed samples were incubated at room temperature for 30min to obtain Y-type DNA building blocks (Y0). Another Y-type DNA construct (Y1-SteX) was prepared by a similar procedure using three different oligonucleotide strands (Y1 a-SteX, Y1b-SteX, Y1c-SteX, X referring to 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 hybridize by base complementary pairing at a concentration ratio of 1 to 3 to give a dendrimer of dendritic DNA (designated G1-SteX). Then, dendritic DNA macromolecules G1-SteX (500. Mu.L, 500 nM) were mixed with ascorbic acid (500. Mu.L, 1.25 mM) and Cu (Ac) was immediately added ₂ (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 absence of light. Wherein, the dendritic DNA macromolecules (G1-SteX) and ascorbic acid are both buffered with MOPS buffer (10mM,pH 7.5,150mM NaAc,1mM MgCl) ₂ ) Dissolving and diluting to the required concentration. Cu (Ac) ₂ Dissolved and diluted to the desired concentration with ultrapure water (18.2 M.OMEGA.cm).

Embodiment two: adhesive end to Pb of different lengths ²⁺ Effect of quenching DNA-Cu NMs fluorescence

DNA-Cu NMs were prepared by the method of example I, and were prepared using dendritic DNA molecules (G1-SteX, DNA nucleic acid single strand using the sequences shown in Table 1) having cohesive ends of different lengths as templates, the cohesive ends were 0, 6, 13, 20, 27 bases in length, respectively, with the same other conditions. DNA-Cu NMs (G1-SteX) for Pb ²⁺ The schematic diagram of the fluorescence quenching detection is shown in fig. 1.

1. Size of DNA-Cu NMs with different base numbers at the sticky end

DNA-Cu NMs with different lengths of viscous terminal dendritic DNA macromolecules (G1-SteX) as templates, and their sizes are represented by transmission electron microscopy, and the results are shown in figure 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 G1 cohesive end bases increases from 0 to 6, 13, 20 and 27, the average size of DNA-Cu NMs increases from 19nm to 131nm, 160nm, 220nm and 264nm, respectively. Furthermore, cu NMs exhibit a more dense morphology and ordered arrangement after the introduction of G1 at the cohesive ends.

2. Fluorescence intensity of DNA-Cu NMs with different base numbers at the sticky end

The fluorescence detection condition is 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 is detected.

The fluorescence intensity results of DNA-Cu NMs prepared by using DNA with different base numbers at the sticky end as a template are shown in FIG. 3. As the length of the G1 cohesive end increases, the fluorescence intensity of Cu NMs gradually increases. The fluorescence intensity of DNA-Cu NMs was increased by nearly 6-fold when increasing from 0 to 27 bases.

3. Adhesive end pair Pb of different lengths ²⁺ 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 62.5nM. 980. Mu.L Cu NMs (62.5 nM) and 20. Mu.L Pb ²⁺ (100 nM) and incubating for 20min at room temperature.

Adopting a fluorescence photometer to detect, wherein the detection conditions are as follows: the excitation wavelength was 340nm and the emission signal at 590nm was collected.

Using adhesive end pairs of different lengths for Pb ²⁺ The effect of quenching DNA-Cu NMs fluorescence is shown in FIG. 4. G1-Ste13-Cu NMs vs Pb ²⁺ The detection is most sensitive, and therefore G1-Ste13-Cu NMs are chosen as optimal.

Embodiment III: detection of Pb in serum by using DNA-Cu NMs ²⁺ Content method

Detection of Pb in serum by using DNA-Cu NMs prepared in example ²⁺ Content, preparation of DNA-Cu NMs and Pb ²⁺ The detection principle diagram of (2) is shown in figure 1.

1、Pb ²⁺ Establishment of a Standard Curve

(1)Pb ²⁺ Preparation of standard solution:

Pb ²⁺ MOPS buffer for Standard solution (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 62.5nM. 980. Mu.L of DNA-Cu NMs and 20. Mu.L of Pb at different concentrations ²⁺ The standard solutions were mixed and incubated at room temperature for 20min. Fitting a Stern-Volmer diagram, F ₀ /F＝1+K _sv [C]The quenching constant (K _sv ) The method comprises the steps of carrying out a first treatment on the surface of the In F, F ₀ Respectively with Pb ²⁺ And without Pb ²⁺ Fluorescent light of (C) Pb ²⁺ Concentration in nM; k (k) _q Is the quenching rate constant, τ ₀ Fluorescence lifetime for Cu NMs. The calculated fluorescence quenching rate signal is brought into a standard curve, and Pb in the detection sample is calculated ²⁺ Is a concentration of (3).

(2) Fluorescence spectrum detection:

adopting a fluorescence photometer to detect, wherein the detection conditions are as follows: the excitation wavelength was 340nm and the emission signal at 590nm was collected.

(3) Determination of linear relation and detection limit:

pb addition by DNA-Cu NMs ²⁺ The fluorescence intensity after the reaction is F ₁ Pb-free DNA-Cu NMs ²⁺ Fluorescent intensity of (F) ₀ The standard curve is plotted as in fig. 5. The standard curve is F ₀ /F＝0.9905+0.01096C，R ² ＝0.989，K _SV ＝0.01096 nM ^-1 . The linear range was 2.0-100nM with a detection limit of 0.75nM.

2. Detection of Pb in serum ²⁺ Content method

(1) Sample pretreatment: adding Pb with different concentration into serum ²⁺ (0.1, 0.4, 1.6 mM). Pb addition ²⁺ After 20-fold dilution of the FBS sample, the sample was centrifuged at 10000rpm for 10min, and the supernatant was collected. With MOPS buffer (10mM,pH 7.5,150mM NaAc,1mM MgCl) ₂ ) The prepared G1-Ste13-Cu NMs were diluted to 62.5nM. 980. Mu.L Cu NMs and 20. Mu.L Pb with different concentrations ²⁺ Is incubated at room temperature for 20min.

(2) Fluorescence spectrum detection: adopting a fluorescence photometer to detect, wherein the detection conditions are as follows: the excitation wavelength was 340nm and the emission signal at 590nm was collected.

3. Comparison of stability of dendritic DNA-Cu NMs and dsDNA-Cu NMs in serum

The stability in serum was compared using G1-Ste13-Cu NMs and dsDNA-Cu NMs. Wherein, dsDNA-Cu NMs takes DNA double chains without sticky ends (dsDNA-a and dsDNA-b are hybridized in the table I) as templates, the sequences of the templates are shown in the table 2, and ascorbic acid is taken as a reducing agent; 500. Mu.L of system contains 500nM dsDNA,1mM ascorbic acid, 100. Mu.M Cu ²⁺ MOPS (10 mM, pH 7.5), naAc (150 mM). The above-mentioned materials are mixedThe solutions were mixed and incubated for 20min to complete the preparation of dsDNA-Cu NMs (Chen, J.; liu, J.; fang, Z.; zeng, L. Random Dsdna-Templated Formation of Copper Nanoparticles as Novel Fluorescence Probes for Label-Free Lead ion detection. Chem. Commun.2012,48, 1057-1059). 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 500nM. 100uL of G1-Ste13-Cu NMs or dsDNA-Cu NMs was added to 700uL of serum, incubated for several days, sampled at regular time, and fluorescence changes were observed.

TABLE 2 preparation of nucleic acid template sequences for dsDNA-Cu NMs

Detection of Pb in serum using DNA-Cu NMs ²⁺ The stability of the content is shown in FIG. 6. The fluorescence intensity did not change much after 6d of storage of G1-Ste13-Cu NASs with serum. However, dsDNA-Cu NMs remained stable for only 4h, only 24% of fluorescence intensity after 1d, and unstable in complex matrices such as serum.

Example 4: accuracy and specificity of the method

1. Accuracy of the method

Addition of Standard Pb to serum samples ²⁺ The concentrations of the solutions were 5, 20 and 80nM, respectively, and each sample was then tested 3 times using G1-Ste13-Cu NMs as probe. Measured Pb ²⁺ Average concentrations are 4.6, 19.3 and 72.88nM respectively, recovery rate is between 91.1% and 96.5%, and Relative Standard Deviation (RSD) value is less than 4.0%, which indicates that the method has better accuracy and precision.

2. Specificity of the method

The effect of common metal ions on detection was examined as shown in fig. 7. First, 980. Mu.L of Cu NMs and 20. Mu.L of various metal ions (Al ³⁺ 、Fe ³⁺ ,Ca ²⁺ ,Cd ²⁺ ,Hg ²⁺ ,Cu ²⁺ ,Ni ³⁺ ,Co ²⁺ ,Mg ²⁺ ,Mn ²⁺ ,Zn ²⁺ ,Pb ²⁺ ) Mixing. After 20min incubation, fluorescence quenching results were analyzed. All measurements were performed three times and the standard deviation was plotted as an error bar. The results show that they do not affect Pb ²⁺ Is detected.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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 Jiangnan

<120> method for detecting Pb2+ in complex matrix based on DNA-Cu NMs fluorescence

<130> BAA201524A

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Claims (9)

1. Fluorescence detection Pb ²⁺ Characterized in that dendritic DNA macromolecules-Cu NMs are used as Pb ²⁺ Fluorescent detection probe of Pb ²⁺ By 5d ¹⁰ (Pb ²⁺ )-3d ¹⁰ (Cu ⁺ ) Is associated with Cu on the surface of DNA macromolecules-Cu NMs ⁺ The DNA macromolecule-Cu NMs are decomposed into Cu NMs with smaller size by the reaction, so that the aggregation-induced fluorescence enhancement effect of the DNA macromolecule-Cu NMs is destroyed;

the preparation method of the dendritic DNA macromolecule-Cu NMs comprises the following steps:

(1) Mixing three equal amounts of oligonucleotide chains Y0a, Y0b and Y0c in MOPS buffer to obtain a mixed solution; then, heating the mixed solution to 90 ℃, cooling to 4 ℃, and incubating the annealed sample at room temperature to obtain a Y-type DNA building block Y0;

(2) Referring to step (1), three other equal amounts of oligonucleotide chains Y1a, Y1b, Y1c produce another Y-type DNA building block Y1; y1 is designated Y1-SteX, wherein X represents the number of sticky end bases, 6, 13, 20 or 27;

(3) The Y0 and the Y1-SteX are subjected to base complementation pairing, and the Y0 and the Y1-SteX are hybridized according to the concentration ratio of 1 to 3 to obtain dendritic DNA macromolecules; then, the dendritic DNA macromolecules were mixed with ascorbic acid and Cu (Ac) was immediately added ₂ The above solution was incubated to complete the preparation of dendritic DNA macromolecules-Cu NMs.

2. A fluorescence detection of Pb as claimed in claim 1 ²⁺ Is characterized by being used for detecting Pb in serum and cell lysate ²⁺ Is contained in the composition.

3. A fluorescence detection of Pb as claimed in claim 1 ²⁺ Is characterized by comprising the following steps:

（1）Pb ²⁺ establishment of a fluorescence detection standard curve:

pb at different concentrations ²⁺ Mixing and incubating the standard solution and the dendritic DNA macromolecule-Cu NMs solution, detecting by adopting a fluorescence spectrophotometer after the mixed reaction, and establishing fluorescence quenching rate signals of the dendritic DNA macromolecule-Cu NMs and different Pb ²⁺ A standard curve of correspondence between concentrations;

(2) Calculating Pb in the sample to be detected by an external standard method ²⁺ Is contained in the composition.

4. A fluorescence detection of Pb according to any of claims 1-3 ²⁺ Is characterized in that 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.

5. A fluorescence detection of Pb as claimed in claim 1 ²⁺ Characterized in that the MOPS buffer concentration is 1-20mM MOPS,15-300mM NaAc,0.1-2 mM MgCl ₂ the pH is 7.5-8.0.

6. A fluorescence detection of Pb as claimed in claim 3 ²⁺ The method is characterized in that serum or cell lysate to be detected is filtered, mixed and incubated with dendritic DNA-Cu NMs, and fluorescence detection is carried out on the mixed solution.

7. A fluorescence detection of Pb as claimed in claim 3 ²⁺ Is characterized in that the concentration of the DNA macromolecule-Cu NMs solution is 30-600 nM.

8. A method for preparing dendritic DNA macromolecules-Cu NMs, comprising the steps of:

(1) Mixing three equal amounts of oligonucleotide chains Y0a, Y0b and Y0c in MOPS buffer to obtain a mixed solution; then, heating the mixed solution to 90 ℃, cooling to 4 ℃, and incubating the annealed sample at room temperature to obtain a Y-type DNA building block Y0;

(2) Referring to step (1), three other equal amounts of oligonucleotide chains Y1a, Y1b, Y1c produce another Y-type DNA building block Y1; y1 is named as Y1-SteX, wherein X represents a number of bases, which is 0, 6, 13, 20 or 27;

(3) The Y0 and the Y1-SteX are subjected to base complementation pairing, and the Y0 and the Y1-SteX are hybridized according to the concentration ratio of 1 to 3 to obtain dendritic DNA macromolecules; then, the dendritic DNA macromolecules were mixed with ascorbic acid and Cu (Ac) was immediately added ₂ The above solution was incubated to complete the preparation of dendritic DNA macromolecules-Cu NMs.

9. The dendritic DNA macromolecule-Cu NMs prepared by the method according to claim 8 and application thereof in aptamer biosensors or cell imaging.

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