CN105598466A - A kind of synthesis method of fluorescent copper nanocluster - Google Patents

Abstract

The invention relates to a synthesis method of a fluorescent copper nanocluster, which belongs to the technical field of biochemistry and comprises the steps of firstly, synthesizing Cu-containing nano clusters²⁺The solution is mixed with lysozyme solution evenly, hydroxylamine hydrochloride is added into the lysozyme solution, the pH value is adjusted to 10-13,reacting for 6-24 h, wherein the Cu is²⁺The molar ratio of the lysozyme to the hydroxylamine hydrochloride is 1: 1-8: 40-400. the invention has the beneficial effects that the synthesized fluorescent copper nano cluster has the advantages of bright emitted fluorescence, good water solubility, high stability, good biocompatibility, low toxicity and the like.

Description

A kind of synthesis method of fluorescent copper nanocluster

technical field

The invention relates to a method for synthesizing fluorescent copper nanoclusters, belonging to the technical field of biochemistry.

Background technique

In recent years, ultrasmall fluorescent metal nanoclusters containing a few to tens of metal atoms, linking atoms and large-sized nanoparticles, have attracted extensive attention due to their size-dependent unique physical and chemical properties. Since the size of metal nanoclusters is similar to the Fermi wavelength of electrons, it exhibits discrete energy levels and molecular-like properties, such as size-tunable electronic transition states and strong fluorescence properties. Metal nanoclusters are considered as novel, potential, and practical nanomaterials with practical applications in many fields, such as: cell imaging, ion sensing, and catalysis. Compared with traditional fluorescent quantum dots, metal nanoclusters have many superior properties, such as: low toxicity, ultra-small size, good biocompatibility and multifunctional surface chemical properties, which make them more and more popular in the field of biochemistry. Much attention.

In the past ten years, extensive research on fluorescent metal nanoclusters has mainly focused on the synthesis of fluorescent Au, Ag, and Pt nanoclusters, applying various types of templates, such as: peptides, proteins, DNA, polymers, dendrimers Polymers and biothiol small molecules. However, Au, Ag, and Pt are noble metals, and thus, the synthesis of Au, Ag, and Pt nanoclusters is very expensive. As we all know, Cu, a non-precious metal element, is abundant on the earth, and it is cheap and easy to get in daily life. As a potential nanomaterial, copper nanoclusters can be applied in many research fields such as catalysis, sensing, cell labeling and cell imaging. However, since the synthesis of ultra-small sized copper nanoclusters is very difficult, and even the synthesized copper nanoclusters are easily oxidized in air, the preparation of ultra-small sized and highly stable copper nanoclusters is still a major challenge at present. , therefore, the synthesis of Cu nanoclusters is still at a preliminary stage compared with the synthetic methods of Au and Ag nanoclusters.

Contents of the invention

The invention synthesizes a novel fluorescent copper nanocluster, and the fluorescent copper nanocluster has the advantages of bright emission of fluorescence, good water solubility, high stability, good biocompatibility, low toxicity and the like.

The invention provides a method for synthesizing fluorescent copper nanoclusters. The method for synthesizing is to firstly mix the solution containing Cu ²⁺ with the lysozyme solution, then add hydroxylamine hydrochloride to it, adjust the pH to 10-13, and react 6 ~24h, the molar ratio of Cu ²⁺ , lysozyme and hydroxylamine hydrochloride is 1:1~8:40~400.

The reaction temperature in the present invention is preferably 20-45°C.

The beneficial effect of the invention is that the synthesized fluorescent copper nano-cluster has the advantages of bright emission of fluorescence, good water solubility, high stability, good biocompatibility, low toxicity and the like.

Description of drawings

9 pieces of accompanying drawings of the present invention,

Fig. 1 is the fluorescence intensity of the fluorescent copper nano-clusters synthesized in embodiments 2-5;

Fig. 2 is the fluorescence intensity of the fluorescent copper nano-cluster synthesized by embodiments 6-12;

Fig. 3 is the fluorescence intensity of the fluorescent copper nano-cluster synthesized by embodiments 13-17;

Fig. 4 is the fluorescence intensity of the fluorescent copper nano-cluster synthesized in Examples 18-21;

Fig. 5 is the fluorescence intensity of the fluorescent copper nano-cluster synthesized in Examples 22-27;

Fig. 6 is the UV-vis absorption spectrogram of the fluorescent copper nanocluster and lysozyme synthesized in Example 10;

Fig. 7 is the fluorescence excitation and emission spectrogram of the fluorescent copper nanocluster synthesized in embodiment 10;

Fig. 8 is the effect of H2O2 of different molar concentrations _on the fluorescent intensity of the fluorescent copper nanoclusters synthesized in Example ₁₀ ;

Fig. 9 is a fluorescence emission spectrum diagram of the fluorescent copper nanocluster synthesized in Example 10 after being stored in the dark at 4°C for 2 months and the fluorescent copper nanocluster of Example 10 after reacting for 16 hours.

detailed description

The following non-limiting examples can enable those skilled in the art to understand the present invention more fully, but do not limit the present invention in any way.

In the following examples, unless otherwise specified, the experimental methods used are conventional methods, and the reagents used can be purchased from chemical or biological reagent companies.

The specific implementation manner of the present invention will be described in detail below in conjunction with the technical solutions.

Embodiment 1-5

A kind of synthesis method of fluorescent copper nanocluster, described synthesis method is first 5mL of Cu(NO ₃ ) ₂ solutions with different concentrations and 5mL concentration of 20mg/mL lysozyme solution were stirred for 10min at 25°C, and then 1mL concentration of 8M Hydroxylamine hydrochloride was added therein, the pH was adjusted to 12 with NaOH, and the reaction was stirred at 25°C for 16 hours to obtain fluorescent copper nanoclusters;

See Table 1 for the concentrations of the Cu(NO ₃ ) ₂ solutions in Examples 1-5.

Table 1 embodiment 1～5Cu(NO ₃ ) The concentration of ₂ solutions

Embodiment 6-12

A kind of synthesis method of fluorescent copper nanocluster, described synthesis method is first 5mL concentration is the Cu(NO ₃ ) ₂ solution of 5mM and 5mL concentration is 20mg/mL lysozyme solution 25 ℃ and stirs 10min, then 1mL different concentrations Hydroxylamine hydrochloride was added therein, the pH was adjusted to 12 with NaOH, and the reaction was stirred at 25°C for 16 hours to obtain fluorescent copper nanoclusters;

The concentrations of hydroxylamine hydrochloride in Examples 6-12 are shown in Table 2.

The concentration of table 2 embodiment 6～12 hydroxylamine hydrochloride

Examples 13-17

A kind of synthetic method of fluorescent copper nanocluster, described synthetic method is that 5mL concentration is the Cu(NO ₃ ) ₂ solution of 5mM and 5mL concentration is the lysozyme solution 20mg/mL 25 ℃ of stirring 10min, then 1mL concentration is Add 8M hydroxylamine hydrochloride to it, adjust different pH values, stir and react at 25°C for 16 hours, and obtain fluorescent copper nanoclusters;

See Table 3 for the pH values of Examples 13-17.

The pH value of table 3 embodiment 13～17

Example 13 Example 14 Example 15 Example 16 Example 17 pH value 4 6 8 10 12

Examples 18-21

A kind of synthetic method of fluorescent copper nanocluster, described synthetic method is that 5mL concentration is the Cu(NO ₃ ) ₂ solution of 5mM and 5mL concentration is the lysozyme solution 20mg/mL 25 ℃ of stirring 10min, then 1mL concentration is Add 8M hydroxylamine hydrochloride, adjust the pH to 12 with NaOH, stir and react at different temperatures for 16 hours to obtain fluorescent copper nanoclusters;

See Table 4 for the reaction temperatures of Examples 18-21.

The temperature of reaction of table 4 embodiment 18～21

Examples 22-27

A kind of synthetic method of fluorescent copper nanocluster, described synthetic method is that 5mL concentration is the Cu(NO ₃ ) ₂ solution of 5mM and 5mL concentration is the lysozyme solution 20mg/mL 25 ℃ of stirring 10min, then 1mL concentration is Add 8M hydroxylamine hydrochloride, adjust the pH to 12 with NaOH, and stir at 25°C for different times to obtain fluorescent copper nanoclusters;

See Table 5 for the reaction times of Examples 22-27.

The reaction time of table 5 embodiment 22～27

Effect Example 1

Test the UV-vis absorption spectrum of the fluorescent copper nanocluster solution synthesized in Example 10 and the lysozyme solution, the results are shown in Figure 6, a is the UV-vis absorption spectrum of the fluorescent copper nanocluster synthesized in Example 10, and b is the bacteriolysis The UV-vis absorption spectrogram of enzyme is obtained by Fig. 6. Compared with the obvious absorption peak of lysozyme at 280nm, the fluorescent copper nanocluster synthesized in Example 10 has no obvious absorption peak in the whole UV-vis light region. The tiny hump that appears around 280nm is due to unreacted lysozyme. In addition, there is no obvious surface plasmon resonance absorption band at 560-600nm, which proves that no large-sized copper nanoparticles are formed.

Effect example 2

The fluorescent copper nanocluster solution synthesized in Example 10 is tested for fluorescence excitation and emission spectra, the results are shown in Figure 7, a is the fluorescence excitation spectrum, and b is the fluorescence emission spectrum, as shown in Figure 7, the Stokes shift is 262nm, which proves that the emission spectrum can Effectively avoid interference from excitation light sources. Compared with organic dyes, the large-scale Stokes shift can effectively avoid the crossover between excitation and emission spectra.

Effect example 3

Mix H2O2 with concentrations of _0nM , 10nM, 100nM, _500nM , 1μM, 10μM, 100μM, 500μM, 1mM with an equal volume of fluorescent copper nanoclusters obtained in Example 10, react at 25°C for 12h, and measure their fluorescence respectively Intensity value, investigate the influence of different molar concentrations of H ₂ O ₂ on the fluorescence intensity of the fluorescent copper nanoclusters obtained in Example 10, the results are shown in Figure 8, from Figure 8, H ₂ O ₂ on the fluorescent copper nanoclusters obtained in Example 10 The fluorescence intensity value of the cluster has almost no effect, which proves that the fluorescent copper nanocluster obtained in Example 10 has strong oxidation resistance.

Effect Example 4

The fluorescence emission spectrum of the fluorescent copper nanocluster synthesized in Example 10 after being stored in the dark at 4°C for 2 months was compared with the fluorescence emission spectrum of the fluorescent copper nanocluster of Example 10 after 16 hours of reaction. The fluorescence emission spectrum of the fluorescent copper nanocluster of Example 10, b is the fluorescence emission spectrum of the fluorescent copper nanocluster synthesized in Example 10 after being stored in the dark at 4°C for 2 months, as shown in Figure 9, the fluorescence intensity values of the two are almost There is no change, which proves that the fluorescent copper nanoclusters synthesized in Example 10 have high stability.

Claims (2)

1. a synthetic method for fluorescence copper nano-cluster, is characterized in that: described synthetic method is for first will be containing Cu²⁺'sSolution and lysozyme soln mix, then hydroxylamine hydrochloride is added wherein, regulate pH to 10～13, reaction 6～24h,Described Cu²⁺, lysozyme and hydroxylamine hydrochloride mol ratio be 1:1～8:40～400.

2. synthetic method according to claim 1, is characterized in that: described reaction temperature is 20～45 DEG C.