CN111318722A

CN111318722A - Fluorescent copper nanoparticles, preparation method thereof and application thereof in detecting content of riboflavin and sulfur ions

Info

Publication number: CN111318722A
Application number: CN202010344539.4A
Authority: CN
Inventors: 王欢
Original assignee: Qinghai Nationalities University
Current assignee: Qinghai Nationalities University
Priority date: 2020-04-27
Filing date: 2020-04-27
Publication date: 2020-06-23
Anticipated expiration: 2040-04-27
Also published as: CN111318722B

Abstract

The invention provides a preparation method of fluorescent copper nanoparticles, which is characterized in that anhydrous copper sulfate is used as a copper source, ascorbic acid is added, and the fluorescent copper nanoparticles are prepared by adopting a low-temperature rotary evaporation closed system liquid phase chemical reduction method. The invention also provides fluorescent copper nanoparticles prepared by the method and application of the fluorescent copper nanoparticles in detection of VB2 and sulfur ion content. The invention firstly synthesizes the copper nano solution with blue fluorescence, and because the copper nano particle has very strong fluorescence at about 440nm and the riboflavin has strong ultraviolet absorption at about 450nm, the copper nano particle and the riboflavin are almost completely finishedTotally coincident, a resonance energy transfer (FERT) process is supposed to occur between the copper nano-meter and the riboflavin, so that a fluorescence resonance energy transfer system between the copper nano-meter and the riboflavin is established, and the riboflavin can be detected. In the copper nano-and riboflavin system, the S is^2‑The reaction with VB2 can quench the fluorescence of the copper nano-particles and the riboflavin system, so that the copper nano-particles and the riboflavin system can be used for detecting sulfur ions.

Description

Fluorescent copper nanoparticles, preparation method thereof and application thereof in detecting content of riboflavin and sulfur ions

Technical Field

The invention relates to a fluorescent copper nano particle, a preparation method thereof and application thereof in detecting riboflavin and sulfur ion content.

Background

In recent years, fluorescence resonance energy transfer (fluorescence resonance energy transfer) is widely used in the field of biosensing, and refers to a non-radiative photophysical process of energy transfer between a fluorescence donor group and an acceptor, wherein the fluorescence resonance energy transfer occurs between the donor and the acceptor through the interaction of a dipole and a dipole. The conditions for fluorescence resonance energy transfer are harsh, i.e., they occur when the emission spectrum of the donor overlaps the absorption spectrum of the acceptor by more than 30% and the distance between the two is less than 10 nm. At a certain excitation wavelength, the emission of the fluorescent donor can be absorbed by a nearby fluorescent acceptor, which results in a reduction in the fluorescence intensity of the donor and the lifetime of the excited state, the acceptor is excited into an electronically excited state, and finally, the acceptor returns to the ground state, so that the emitted photons come mainly from the acceptor, resulting in a significant stokes shift. The fluorescence resonance energy transfer method has the advantages of simple equipment, high sensitivity, less required samples, high analysis speed and the like, and has been used for measuring and detecting small molecules such as metal ions, DNA, glucose and the like in recent years.

Nanoparticles (NPs) have a size of about 1 to 100nm, have a specific small-size property, change colorimetric, fluorescent and electrochemical properties according to substances in the environment, and have been widely used in many fields including environmental monitoring, biomedical molecular detection, and the like. The fluorescent nano material has unique physical, chemical and optical properties such as low toxicity, excellent light stability, good water solubility, good biocompatibility and the like. Compared with fluorescent noble metal nano materials, the fluorescent copper nano has the advantages of high catalytic activity, good conductivity, low cost and the like, and is a better fluorescent probe. The synthesis method of the copper nanoparticle adopts a chemical reduction method that copper chloride is taken as a precursor and sodium formaldehyde sulfoxide is taken as a reducing agent to synthesize the copper nanoparticle by ZeenatArif and the like, adopts oleic acid as a capping agent, and adopts surfactants such as polyvinyl alcohol/polyvinyl pyrrolidone and the like to prevent the formation of oxides and agglomeration. Chen Shenhua et al uses poly (thymine) template to synthesize fluorescent copper nano-particle hydrogel and provides a visual and portable organophosphorus pesticide detection strategy.

Riboflavin, also known as vitamin B2, is an important member of the water-soluble vitamin family, a micronutrient readily absorbed by the human body, and consists of promethazine and ribitol. Riboflavin is the core module of Flavin Mononucleoside (FMN) and Flavin Adenine Dinucleotide (FAD) coenzymes and plays an important role in carbohydrate, fat and protein metabolism. An excess of riboflavin poses a threat to human health because riboflavin is a highly potent photosensitizer and causes oxidative damage to tissues and DNA when the riboflavin is in excess. Lack of riboflavin in the diet can cause health problems such as stomatitis, reddened tongue, sore throat, inflammation of the corners of the mouth, itchy and watery eyes, oily scaly rash, nasolabial sulcus, and the like. Therefore, the method is crucial to the analysis and detection of the riboflavin content in daily diet and medicines, and at present, many methods for detecting riboflavin are reported, such as a high performance liquid chromatography, a fluorescence method, a colorimetric method, an electrochemical method and a capillary electrophoresis method. Among the detection methods, the fluorescence method has the advantages of simple operation, high sensitivity, low cost and the like, and the previously reported detection method has a complex system and a narrow linear range, so that the invention designs a rapid, simple and sensitive method for fluorescence detection of riboflavin.

Inorganic anions play a crucial role in industrial, biological and environmental processes. Among various anions, sulfide ions have very high toxicity and are harmful to human health and ecological environment. On the one hand, most environmental sulfides are widely distributed as important environmental indicators in tap water and wastewater discharged from industry, and can cause mucosal irritation, loss of consciousness and respiratory paralysis if a person is excessively exposed to the sulfides. On the other hand, S^2-Protonated HS-and H₂S, has proven to be a more toxic and harmful substance than sulfur ions. H₂S is an important endogenous gas signaling molecule that regulates cardiovascular, neurological, immunological, endocrine, and gastrointestinal functions. When the concentration of blood sulfide is in the range of 10-100. mu.M, the concentration of hydrogen sulfide in the central nervous system is in the range of 50-160. mu.M. H₂Abnormal S level and Alzheimer 'S disease, Down' S syndrome, diabetes, liver cirrhosis, etcThe disease is related. Therefore, it is necessary to develop a sensor for ultra-sensitive detection of sulfide ions. The currently reported methods for detecting sulfide ions include: the fluorescence method, the ion chromatography, the electrochemiluminescence method, the thin layer chromatography, the colorimetric method and the like have certain disadvantages in application, such as expensive instruments, complex sample processing process, difficult operation and the like, wherein the fluorescence method is relatively simple to operate and high in sensitivity, a plurality of fluorescence probes are compounded like carbon dots and noble metals to detect sulfur ions, but the cost of raw materials is high, so that the inorganic fluorescence nanoprobes are paid more and more attention due to the fact that the inorganic fluorescence nanoprobes are simple to prepare and low in cost.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a fluorescent copper nanoparticle, a preparation method thereof and application thereof in detecting the content of riboflavin and sulfur ions.

The invention provides a preparation method of fluorescent copper nanoparticles, which comprises the steps of taking anhydrous copper sulfate as a copper source, adding ascorbic acid, and preparing the fluorescent copper nanoparticles by adopting a low-temperature rotary evaporation closed system liquid-phase chemical reduction method; preferably, a closed shading system of low-temperature rotary evaporation is adopted.

After shading, the yield of the fluorescent copper nanoparticles can be improved from 5.0% to 5.2% compared with the situation of not shading.

Preferably, the molar ratio of the anhydrous copper sulfate to the ascorbic acid is 1: 10.

preferably, the NaOH solution is added into anhydrous copper sulfate, then ultrapure water is added, the mixture is stirred, the mixture is transferred into a rotary evaporation bottle wrapped by an opaque object, a condensation switch and a vent plug are closed, the ascorbic acid solution is added, the mixture is rotated for 10min at the temperature of 30-90 ℃, the pH value is adjusted to 5-10 by using 0.5mol/L NaOH solution, and the rotation is continued for 3-17 hours at the temperature of 30-90 ℃, so that the fluorescent copper nanoparticle solution is obtained.

More preferably, the method comprises adding NaOH solution into anhydrous copper sulfate, adding ultrapure water, stirring, transferring into a rotary evaporation bottle wrapped with an opaque substance, closing a condensation switch and a vent plug, adding ascorbic acid solution, rotating at 50 ℃ for 10min, adjusting pH to 8-9 with 0.5mol/L NaOH solution, and rotating at 50 ℃ for 15 hours to obtain the fluorescent copper nanoparticle solution.

The invention provides fluorescent copper nanoparticles prepared by the preparation method.

The invention provides application of the fluorescent copper nanoparticles in detection of VB2 and sulfur ion content.

The invention provides application of the fluorescent copper nanoparticles in detecting VB2 in fruits and vegetables and detecting the content of sulfur ions in serum.

The invention provides a method for detecting VB2 content, which comprises the steps of taking 36.025 mu M of fluorescent copper nanoparticle solution prepared by claim 4, sequentially adding 0.1M PBS (pH 7.0-8.0) and solution to be detected, standing for 4-5min, carrying out fluorescence detection, detecting fluorescence intensity under 525nm emission wavelength under the conditions that the excitation wavelength is 360nm and the slit width is 10 × 10nm, carrying out contrast experiment by taking series of VB2 standard solutions with known concentrations as contrast groups, drawing a standard curve equation by taking the concentration of VB2 standard solution as a horizontal coordinate and the fluorescence intensity of riboflavin solution at 525nm as a vertical coordinate, substituting the fluorescence intensity of the solution to be detected at 525nm into the standard curve equation to obtain VB2 in the solution to be detected₂The concentration of (c).

Preferably, the pH is 7.5.

The invention provides a method for detecting the content of sulfur ions, which comprises the following steps: 36.025 μ M of the fluorescent copper nanoparticle solution prepared in claim 4 was added to a 0.1M PBS solution with pH 7.0-8.0, followed by VB of known concentration₂Shaking the standard solution, standing for 4-5min, and adding the S to be detected^2-Standing the solution for 30-35min, detecting S at emission wavelengths of 440nm and 525nm under the conditions of excitation wavelength of 360nm and slit width of 10 × 10nm^2-The fluorescence intensity of (a); and using a series of known concentrations of S^2-The standard solution is used as a control group to carry out a control experiment, and S is used^2-Taking the concentration of the standard solution as an abscissa and taking the fluorescence intensity ratio of F525/F440 as an ordinate to draw a standard curve equation; substituting the fluorescence intensity ratio of F525/F440 of the solution to be detected into a standard curve equation to obtain S in the solution to be detected^2-The concentration of (c).

Preferably, the pH is 7.5.

The invention takes Ascorbic Acid (AA) as a reducing agent to prepare copper nano particles (CuNPs) with blue fluorescence, and the copper nano particles are used for measuring the content of riboflavin and sulfur ions, such as measuring the content of riboflavin in vegetables and fruits and measuring the content of sulfur ions in serum.

The invention firstly synthesizes the copper nano solution with blue fluorescence, and establishes a novel system, because the copper nano particles have strong fluorescence at about 440nm, the riboflavin has strong ultraviolet absorption at about 450nm, the two almost completely coincide, and the resonance energy transfer (FERT) process is supposed to occur between the copper nano particles and the riboflavin, so the fluorescence resonance energy transfer system between the copper nano particles and the riboflavin is established, and the system can be used for detecting the riboflavin. In the copper nano-and riboflavin system, the S is^2-The reaction with VB2 is carried out, so that the fluorescence of the copper nano-meter and the riboflavin system is quenched, and the mechanism for detecting the riboflavin and the sulfur ions based on the fluorescence of the copper nano-meter is shown in FIG. 1.

FIG. 1 shows the fluorescence detection VB2 and S^2-Schematic process diagram of (1).

The invention designs a fluorescence resonance energy transfer system and is used for riboflavin (VB2) and sulfur ions (S) in orange juice and rabbit serum^2-) Detection of (3). In the fluorescence sensing system, copper nanoparticles (CuNPs) serve as fluorescent probes. Fluorescent copper nanoparticles with blue fluorescence are prepared by a liquid phase chemical reduction method of low-temperature rotary evaporation. In the whole preparation process, anhydrous copper sulfate is used as a copper source, and Ascorbic Acid (AA) is used as a reducing agent, a protective agent and a stabilizing agent. Because the copper nanoparticles have very strong fluorescence emission peak at about 440nm, and VB2 has wide ultraviolet-visible absorption peaks at about 370nm and 450nm, the two are almost completely coincided, and CuNPs and VB are presumed to be₂There may be resonance energy transfer (FERT) occurring in between. Therefore, the invention designs a fluorescence resonance energy transfer system between CuNPs and VB2 and is used for detecting the riboflavin. When the direction is from CuNPs to VB₂Further introducing S into the system^2-Due to S^2-And VB₂There is a specific reaction between them, so thatObtaining CuNPs-VB₂The fluorescence intensity of VB2 in the system is linearly quenched, and the fluorescence intensity of CuNPs is almost unchanged. Therefore, a dual-signal ratio type fluorescence sensing system is further constructed to detect the sulfur ions. The linear range of riboflavin obtained is between 0.99nM and 34.641nM, the limit of detection is 0.455nM (S/N-3) and the correlation coefficient is R²0.9981 (n-3), the method was used for vitamin B2 tablets with VB₂The content measurement of the tablets and the fruits and vegetables rich in VB2 have the relative standard deviation lower than 0.35 percent and the standard recovery rate of 98 to 109.5 percent. The linear range of the detection of the sulfide ion is 5.33 mu M-40.6 mu M, the detection limit is 0.955 mu M (S/N is 3), and the correlation coefficient is R²The content of sulfur ions in serum is detected at 0.9962(n is 3), and the standard recovery rate is 97.5-104.4%, so that the system has potential practical value for VB2 and S2-detection in actual samples. The invention establishes a rapid and simple method for simultaneously measuring VB2 and S2 by taking copper nanoparticles as fluorescent probes.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

In FIG. 2, A is the UV-VIS absorption spectrum and fluorescence emission spectrum of the aqueous solution of CuNPs; the inset is a photograph under daylight (left) and under 365nm ultraviolet light (right); and the B picture is the emission peak of the CuNPs aqueous solution under the excitation wavelength of 260 nm-400 nm and the excitation peak under the emission wavelength of 440 nm.

FIG. 3 is an infrared absorption spectrum of CuNPs.

In FIG. 4, Panel A is a Transmission Electron Microscope (TEM) image of CuNPs prepared; and B is a particle size distribution diagram of CuNPs.

In FIG. 5, A and B are graphs showing the effect of different buffer systems on the fluorescence intensity of copper nanoparticles; and the graph C and the graph D show the effect of different pH values of the PBS buffer system on the fluorescence intensity of the copper nanometer.

FIG. 6 shows fluorescence intensities of copper nano-solutions of different concentrations.

FIG. 7 shows fluorescence intensity of copper nano-solution at different times.

In FIG. 8, panel A shows the change of fluorescence intensity of copper nanoparticles with time when VB2 was added at a certain concentration; b is shown when a certain concentration of S is added^2-The change in the VB2 fluorescence intensity with time.

FIG. 9 shows the ultraviolet-visible absorption spectrum of riboflavin and the fluorescence emission spectrum of CuNPs.

FIG. 10 shows the UV-VIS absorption spectrum of riboflavin with sulfide ions.

In FIG. 11, graph A is a graph showing the change in fluorescence intensity with the concentration of VB 2; the B plot is a linear fit curve of fluorescence intensity at wavelength 525nm as a function of VB2 concentration.

In FIG. 12, graph A shows fluorescence intensity as a function of S^2-A curve of concentration change; b is the fluorescence intensity with S^2-A linear fit curve of the concentration change.

FIG. 13 shows the quenching rate of various interfering substances on the fluorescence intensity of CuNPs solution.

FIG. 14 shows the quenching rate of each interfering substance on the fluorescence intensity of riboflavin.

Detailed Description

1 instruments and reagents

1.1 instruments

Cary Eclipse fluorescence analyzer (RF-5301PC, Shimadzu, Japan); a dual-beam uv-vis spectrophotometer (TU-1901, beijing prosperous instrument, llc); fourier Infrared spectrometer (IRPrestige-21, Shimadzu, Japan); a rotary evaporator (EYEL4, Shanghai Ailang instruments, Inc.); acidimeters (seven excellence, mettlettolido corporation); electronic balance (XP205, mettlerlatolite inc).

1.2 reagents

Anhydrous cupric sulfate (CuSO)₄) Purchased from national drug group chemical agents, ltd; ascorbic Acid (AA), sodium sulfide (Na)₂S) purchased from Shanghai Guanuo chemical science and technology, Inc.; sodium hydroxide (NaOH), dipotassium hydrogen phosphate (K)₂HPO₄) Potassium dihydrogen phosphate (KH)₂PO₄) And potassium chloride (KCl) from Dalocene chemical of TianjinA factory; rabbit serum was from the university pharmacological laboratory; all the reagents are analytically pure; the water used for the experiments was ultrapure water, a Milli-Q system (Milli-Q, MilliporeCorp,>18.25MΩ.cm)。

2 experimental part

2.1 preparation of copper NanolsCuNPs

Weighing 32mg of anhydrous copper sulfate, adding 2mL of 0.5mol/L NaOH solution, adding 20mL of ultrapure water, stirring for 2min, transferring into a rotary evaporation bottle (the rotary evaporation bottle is wrapped by a black bag), closing a condensation switch, closing an exhaust plug, adding 20mL of 0.1mol/L ascorbic acid solution, enabling the solution to be changed into yolk color, rotating at 50 ℃ for 10min, adjusting the pH value to 8-9 by using 0.5mol/L NaOH solution, continuing rotating at 50 ℃ for 15 hours, enabling the solution color to be finally changed into green brown, forming CuNPs, and keeping the solution in a refrigerator at 4 ℃ in a dark place.

2.2 formation of fluorescence resonance energy

VB2 was detected fluorescently in PBS buffer at pH 7.5(0.1M) at room temperature. The specific process is as follows: mu.l (36.025mM) of CuNPs solution was transferred to a quartz cuvette and 3mL (0.1M, pH 7.5) of PBS buffer solution was added, and 10. mu.L of VB2 solutions with concentrations of 0.99, 3.846, 7.407, 11.5, 15.966, 20.635, 25.373 and 34.641nM were added, and the fluorescence intensity was measured under the following conditions to determine the linear range and the detection limit: the excitation wavelength is 360nm, the wavelength range is 380-620nm, and the slit width is 10 nm.

2.3 VB in actual samples₂Detection of (2)

The actual sample selected in the experiment is a medicine vitamin B2 tablet, vegetables and fruits such as orange and carrot are pre-treated, firstly crushed, centrifuged, supernatant is taken, concentration required by the experiment is prepared, and the sample is refrigerated in a refrigerator for standby.

The detection method comprises the following steps of taking 25 mu L (36.025uM) of CuNPs solution, sequentially adding 3mL of PBS (pH 7.5) and 300 mu L of the actual sample stock solution, standing for 4min, carrying out fluorescence detection, and detecting the fluorescence intensity under the emission wavelength of 525nm under the conditions that the excitation wavelength is 360nm and the slit width is 10 × 10nm, wherein the resonance energy transfer effect between the CuNPs and VB2 is utilized to detect a sample rich in vitamin B2.

2.4 fluorescent detection of sulfide ions (S)^2-)

At room temperature, the process of detecting the sulfur ions based on the CuNPs-VB2 fluorescent probe is as follows: mu.L (36.025. mu.M) of CuNPs solution was pipetted, 3mL (0.10M, pH 7.5) of PBS solution was added, and then 300. mu.L (0.1. mu.M) of VB was added₂Shaking the standard solution, mixing, standing for 4min, adding S with different concentrations^2-Standing the solution for 30min, detecting the fluorescence intensity under each condition, determining the linear detection range and the detection limit, and detecting S with different concentrations added in a CuNPs-VB2 system under the conditions that the excitation wavelength is 360nm and the slit width is 10 × 10nm and the emission wavelengths are 440nm and 525nm^2-The fluorescence intensity of (2).

2.5 Sulfur ions (S) in the actual sample^2-) Determination of the content

The actual sample was selected from rabbit serum, blood from rabbits collected in the institute's pharmacological laboratory, centrifuged, and refrigerated for use. The actual samples were used in the experimental conditions under item 2.4.

3 results and discussion

In the invention, the fluorescent copper nanoparticles with high quantum yield are prepared by using a simple, green and cheap closed reduction reaction. Throughout the reaction, the environmentally friendly ascorbic acid serves as both a reducing agent and a protecting agent, as well as a stabilizing agent and an encapsulating agent. The prepared CuNPs are characterized by UV-Vis absorption spectrum, fluorescence spectrum, infrared absorption spectrum, transmission electron microscope and the like.

3.1 UV-Vis absorption Spectroscopy and fluorescence emission Spectroscopy of CuNPs

The synthesized CuNPs are optically characterized, the result is shown in figure 2(A), line a is the UV-Vis absorption spectrum of the prepared CuNPs, and the result shows that continuous absorption exists in ultraviolet and visible light regions, and the maximum absorption wavelength is 295 nm. Line b is the fluorescence emission spectrum of the prepared CuNPs, and the maximum fluorescence emission wavelength of the CuNPs is obtained at 440nm when the excitation wavelength is 360 nm. The prepared CuNPs aqueous solution is grey-green under sunlight and bright blue under the excitation of a 365nm ultraviolet lamp. The synthesized CuNPs showed an excitation-dependent fluorescence behavior as in fig. 2(B), as do most fluorescent nanoparticles. When the wavelength is 360nm, the fluorescence emission of CuNPs is strongest at 440 nm. The synthesized CuNPs have narrow and symmetrical emission bands, and the uniformity and monodispersity thereof are confirmed.

In FIG. 2, A is the UV-VIS absorption spectrum and fluorescence emission spectrum of the aqueous solution of CuNPs; and the B picture is the emission peak of the CuNPs aqueous solution under the excitation wavelength of 260 nm-400 nm and the excitation peak under the emission wavelength of 440 nm.

3.2 Infrared absorption Spectrum of CuNPs

FIG. 3 is an infrared absorption spectrum of CuNPs.

The infrared absorption spectrum of CuNPs is shown in FIG. 3, which shows that the wavelength of CuNPs is 3334cm^-1The absorption band has a characteristic O-H stretching vibration at 1633cm^-1The peak is C ═ C characteristic absorption peak, 1333cm^-1The peak is a characteristic peak of C-OH stretching vibration. These results indicate that ascorbic acid is present as a coating agent on the surface of CuNPs.

3.3 TEM features of CuNPs

In fig. 4, a diagram shows Transmission Electron Microscope (TEM) imaging of the prepared CuNPs, and the results show that the CuNPs have good spherical structures and good dispersibility. The average particle size of CuNPs is 10nm through random point selection statistics, and the CuNPs have a narrow particle size distribution range of 8-13nm, as shown in a B diagram in figure 4.

3.4 Effect of different buffer solutions on the fluorescence intensity of CuNPs

It is known that the pH of the buffer system and buffer solution has a certain influence on the fluorescence intensity of the phosphor. Therefore, in the present invention, five buffer systems of phosphate, borate, birutan-Robinson (Britton-Robinson), boric acid-potassium chloride-sodium hydroxide and Tris-HCL are selected respectively for research, and at the same pH (pH 7.5), 25 μ L (36.025 μ M) of the prepared CuNPs aqueous solution is taken, 3mL of five buffer solutions are added respectively, the slit width is 10nm at the excitation wavelength of 360nm, and the fluorescence intensity of the five buffer solutions at the emission wavelength of 440nm is measured, and as a result, as shown in fig. 5(a and B), it is obvious that the most preferable of the five buffer solutions is Phosphate Buffer Solution (PBS), and therefore, the most preferable buffer solution selected in the following experiments is phosphate buffer solution.

We further optimized the pH of the phosphate system chosen above. To 25. mu.L (36.025. mu.M) of CuNPs, 3mL of 0.1M PBS at pH 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, respectively, were added, and the slit widths were all 10nm at an excitation wavelength of 360nm, and the fluorescence intensities at copper nm at different pH's at an emission wavelength of 440nm were measured, respectively, as shown in FIG. 5(C and D). The experiment result shows that the fluorescence of the copper nanometer is strongest when the pH value is 7.5. This phenomenon occurs, probably because the stability of the copper nanoparticles is impaired under alkaline conditions.

3.5 selection of the concentration of CuNPs solution

It is well known that the concentration of the luminophore plays a crucial role in the fluorescence intensity in the investigation system. Therefore, the concentration of the copper nano solution is further optimized on the basis of the optimal condition of 3.4. Separately, 7.253 μ M (5 μ L), 14.482 μ M (10 μ L), 21.687 μ M (15 μ L), 28.868 μ M (20 μ L), 36.025 μ M (25 μ L) and 40.308 μ M (28 μ L) copper nanoparticle solutions were prepared, 3mL of PBS (pH 7.5) buffer solutions were added, the slit widths were all 10nm at an excitation wavelength of 360nm, and the fluorescence intensities of the solutions at an emission wavelength of 440nm were measured, respectively, as shown in fig. 6. The experimental results showed that when the concentration was increased to 40.308 μ M, the fluorescence emission spectrum almost overlapped with the fluorescence emission peak of 36.025 μ M copper nanoparticles, which is probably attributable to self-adsorption of the phosphor. Therefore, in the following experiments, the optimum concentration of the luminophore was chosen to be 36.025. mu.M.

3.6 stability Studies of CuNPs

The stability of the luminophor plays a role in determining the type of research on fluorescence sensing, so the applicant examines the stability of the prepared copper nanoparticles. The fluorescence intensity was measured every 5 days under optimal conditions, i.e., 0.1M PBS at pH 7.5, 36.025 μ M copper nanosol, as shown in FIG. 7. As can be seen in the figure, the fluorescence intensity of the copper nano solution is almost unchanged within 50 days, which shows that the copper nano particles prepared by the invention are relatively stable and can meet the research of a fluorescence sensing system.

FIG. 7 shows fluorescence intensity of copper nano-solution at different times.

3.7 reaction time optimization

In the fluorescent sensing research, the reaction time between the luminophor and the detection object is also the main focus of the sensing research, and the reaction time has direct influence on the detection limit. Therefore, the present invention further optimizes the reaction time between the luminophore and the detector on the basis of the optimal conditions of 3.5. Under the conditions that the excitation wavelength is 360nM, the slit width is 10nM, and the emission wavelength is 440nM, when 24.823nM VB2 solution is added into the copper nanoparticle solution, the change of the fluorescence intensity of the copper nanoparticle solution with time is shown as a graph A in FIG. 8. As is apparent from the graph a in fig. 8, the fluorescence intensity of the copper nanoparticle solution continuously decreases with the continuation of the reaction time, and when the reaction time reaches 4min, the fluorescence intensity of the copper nanoparticle solution hardly decreases, which means that the energy transfer between the donor and the acceptor is balanced. Therefore, we chose 4min as the optimal reaction time between VB2 and copper nanoparticles. FIG. 8B shows that 52.724 μ M S is continuously added into the resonance energy transfer system under the wavelength range of the excitation wavelength 360nm, the slit width 10nm, 380-620nm^2-Solution, VB2 fluorescence intensity at an emission wavelength of 525nm with the introduction of S^2-And then the reaction time between the two is changed. The research result shows that the corresponding VB2 fluorescence intensity is continuously reduced along with the continuous increase of the reaction time of the two, and when the reaction time reaches 30min, the VB2 fluorescence intensity is hardly reduced any more, so that S^2-The optimum reaction time with VB2 was 30 min.

In FIG. 8, graph A shows the copper concentration of VB2The change of the fluorescence intensity of the rice grains with time; b is shown when a certain concentration of S is added^2-The change in the VB2 fluorescence intensity with time.

3.8 discussion of detection mechanism

The ultraviolet-visible absorption spectrum of riboflavin and the fluorescence emission spectrum of the prepared CuNPs solution are shown in fig. 9. As can be seen from FIG. 9, the UV-visible absorption spectrum of riboflavin has two characteristic absorption peaks at about 360nm and 450nm, and the prepared CuNPs aqueous solution has a strong fluorescence emission peak at about 440nm under the excitation wavelength of 360 nm. From the figure, it can be clearly observed that the UV-visible absorption spectrum of riboflavin overlaps with the fluorescence emission spectrum of the CuNPs solution over a significantly larger area. From this, it is presumed that Fluorescence Resonance Energy Transfer (FRET) may occur between the two, and CuNPs serve as a donor and riboflavin serves as an acceptor. Based on the mechanism, the invention adopts the fluorescence sensing analysis technology to detect the VB2 content in the actual sample.

In the research, the S is introduced into the CuNPs-VB2 resonance energy transfer system^2-When S is present^2-The linear reduction of the fluorescent signal of VB2 can be realized, and the influence on the fluorescence intensity of the CuNPs solution is not large, thereby showing that S^2-Has stronger interaction with VB2 than S^2-Interaction with CuNPs. To further investigate the mechanism of action between the two, S is investigated here^2-VB2 and S^2-And VB2 were investigated for UV-visible absorption spectra as shown in FIG. 10. The research result shows that when S^2-After mixing with VB2, a blue shift of the UV absorption peak at 370nm was clearly observed, probably due to S^2-Has special interaction with amide groups in the molecular structure of riboflavin, and the interaction can enable S to react^2-The ring structure in the molecular structure of VB2 is destroyed, so that the unsaturation degree of the molecular structure is reduced, and the ultraviolet absorption peak at 370nm is caused to move towards the short wave direction. Based on the mechanism, the invention further constructs a ratio type fluorescent probe pair S on the basis of fluorescence resonance energy transfer^2-Sensing studies of (2).

FIG. 10 shows the UV-VIS absorption spectrum of riboflavin with sulfide ions.

3.9VB2 and S^2-Obtaining a linear relationship

3.9.1VB2 linear relation acquisition

In FIG. 11, panel A shows the trend of the fluorescence intensity of CuNPs solution with different riboflavin concentrations when riboflavin concentrations were 0.99, 3.846, 7.407, 11.5, 15.966, 20.635, 25.373 and 34.641nM, respectively. The excitation wavelength of the CuNPs solution is 360nm, and the maximum fluorescence emission wavelength of the solution can be observed at about 440 nm. When riboflavin exists, the excited state CuNPs transfers the energy of the CuNPs donor to the adjacent acceptor riboflavin through a remote non-radiative dipole-dipole coupling effect, so that the fluorescence of the adjacent acceptor riboflavin is excited to generate autofluorescence at 525 nm. When the riboflavin concentration is increased from 0.99nM to 34.641nM, the fluorescence intensity of CuNPs gradually decreases linearly from high to low, and conversely the fluorescence of riboflavin at 525nM gradually increases linearly. This phenomenon further demonstrates that resonance energy transfer occurs between CuNPs and riboflavin. And B is a standard curve obtained by fluorescence resonance energy transfer between the fluorescence intensity of the riboflavin solution at 525nm in the system and the corresponding concentration. The linear range of the system is 0.99nM-34.641nM, the detection limit is 0.125nM (S/N-3), and the standard curve is Y-23.729 [ VB2 ]]+127.241 where Y represents the fluorescence intensity of the riboflavin solution at 525nm, [ VB2]Representing the concentration of VB2 with a correlation coefficient of R²0.9981(n 3). The sensing system of CuNPs-VB2 has a wide linear range and a low detection limit compared with the previously reported detection method of riboflavin, and the results are shown in Table 1. Therefore, the CuNPs-VB2 system has potential application prospect in the detection of practical samples.

TABLE 1 comparison of analytical Performance of different sensing of Riboflavin

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2Y.Wang,P.H.Zhu,T.Tian,J.Tang,L.Wang,X.Y.Hu,Synchronous Fluorescenceas a Rapid Method for the Simultaneous Determination of Folic Acid andRiboflavin in Nutritional Beverages,Journal ofAgricultural&Food Chemistry,59(2011)12629-12634.

3G.Liu,Y.M.Wang,D.M.Sun,Simultaneous determination of vitamins B2,B6and C using silver-doped poly(L-arginine)-modified glassy carbon electrode,Journal of Analytical Chemistry,71(2016)105-112

4Q.Ma,J.Song,S.Zhang,M.Wang,Y.Guo,C.Dong,Colorimetric detectionofriboflavin by silver nanoparticles capped withβ-cyclodextrin-graftedcitrate,Colloids&Surfaces B Biointerfaces,148(2016)66-72.

3.9.2 fluorescence detection S^2-

In FIG. 12, graph A represents S^2-The fluorescence intensity of the riboflavin solution was varied by adding different concentrations of S at concentrations of 0, 5.33, 10.16, 15.19, 20.92, 25.64, 30.96, 35.28, 40.6, 45.91, 49.2, 54.8, and 60. mu.M^2-A trend graph of the change. B is the ratio of the fluorescence intensity of the riboflavin solution at the emission wavelength of 525nm to the fluorescence intensity of the riboflavin solution at the emission wavelength of 440 in the system and the corresponding S^2-Standard curve between concentrations. Due to S^2-The interaction with riboflavin is stronger than that with CuNPs, and the experimental result shows that S^2-The fluorescence intensity of VB2 can be linearly reduced, and the influence on CuNPs is almost avoided, so that the ratio type fluorescence sensing probe can be constructed by the sensing of the invention. Based on the ratio of F525/F440 and S^2-The concentration achieved a linear range of 5.33 μ M to 40.6 μ M with a limit of detection of 3.547 μ M (S/N-3), standardCurve is Y ═ 0.0084[ S ]^2-]+1.5639 wherein Y represents the fluorescence intensity ratio of F525/F440, [ S ]^2-]Represents S^2-With a correlation coefficient of R²0.9962(n is 3). The sensing system of CuNPs-VB2 compares to previously reported S^2-The detection method of (3) has a wide linear range and a low detection limit, and the results are shown in Table 2. Therefore, the reliability of the CuNPs-VB 2-system to be researched in a complex biological sample can be shown, and the feasibility of the method in practical application can be effectively shown.

TABLE 2S^2-Comparison of analytical Performance of different Sensors

References 5 to 8 are respectively:

5Gore A H,Vatre S B,Anbhule P V,et al.Direct detection of sulfideions[S2-]in aqueous media based on fluorescence quenching of functionalizedCdS QDs at trace levels:analytical applications to environmental analysis[J].The Analyst,2013,138(5):1329.

6Li C Y,Kong X F,Li Y F,et al.Ratiometric and colorimetricfluorescent chemosensor forAg+based on tricarbocyanine[J].Dyes and Pigments,2013,99(3):903-907.

7Park K S,Kim M I,Cho D Y,et al.Biosensors:Label-Free ColorimetricDetection of Nucleic Acids Based on Target-Induced ShieldingAgainst thePeroxidase-MimickingActivity of Magnetic Nanoparticles(Small 11/2011)[J].Small,2011,7(11):1497-1497.

8Hatamie A,Zargar B,Jalali A.Copper nanoparticles:A new colorimetricprobe for quick,naked-eye detection ofsulfide ions in water samples[J].Talanta,2014,121:234-238.

3.10 fluorescence detection of VB2 and S^2-Selectivity of (2)

3.10.1 selectivity of fluorescence detection of VB2

The selectivity of the method was further investigated in order to test the investigational properties of the inventive sensor. When other interferents with a concentration of more than 100 times higher than riboflavin are added to the CuNPs solution: alanine (Alanine), Methionine (Methionine), Arginine (Arginine), Glucose (Glucose), Lactose (Lactose), Sucrose (Sucrose), Na⁺，Mg²⁺，K⁺，SO₄ ^2-，Ca²⁺And Ascorbic Acid (AA), which show only a small interference with the fluorescence intensity of the CuNPs solution, as shown in fig. 13. Research results show that the CuNPs have good selectivity for detecting riboflavin.

3.10.2 fluorescence detection S^2-Selectivity of (2)

When 100-fold concentrations of other interferents were added to the CuNPs-VB 2-system: trisodium citrate (citrate), sodium potassium tartrate (tartrate), SO₃ ^2-、CO₃ ^2-、F^-、NO₃ ^-、Cl^-、SO₄ ^2-、S₂O₃ ^2-、NO₂ ^-、Br^-、HCO^3-、I^-Glutathione (glutathione), L-Phenylalanine (L-Phenylalanine), as shown in FIG. 14. The research result shows that the interference of 100 times concentration only shows little interference on the fluorescence intensity of the VB2 solution, thereby indicating S^2-The selectivity requirement of a CuNPs-VB 2-ratio sensing system can be met. The sensing exhibits high selectivity and interference rejection.

3.11 VB2 and S in actual samples^2-Detection of (2)

3.11.1 detection of VB2 in actual samples

To further investigate the feasibility of the sensing method of the present invention. The optimized sensing system is applied to the sensing research of practical samples, specifically the content of riboflavin in a vitamin B2 tablet and the content of VB2 in orange juice and carrot juice, and the experimental results are shown in tables 3 and 4. The experimental result shows that the recovery rate of the actual sample is between 98 and 109.5 percent. Therefore, the method can be well applied to the detection of the riboflavin content in the actual sample.

TABLE 3 detection of riboflavin content in vitamin B2 tablets

TABLE 4 detection of Riboflavin content in vegetables and fruits

3.11.2 actual sample S^2-Detection of (2)

To further investigate the feasibility of ratiometric fluorescence sensing. The applicant applied this sensing study to S in serum^2-The results of the tests are shown in Table 5. Research results show that the average recovery rate of actual samples is 97.5-104.4%. It can be seen that the method of the present invention can be well applied to S in real samples^2-And (5) detecting the content. The sensing system has high selectivity, high sensitivity and high accuracy.

TABLE 5 serum S^2-Detection of content

4. Conclusion

The invention successfully prepares the blue-emitting fluorescent copper nanoparticles with high quantum yield by using a closed system liquid-phase chemical reduction method, the preparation method is simple, no stabilizer is needed, the prepared copper nanoparticles have good stability and biocompatibility, and the invention aims to construct a complex biosensing researchLays a certain foundation. The nanoparticle can generate resonance energy transfer with riboflavin, and therefore, the nanoparticle can be used as an excellent probe for detecting VB 2. Based on riboflavin and S^2-Further constructs the pair S^2-The dual signal ratio type fluorescence detection method of (1). The fluorescent copper nanoparticles are used for constructing riboflavin and S^2-The method is simple and low in cost, and does not need to carry out chemical labeling and modification on the probe. The sensing system of the invention has high selectivity, high sensitivity and high selectivity.

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The preparation of the fluorescent copper nanoparticles and the application method in detecting VB2 and the content of sulfur ions are as follows:

preparation of 1 copper nano solution CuNPs

Weighing 30-40mg of anhydrous copper sulfate, adding 2-6mL of 0.1-1mol/L NaOH solution, adding 20mL of ultrapure water, stirring for 2min, transferring into a rotary evaporation bottle (the rotary evaporation bottle is wrapped by a black bag), closing a condensation switch, closing an exhaust plug, adding 20mL of 0.06-0.12mol/L ascorbic acid solution, enabling the solution to become yolk color, rotating for 10min at 30-90 ℃, adjusting the pH value to be 5-10 by using 0.5mol/L NaOH solution, continuing to rotate for 3-17 hours at 30-90 ℃, enabling the solution color to become green-brown finally, forming CuNPs, and keeping the solution in a refrigerator at 4 ℃ in a dark place.

2VB₂And detection of the concentration of sulfide ions

2.1VB₂Detection of concentration

Pretreating oranges, firstly mashing the oranges, centrifuging the oranges, taking supernatant, preparing the concentration required by the test, and refrigerating the supernatant in a refrigerator for later use.

25 μ L (36.025 μ M) of CuNPs solution was added to 3mL PBS (pH 7.5), 300 μ L (0.1 μ M) of VB2 standard solution and 300 μ L of test solutionStanding for 4min, performing fluorescence detection, detecting fluorescence intensity at emission wavelength of 525nm under the conditions that excitation wavelength is 360nm and slit width is 10 × 10nm, performing contrast experiment by using series of VB2 standard solutions with known concentration as contrast group, drawing a standard curve equation by using the concentration of VB2 standard solution as abscissa and the fluorescence intensity of riboflavin solution at 525nm as ordinate, substituting the fluorescence intensity of the solution to be detected at 525nm into the standard curve equation to obtain VB in the solution to be detected₂The concentration of (c).

2.2 detection of the concentration of sulfide ions:

at room temperature, the process of detecting the sulfur ions based on the CuNPs-VB2 fluorescent probe is as follows: mu.L (36.025. mu.M) of CuNPs solution was pipetted, 3mL (0.10M, pH 7.5) of PBS solution was added, and then 300. mu.L (0.1. mu.M) of VB was added₂Shaking the standard solution, standing for 4min, and adding S to be detected^2-Standing the solution for 30min, and detecting S at emission wavelengths of 440nm and 525nm under the conditions of excitation wavelength of 360nm and slit width of 10 × 10nm^2-The fluorescence intensity of (2). And using a series of known concentrations of S^2-The standard solution is used as a control group to carry out a control experiment, and S is used^2-The concentration of the standard solution was plotted on the abscissa and the fluorescence intensity ratio of F525/F440 was plotted on the ordinate to prepare a standard curve equation. Substituting the fluorescence intensity ratio of F525/F440 of the solution to be detected into a standard curve equation to obtain S in the solution to be detected^2-The concentration of (c).

According to the practical situation, about 3mL of solution is easily irradiated by a fluorescent lamp, too little irradiation is not achieved, too much solution is easily spilled out of a corrosion instrument, the volume of the required copper nano solution is obtained by optimizing the copper nano concentration by taking the solution as the mother solution, and 300 muL (0.1 muM) of VB2 standard solution is selected within the riboflavin concentration range.

Example 1

Preparation of 1 copper nano solution CuNPs

Weighing 32mg of anhydrous copper sulfate, adding 2mL of 0.5mol/L NaOH solution, adding 20mL of ultrapure water, stirring for 2min, transferring into a rotary evaporation bottle (the rotary evaporation bottle is wrapped by a black bag), closing a condensation switch, closing an exhaust plug, adding 20mL of 0.1mol/L ascorbic acid solution, enabling the solution to become egg yellow in color, rotating at 50 ℃ for 10min, adjusting the pH value to 8 by using 0.5mol/L NaOH solution, continuing rotating at 50 ℃ for 15 hours, enabling the solution to become green brown finally, forming CuNPs, and keeping the solution in a refrigerator at 4 ℃ in a dark place.

2VB₂Detection of concentration

Adding 3ml LPBS (pH 7.5) and 300. mu.L of solution to be detected into 25. mu.L (36.025uM) of CuNPs solution, standing for 4min, detecting fluorescence intensity at 525nm emission wavelength under the conditions of excitation wavelength of 360nm and slit width of 10 × 10nm, and substituting into standard curve equation of Y23.729 [ VB2 ]]+127.241 where Y represents the fluorescence intensity of the riboflavin solution at 525nm, [ VB2]Represents the concentration of VB2 to obtain VB₂The concentration of (c).

Example 2

Preparation of 1 copper nano solution CuNPs

Weighing 30mg of anhydrous copper sulfate, adding 4mL of 0.1mol/L NaOH solution, adding 20mL of ultrapure water, stirring for 2min, transferring into a rotary evaporation bottle (the rotary evaporation bottle is wrapped by a black bag), closing a condensation switch, closing an exhaust plug, adding 20mL of 0.06mol/L ascorbic acid solution, enabling the solution to become yolk color, rotating at 90 ℃ for 10min, adjusting the pH value to be 5 by using 0.5mol/L NaOH solution, continuing rotating at 90 ℃ for 17 hours, enabling the solution color to become green brown, forming CuNPs, and keeping the solution in a refrigerator at 4 ℃ in a dark place.

2VB₂Detection of concentration

Adding 25 μ L (36.025uM) of CuNPs solution into 3mLPBS (pH 7.5) and 300 μ L of solution to be detected, standing for 4min, detecting fluorescence intensity at 525nm emission wavelength under the conditions of excitation wavelength of 360nm and slit width of 10 × 10nm, performing control experiment with series of VB2 standard solutions with known concentration as control group, and taking concentration of VB2 standard solution as abscissa and riboflavin solution at 525nmThe fluorescence intensity is used as the ordinate, and a standard curve equation is drawn. Substituting the fluorescence intensity of the solution to be detected at 525nm into a standard curve equation to obtain VB in the solution to be detected₂The concentration of (c).

Example 3

Preparation of 1 copper nano solution CuNPs

Weighing 40mg of anhydrous copper sulfate, adding 6mL of 1mol/L NaOH solution, adding 20mL of ultrapure water, stirring for 2min, transferring into a rotary evaporation bottle (the rotary evaporation bottle is wrapped by a black bag), closing a condensation switch, closing an exhaust plug, adding 20mL of 0.12mol/L ascorbic acid solution, enabling the solution to become egg yellow in color, rotating at 30 ℃ for 10min, adjusting the pH to 10 by using 0.5mol/L of the LNaOH solution, continuing to rotate at 80 ℃ for 3 hours, enabling the solution to become green-brown finally, forming CuNPs, and keeping the solution in a refrigerator at 4 ℃ in a dark place.

2VB₂The concentration was measured in the same manner as in example 2.

Example 4

Preparation of 1 copper nano solution CuNPs

2 fluorescent detection of sulfide ions (S)^2-) Concentration of

At room temperature, the process of detecting the sulfur ions based on the CuNPs-VB2 fluorescent probe is as follows: mu.L (36.025. mu.M) of CuNPs solution was pipetted, 3mL (0.10M, pH 7.5) of PBS solution was added, and then 300. mu.L (0.1. mu.M) of VB was added₂Shaking the standard solution, standing for 4min, and adding S to be detected^2-Standing the solution for 30min, and detecting S at emission wavelengths of 440nm and 525nm under the conditions of excitation wavelength of 360nm and slit width of 10 × 10nm^2-The fluorescence intensity of (2). Substituting into a standard curve equation: y-0.0084 [ S ]^2-]+1.5639 wherein Y represents the fluorescence intensity ratio of F525/F440, [ S ]^2-]Represents S^2-To obtain S^2-The concentration of (c).

Example 5

Preparation of 1 copper nano solution CuNPs

Weighing 35mg of anhydrous copper sulfate, adding 5mL of 0.6mol/L NaOH solution, adding 20mL of ultrapure water, stirring for 2min, transferring into a rotary evaporation bottle (the rotary evaporation bottle is wrapped by a black bag), closing a condensation switch, closing an exhaust plug, adding 20mL of 0.08mol/L ascorbic acid solution, enabling the solution to become egg yellow in color, rotating at 70 ℃ for 10min, adjusting the pH value to 8 by using 0.5mol/L NaOH solution, continuing rotating at 80 ℃ for 12 hours, enabling the solution to become green brown finally, forming CuNPs, and keeping the solution in a refrigerator at 4 ℃ in a dark place.

2 fluorescent detection of sulfide ions (S)^2-) Concentration of

Example 6

Preparation of 1 copper nano solution CuNPs

Weighing 36mg of anhydrous copper sulfate, adding 3mL of 0.3mol/L NaOH solution, adding 20mL of ultrapure water, stirring for 2min, transferring into a rotary evaporation bottle (the rotary evaporation bottle is wrapped by a black bag), closing a condensation switch, closing an exhaust plug, adding 20mL of 0.09mol/L ascorbic acid solution, enabling the solution to become yolk color, rotating at 80 ℃ for 10min, adjusting the pH value to 6 by using 0.5mol/L NaOH solution, continuing to rotate at 60 ℃ for 16 hours, enabling the solution to become green brown finally, forming CuNPs, and keeping the solution in a refrigerator at 4 ℃ in a dark place.

2 fluorescent detection of sulfide ions (S)^2-) The method of concentration was the same as in example 5.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A preparation method of fluorescent copper nanoparticles is characterized by comprising the following steps: anhydrous copper sulfate is used as a copper source, ascorbic acid is added, and the fluorescent copper nanoparticles are prepared by adopting a low-temperature rotary evaporation closed system liquid-phase chemical reduction method.

2. The method of claim 1, wherein: the molar ratio of the anhydrous copper sulfate to the ascorbic acid is 1: 10.

3. the production method according to claim 1 or 2, characterized in that: adding NaOH solution into anhydrous copper sulfate, adding ultrapure water, stirring, transferring into a rotary evaporation bottle wrapped by an opaque substance, closing a condensation switch and an exhaust plug, adding ascorbic acid solution, rotating at 30-90 ℃ for 10min, adjusting the pH value to 5-10 by using 0.5mol/LNaOH solution, and continuing rotating at 30-90 ℃ for 3-17 hours to obtain a fluorescent copper nanoparticle solution;

preferably, the NaOH solution is added into anhydrous copper sulfate, then ultrapure water is added, the mixture is stirred, the mixture is transferred into a rotary evaporation bottle wrapped by an opaque substance, a condensation switch and an exhaust plug are closed, the ascorbic acid solution is added, the mixture is rotated at 50 ℃ for 10min, the pH value is adjusted to be 8-9 by using 0.5mol/L NaOH solution, and the rotation is continued at 50 ℃ for 15 hours, so that the fluorescent copper nanoparticle solution is obtained.

4. A fluorescent copper nanoparticle characterized by: is prepared by the preparation method of any one of claims 1 to 3.

5. Use of the fluorescent copper nanoparticles of claim 4 for detecting riboflavin and sulfide ion content.

6. The fluorescent copper nanoparticles as claimed in claim 4, for use in detecting riboflavin in fruits and vegetables and detecting the content of sulfur ions in serum.

7. A method for detecting riboflavin content is characterized by comprising the steps of taking 36.025 mu M of fluorescent copper nanoparticle solution prepared according to claim 4, sequentially adding 0.1M PBS with pH of 7.0-8.0 and solution to be detected, standing for 4-5min, carrying out fluorescence detection, detecting fluorescence intensity under 525nm emission wavelength under the conditions that the excitation wavelength is 360nm and the slit width is 10 × 10nm, carrying out contrast experiment by taking series of VB2 standard solutions with known concentrations as contrast groups, drawing a standard curve equation by taking the concentration of VB2 standard solution as a horizontal coordinate and the fluorescence intensity of riboflavin solution at 525nm as a vertical coordinate, substituting the fluorescence intensity of the solution to be detected at 525nm into the standard curve equation to obtain VB2 in the solution to be detected₂The concentration of (c).

8. The method for detecting riboflavin content according to claim 7, wherein: the pH was 7.5.

9. A method for detecting the content of sulfur ions is characterized in that: the method comprises the following steps: adding 36.025 μ M of the fluorescent copper nanoparticle solution prepared according to claim 4 into 0.1M PBS solution with pH of 7.0-8.0, adding riboflavin standard solution with known concentration, shaking, mixing, standing for 4-5min, and adding S to be measured^2-Standing the solution for 30-35min, detecting S at emission wavelengths of 440nm and 525nm under the conditions of excitation wavelength of 360nm and slit width of 10 × 10nm^2-The fluorescence intensity of (a); and use series of knownConcentration of S^2-The standard solution is used as a control group to carry out a control experiment, and S is used^2-Taking the concentration of the standard solution as an abscissa and taking the fluorescence intensity ratio of F525/F440 as an ordinate to draw a standard curve equation; substituting the fluorescence intensity ratio of F525/F440 of the solution to be detected into a standard curve equation to obtain S in the solution to be detected^2-The concentration of (c).

10. The method for detecting the content of the sulfur ions according to claim 9, wherein: the pH was 7.5.