CN114456806A

CN114456806A - Near-infrared fluorescent nano probe capable of identifying palladium ions as well as preparation method and application thereof

Info

Publication number: CN114456806A
Application number: CN202210178686.8A
Authority: CN
Inventors: 杜方凯; 屈雲霆; 刘金瑞
Original assignee: Guangxi University for Nationalities
Current assignee: Guangxi University for Nationalities
Priority date: 2022-02-25
Filing date: 2022-02-25
Publication date: 2022-05-10
Anticipated expiration: 2042-02-25
Also published as: CN114456806B

Abstract

The invention discloses a near-infrared fluorescent nano probe capable of identifying palladium ions and a preparation method and application thereof, belonging to the technical field of material science. According to the invention, the magnolia denudata leaves are used as raw materials, the near-infrared carbon dots are synthesized by adopting a solvothermal method, the synthesized near-infrared carbon dots show stable near-infrared emission, the peak value is about 678nm, and the peak value has narrow half-peak width. After the near-infrared carbon dots and the nonionic surfactant are dissolved in the ethanol solution to prepare the near-infrared fluorescent nano probe, the near-infrared fluorescent nano probe shows excellent performances including high selectivity and excellenceHeterogeneous water solubility, excellent stability and low cytotoxicity, and the fluorescence intensity of the nanoprobe is Pd within the range of 0-200 mu M²⁺Selective quenching, simple application to Pd²⁺And the cell identification and analysis can be performed, and cell imaging and actual water sample analysis can be performed, so that the method has a good application prospect.

Description

Near-infrared fluorescent nano probe capable of identifying palladium ions as well as preparation method and application thereof

Technical Field

The invention relates to the technical field of material science, in particular to a near-infrared fluorescent nano probe capable of identifying palladium ions and a preparation method and application thereof.

Background

In recent years, with rapid development of industrial production, divalent palladium ion Pd²⁺Has become an important metal catalyst. Because of its excellent catalytic performance, it has been widely used in the fields of medicine, jewelry, automobiles, fuel cells, and the like. However, Pd²⁺The wide use in daily life also increases the amount of palladium ions discharged. Pd²⁺Can react with proteins, amino acids, and other macromolecules (including DNA) that may interfere with a variety of cellular activities. Increasing the environmental and health risks that are difficult to ignore due to their potential toxicity. Thus, a rapid and effective Pd was sought²⁺The detection method has very important significance.

Conventionally, to detect Pd²⁺Various methods such as an X-ray fluorescence method, an Atomic Absorption Spectrometry (AAS), a plasma emission method including an inductively coupled plasma mass spectrometry (ICP-MS) and an inductively coupled plasma atomic emission spectrometry (ICP-AES), and the like are designed. However, these methods are not only complex and expensive, but also unsuitable for Pd in living systems²⁺Real-time detection of. In response to the above problems, fluorescence methods are beginning to appear more and more.

Carbon dots, a novel fluorescent carbon-based material, were discovered accidentally in 2004. The compounds not only have the characteristics of the traditional organic dye, such as high sensitivity and quick reaction, but also have the advantages of low toxicity, low cost, simple synthesis, good biocompatibility, modification and the like. Wherein, the near infrared carbon dot not only has very low self-absorption and photobleaching, but also has very strong tissue penetration capability and background interference resistance capability. At present, for Pd²⁺Most of the emission peaks of the detected carbon dots are in the blue-green light region, and the emission peaks are very few in the near infrared region. It is reported that Pawar et al constructed a probe for Pd²⁺And trivalent indium ion (In)³⁺) The nitrogen of (2) is doped with carbon dots. Gao et al reported a method for detecting Pd²⁺Trivalent gold ion (Au)³⁺) And divalent platinum ion (Pt)²⁺) The "on-off" fluorescence sensor of (1). However, the above carbon dots all suffer from several significant drawbacks, including broad half-peak width and multi-ion detection. And the current carbon point isHas limitations in application. Therefore, a near-infrared carbon point with high selectivity, narrow half-peak width and larger Stokes shift is developed, and Pd can be specifically identified by synthesizing the near-infrared carbon point on the basis of the infrared carbon point²⁺The near infrared fluorescent nano probe has a very important application prospect.

Disclosure of Invention

Aiming at the problems, the invention provides a near-infrared fluorescent nano probe capable of identifying palladium ions, and a preparation method and application thereof.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a preparation method of a near-infrared fluorescent nano probe capable of identifying palladium ions comprises the following steps:

(1) selecting fresh magnolia denudata leaves, cleaning, drying and crushing into powder;

(2) adding the powder obtained in the step (1) into an organic solvent, and fully and uniformly stirring;

(3) transferring the uniformly stirred solution into a stainless steel reaction kettle, and heating for 3-8h at constant temperature in a reactor at the temperature of 100-200 ℃ to obtain a reactant;

(4) cooling to room temperature, centrifuging the obtained reactant, filtering the obtained supernatant with a filter membrane, and purifying the obtained filtrate by silica gel column chromatography;

(5) petroleum ether and ethyl acetate eluents with different volume ratios are adopted for dynamic elution; finally, collecting the dark green solution, and evaporating under reduced pressure to obtain a dark green colloidal product near-infrared carbon dot;

(6) dissolving the obtained colloid product near-infrared carbon dots and the nonionic surfactant in an ethanol solution, and performing ultrasonic treatment for 10-15 minutes;

(7) after the ultrasonic treatment is finished, removing the ethanol by decompression and rotation to form a green mixed film; adding deionized water for hydration, carrying out ultrasonic treatment for 30-40 minutes, and carrying out freeze drying to obtain the water-soluble nano probe.

Preferably, the organic solvent in step (2) is acetone, absolute ethyl alcohol or methanol.

Preferably, the mass-to-volume ratio of the powder in the step (2) to the organic solvent is 1 g: 10-30 mL.

Preferably, the filter membrane in the step (4) is a polyethersulfone membrane with the thickness of 0.22 μm.

Preferably, the volume ratio of the petroleum ether to the ethyl acetate in the petroleum ether and ethyl acetate eluent in the step (5) is 3: 1-1: 1.

Preferably, the nonionic surfactant in the step (6) is Pluronic F-127, and the concentration of the nonionic surfactant is 40 mu mol. L^-1。

Preferably, the mass ratio of the near-infrared carbon dots to the nonionic surfactant in the step (6) is 1: 8-11.

The invention also provides the near-infrared fluorescent nano probe capable of identifying the palladium ions, which is obtained according to the preparation method.

In addition, the invention also provides application of the near-infrared fluorescent nano probe prepared by the preparation method of the near-infrared fluorescent nano probe capable of identifying palladium ions in palladium ion detection, actual water sample detection and cell imaging.

Preferably, in the process of palladium ion detection, the concentration of the near-infrared fluorescent nanoprobe is 0.5 mg-mL^-1。

By adopting the technical scheme, the invention has the beneficial effects that:

according to the invention, the near-infrared carbon dots are prepared by adopting a solvothermal method, and are functionally modified to obtain the water-soluble near-infrared fluorescent nano probe capable of identifying palladium ions, so that the water-soluble near-infrared fluorescent nano probe has the advantages of wider application, simple preparation process and environmental friendliness. Red luminescence (luminescence center is 678 +/-2 nm) is observed through Photoluminescence (PL) emission spectrum of the near-infrared carbon dots, and the half-peak width is narrow (25nm), which shows that the near-infrared carbon dots have obvious near-infrared fluorescence effect. By targeting Pd with a nanoprobe²⁺The fluorescence response of (2) was analyzed and found to be when Pd was present²⁺When present, the red fluorescence is quenched, and the fluorescence intensity ratio is F/F₀A significant reduction. Remove Pd²⁺In addition, after other metal ions are added, the nano probe solution hardly changes, which shows that the nano probe solution is nanoThe rice probe has good ion selectivity. In Pd²⁺When present, adding other metal ions, the fluorescence intensity ratio is F/F₀Basically, the change does not occur, and the nano probe has good ion interference resistance. The Pd pair of the nano probe is further researched²⁺Response time and Pd at different concentrations²⁺Next, the fluorescence intensity of the nanoprobe changes. It was found that when Pd was added²⁺Fluorescence intensity ratio F/F₀The decrease starts, and after 30 minutes, the fluorescence intensity ratio starts to stabilize. At the same time, Pd at different concentrations²⁺Then, the fluorescence intensity of the nanoprobe gradually decreases as the concentration increases. Therefore, the near-infrared fluorescent nano probe has high selectivity, narrower half-peak width, larger Stokes shift, excellent stability and low cytotoxicity, and can be simply and conveniently used for Pd²⁺And (4) identifying and analyzing. More importantly, the cell imaging and the analysis of an actual water sample can be carried out through verification, and the method has a good application prospect.

Drawings

FIG. 1(a) is a TEM image of NIR-CDs; (b) HR-TEM images as NIR-CDs; (c) HR-TEM images of NIR-CDs lattices; (d) particle size distribution histogram of NIR-CDs;

FIG. 2(a) is an XRD pattern of NIR-CDs; (b) FT-IR spectrum chart of NIR-CDs; (c) XPS measured spectra for NIR-CDs; (d) (e) and (f) high resolution XPS spectra for C1s, O1s and N1s in NIR-CDs, respectively;

FIG. 3 is a Differential Scanning Calorimetry (DSC) thermogram of NIR-CDs;

FIG. 4(a) is the absorption spectrum of NIR-CDs in acetone solution; (b) fluorescence emission spectra of NIR-CDs in acetone solution at different excitation wavelengths; (c) fluorescence emission spectra of NIR-CDs in different solvents; (d) fluorescence emission spectra of NIR-CDs in ethanol solution at different water contents; (e) fluorescence emission spectra of NIR-CDs in DMSO solution at different excitation wavelengths; (f) fluorescence emission spectra of NIR-CDs in DMSO solutions for different water contents (50 mg. multidot.L NIR-CDs)^-1)；

FIG. 5 shows ultraviolet lightNIR-CDs (50 mg. L)^-1) Photographs in ethanol solutions of different water contents;

FIG. 6 is a graph of absolute fluorescence quantum yields of NIR-CDs in (a) acetone and (b) DMSO solutions, respectively, at an excitation wavelength of 413 nm;

FIG. 7 shows NIR-CDs (50 mg. L)^-1) Adding a fluorescence emission spectrum of 40 μ MF-127 (λ ex ═ 413nm) to water;

FIG. 8(a) is a graph of the fluorescence response of NIR-CDs after addition of different surfactants F-127; (b) is F/F₀Line graphs with different concentrations of F-127; (F)₀And F is the fluorescence intensity of NIR-CDs at 678nm in the absence and presence of surfactant F-127, respectively);

FIG. 9(a) is a size distribution of nanoprobes in an aqueous solution measured by the DLS method; (b) the fluorescence emission spectra of the nanoprobe are obtained under different pH values; (c) and (d) the fluorescence intensity ratio F/F0 of the nanoprobe under ion intensity and ultraviolet irradiation, respectively (n is 3);

FIG. 10 is a graph of absolute fluorescence quantum yield of nanoprobes in water at an excitation wavelength of 413 nm;

FIG. 11(a) is a graph showing the fluorescence response of nanoprobes to various metal ions; (b) is at Pd²⁺In the presence of the metal ions, the nano probe has fluorescence response to various metal ions; (the concentration of each metal ion was 200. mu.M, F)₀And F is the absence and presence of Pd, respectively² ⁺The fluorescence intensity of the nanoprobe at 678nm, and the number of parallel experiments n is 3);

FIG. 12 is a photograph of the nanoprobe in daylight (a) and ultraviolet (b) respectively (the concentration of each metal ion is 200 μ M) after the addition of different metal cations;

FIG. 13 shows excitation and emission spectra of the nanoprobe of the present invention;

FIG. 14(a) shows a pair of nanoprobes Pd²⁺Response time (Pd)²⁺ Concentration 200. mu.M); (b) is Pd in different concentrations²⁺Fluorescence spectrum change diagram of the nanoprobe; (c) is F₀(ii) F-1 with different Pd²⁺Fitted curve of concentration (number of parallel experiments n-3); (d) f₀/F-1 for different concentrations of Pd²⁺Linear fitting curve (linear range is 4-18 mu M);

FIG. 15 shows the presence or absence of the quencher Pd²⁺Then, the absorption spectrum of the nanoprobe;

FIG. 16 is a graph of cell viability of Li-7 cells after 24 hours incubation at varying concentrations of nanoprobe;

FIG. 17 is a fluorescence microscope image of Li-7 cells in bright field, red channel and overlay, respectively: (a) blank group, (b) no Pd addition²⁺The cell image of the nanoprobe of (a), (c) adding 80. mu. MPd²⁺Cytographic imaging of post-nanoprobes, (d) addition of 160. mu. MPd²⁺Cell imaging of the latter nanoprobe.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

(1) selecting fresh magnolia denudata leaves, cleaning, drying at 50 ℃ for 12 hours, and crushing into powder;

(2) adding 4.0g of powder obtained in the step (1) into 80mL of acetone solution, and fully and uniformly stirring;

(3) transferring the uniformly stirred solution into a polytetrafluoroethylene lining autoclave with the size of 100mL, and heating at the constant temperature of 120 ℃ for 5 hours to obtain a reactant;

(4) after heating, cooling to room temperature, centrifuging the obtained reactant at 8000r/min for 10 min; the supernatant was filtered using a polyethersulfone membrane (0.22 μm), and the filtrate was purified by silica gel column chromatography;

(5) dynamically eluting by using petroleum ether and ethyl acetate eluent in a volume ratio of 3: 1-1: 1; finally, the dark green solution was collected and evaporated under reduced pressure to give the near infrared carbon dots (NIR-CDs) as a dark green colloidal product in 2% yield;

(6) taking 12.5mg of NIR-CDs and 126mg of a nonionic surfactant Pluronic F-127(Pluronic F-127, also known as Poloxamer407, i.e. Polyethylene-polyoxypropylene glycol, which is a polyoxyethylene polyoxypropylene ether triblock copolymer, known as Poloxamer, polyether or polyoxyethylene polyoxypropylene) and dissolving in 250ml of ethanol solution, and carrying out ultrasonic treatment for 10 minutes; wherein the concentration of the nonionic surfactant is 40 mu mol.L^-1；

(7) Removing ethanol by rotation under reduced pressure to form a green mixed film; adding deionized water for hydration, carrying out ultrasonic treatment for 30 minutes, and carrying out freeze drying to obtain the water-soluble nano probe.

Example 2

(2) adding 4.0g of powder obtained in the step (1) into 80mL of absolute ethyl alcohol solution, and fully and uniformly stirring;

(5) dynamically eluting by using petroleum ether and ethyl acetate eluent in a volume ratio of 3: 1-1: 1; finally, collecting the dark green solution, and evaporating under reduced pressure to obtain a dark green colloidal product NIR-CDs with the yield of 2%;

(6) taking 12.5mg of NIR-CDs and 126mg of nonionic surfactant Pluronic F-127, dissolving in 250ml of ethanol solution, and carrying out ultrasonic treatment for 10 minutes; wherein the concentration of the nonionic surfactant is 40 mu mol.L^-1；

Example 3

(2) adding 4.0g of powder obtained in the step (1) into 90mL of methanol solution, and fully and uniformly stirring;

(7) Removing ethanol by rotation under reduced pressure to form a green mixed film; adding deionized water for hydration, carrying out ultrasonic treatment for 30 minutes, and carrying out freeze drying to obtain the water-soluble near-infrared fluorescent nano probe.

As another aspect of the technical scheme, the invention also provides the near-infrared fluorescent nano probe capable of identifying the palladium ions, which is obtained according to the preparation method.

Because the near-infrared fluorescent nanoprobe is obtained on the basis of the near-infrared carbon dot synthesized by the application, the applicant also performs characterization tests on the morphology structure and the like of the near-infrared carbon dot synthesized by the application and researches the optical property of the near-infrared carbon dot.

Characterization of NIR-CDs

Morphology was first characterized by HR-TEM. As can be seen from FIGS. 1a, 1b and 1d, the prepared NIR-CDs are spheroidal, have a small particle size, an average particle size of 2.9nm, good dispersibility and no significant aggregation. FIG. 1c shows that NIR-CDs have a lattice spacing of 0.18nm, which is in comparison with sp²The diffraction of graphitic carbon is consistent (102), indicating that the prepared NIR-CDs have certain crystallinity.

As can be seen in FIG. 2a, NIR-CDs have broad X-ray diffraction peaks between 10 and 32 degrees due to highly disordered carbon atoms, indicating that the prepared NIR-CDs internally form disordered carbon structures and a large amount of amorphous carbon is present. Two sharp diffraction peaks appear in the figure, which may be that the crystalline structure of cellulose in the leaves is not completely destroyed.

The elemental composition and surface groups of the NIR-CDs were then characterized by FT-IR and XPS. FIG. 2b shows NIR-CDs at 3417cm^-1And 3471cm^-1There are distinct peaks nearby due to O-H and N-H stretching vibrations. 2850cm^-1And 2923cm^-1The double peak at (a) is due to C-H stretching vibration. 1736cm^-1The peak at (a) is due to stretching vibration of O ═ C — OH. 1646cm^-1The peak at (a) is due to the stretching vibration of C ═ O/C ═ C. 1458cm^-1The bond at (a) corresponds to the stretching vibration of C-N. 1064cm^-1And 987cm^-1The double peak at (A) is due to the bending vibrations of C-O-C and N-H. The above results indicate that the prepared NIR-CDs are rich in carbonyl, amide, hydroxyl, ether bond and aromatic ring groups.

XPS characterization of NIR-CDs As shown in FIGS. 2c-f, the XPS survey (FIG. 2c) shows three typical peaks at 285.0eV, 532.5eV and 400.1eV respectively, indicating that NIR-CDs are composed primarily of three elements, carbon (86.67%), oxygen (12.56%), and nitrogen (0.78%). The spectrum of C1s in fig. 2d shows four characteristic peaks, corresponding to C-C/C ═ C (284.7eV), C-N (285.2eV), C-O-C/C-OH (285.9eV) and C ═ O/C ═ N (286.9eV), respectively. The O1s spectrum (fig. 2e) shows two characteristic peaks, corresponding to C-O-C/C-OH (532.3eV) and O ═ C-OH (532.9eV), respectively. The spectrum of N1s (FIG. 2f) has two characteristic peaks, corresponding to pyridine N (398.5eV), amino N (400.1eV) and pyrrole N (400.9 eV). The XPS characterization result of the NIR-CDs shows that the prepared NIR-CDs contain oxygen and nitrogen elements and are rich in carbonyl, hydroxyl, amide, aromatic ring and other groups on the surface.

To further verify the polymer properties of NIR-CDs, studies were made at N₂Under the atmosphere, the heating rate is 10 ℃ min^-1Phase transition of NIR-CDs (fig. 3, Differential Scanning Calorimetry (DSC) thermogram of NIR-CDs). It can be seen that the glass transition temperature (Tg) of the NIR-CDs is-24 deg.C, which results in a viscous colloidal state of the dried NIR-CDs at room temperature. As the temperature increases, the NIR-CDs gradually change to a liquid state, which corresponds to the actual state. Thus, the results discussed above indicate that NIR-CDs have individual polymer properties.

Optical Properties of NIR-CDs

UV-Vis absorption and Photoluminescence (PL) emission spectra of NIR-CDs solutions were recorded to describe their spectral properties. As shown in FIG. 4a, NIR-CDs show extensive absorption in the range of 350-470 nm, and the maximum absorption peak is near 413nm, which is probably from the n-pi of the aromatic fluorophore structure^*And (4) transition.

FIG. 4b shows PL emission of NIR-CDs in acetone solution at different excitation wavelengths, showing stable deep red emission, with peak around 678nm and half-peak width of 25 nm; as the polarity of the solvent was increased (FIG. 4c), NIR-CDs showed a weak shoulder-to-shoulder emission peak at 720 nm. However, the NIR-CDs prepared have poor solubility in water. In addition, under 413nm excitation, PL emission spectra of NIR-CDs in mixed solvents of different volume ratios of water/ethanol were obtained. Solutions of NIR-CDs in pure ethanol were clear liquids and showed significant near infrared fluorescence, whereas the fluorescence intensity gradually decreased with increasing water proportion due to the aggregation-induced quenching (ACQ) effect (fig. 4 d). The photograph in figure 5 shows more visually that the near infrared fluorescence of NIR-CDs in ethanol solution decreases significantly with the addition of water. NIR-CDs in DMSO (dimethyl sulfoxide) solution showed similar fluorescence phenomenon compared to acetone or ethanol solution (fig. 4e and 4f), which provides possibility for biological applications. The absolute quantum yield of NIR-CDs was 19.80% and 22.66% in acetone and DMSO solutions, respectively, as measured by fluorescence steady state/transient state spectroscopy (fig. 6a and 6 b).

At the same time, the Applicant has also observed the nature of the nanoprobes obtained and their Pd pair²⁺The fluorescence response of (a) was studied as follows:

1. properties of the nanoprobe

NIR-CDs were functionally modified in order to enhance fluorescence intensity. Insoluble NIR-CDs were converted into water-soluble micellar nanoprobes by a self-assembly method using a non-ionic surfactant F-127. As shown in FIG. 7, the fluorescence intensity of NIR-CDs in micellar solution increases dramatically compared to neat aqueous solution. Subsequently, the optimum F-127 concentration was investigated. As shown in FIG. 8, the fluorescence intensity of NIR-CDs reached a maximum in water at 40 μ M with increasing F-127 and then began to decrease, probably because the encapsulation pattern changed above the critical micelle concentration. Therefore, in constructing the nanoprobe, the concentration of F-127 was selected to be 40. mu.M. We also tested the size of the micellar probe using DLS. The average particle size of the nanoprobes in water was about 34nm (FIG. 9 a).

The nanoprobes were then tested for stability under different conditions. As shown in FIG. 9b, the fluorescence intensity of the nanoprobe hardly changed from pH1.0 to 9.0, indicating that the nanoprobe has a wide pH stability, indicating that it can be used in an in vivo environment. Furthermore, even at high ionic strength (FIG. 9c) and prolonged UV radiation (FIG. 9d), the fluorescence intensity ratio F/F₀(F₀And F is the fluorescence intensity in the absence and presence of the nanoprobe, respectively) still did not change significantly. The fluorescence steady state/transient state spectrometer (fig. 10) calculated the absolute quantum yield of the nanoprobe in water to be 7.97%. The results show that the nano probe has potential application prospect in the aspects of analysis, detection and biological labeling.

2. Nanometer probe pair Pd²⁺Fluorescence response of

The applicant adopted the prepared nano probe pair Pd²⁺The fluorescence response of (a) is analyzed, and the analysis process is as follows: firstly, by mixing different concentrations of Pd²⁺Stock solution (50. mu.L) was added to the nanoprobe solution, thenThe mixed solution was then diluted with phosphate buffer solution (PBS,50mM, pH7.4) to a final concentration (the final concentration of the nanoprobe was 0.5 mg. multidot.mL)^-1) The mixed solution was stirred for 30 minutes, and then subjected to a test. All fluorescence spectra were collected under excitation with light at 413nm, slit 5.0nm-5.0nm, voltage 700V.

Different metal ions were added to the 0.5mg/mL nanoprobe solution, as shown in FIG. 11a, when Pd was found²⁺When present, the red fluorescence is quenched, and the fluorescence intensity ratio is F/F₀A significant reduction. Remove Pd²⁺In addition, the nanoprobe solution hardly changed after adding other metal ions (fig. 12), indicating that the nanoprobe has good selectivity for ion detection. In Pd²⁺When present (FIG. 11b), other metal ions were added, the fluorescence intensity ratio F/F₀Basically, the change does not occur, and the nano probe has good ion interference resistance. Fig. 13 is an excitation and emission spectrum of the nanoprobe, and it can be known from the figure that the peak value of the emission spectrum of the nanoprobe is much larger than the peak value of the excitation spectrum, and has a larger stokes shift, so that the nanoprobe can be better applied to biology. Table 1 shows part of Pd²⁺The performance of the near infrared sensor, compared with other sensors, shows that the nanoprobe is more Pd than other sensors²⁺The detection sensor has better performance: the nanoprobe not only has excellent selectivity, but also has larger Stokes shift and narrower half-peak width.

TABLE 1 Pd²⁺Performance comparison of detection sensors

^aThe half-peak width is estimated by reference

The invention also further researches the Pd of the nano probe pair²⁺And Pd at different concentrations²⁺Next, the fluorescence intensity of the nanoprobe changes. FIG. 14a shows that when Pd is added²⁺Fluorescence intensity ratio F/F₀The decrease starts, and after 30 minutes, the fluorescence intensity ratio starts to stabilize. Pd of different concentrations²⁺Next (FIGS. 14b and 14c), the fluorescence intensity of the nanoprobe gradually decreased as the concentration increased. And F₀/F-1 and Pd²⁺The concentration is in a good linear relation of 4-18 mu M (figure 14d), the linear regression equation is that y is 0.04573x-0.13988, and the correlation coefficient R²The detection limit LOD was calculated according to the formula to be 85.3nM 0.9855. Conform to the Stern-Volmer equation:

F₀/F＝1+K_sv[Q]＝1+K_qτ₀[Q]

wherein F₀And F are each Pd-free²⁺And has Pd²⁺Fluorescence intensity of NIR-CDs at 678nm, K_svIs the Stern-Volmer quenching constant, K_qIs a quenching rate constant, τ₀Is Pd-free²⁺Average life time, [ Q ]]Is the nanoprobe concentration. Calculating K by the slope of the regression equation_svAnd finally, calculating to obtain K_qIs 1.09X 10¹³L·mol^-1·s^-1This is much greater than the limiting diffusion constant of dynamic quenching, 2X 10¹⁰L·mol^-1·s^-1Does not conform to the dynamic quenching mechanism, therefore, Pd²⁺The fluorescence quenching of the nanoprobe is likely to be by a static quenching mechanism.

For better explanation of Pd²⁺Quenching mechanism in nanoprobe solution to add Pd²⁺And further researching the ultraviolet-visible absorption spectrum and the fluorescence lifetime of the sample before and after. FIG. 15 shows the addition of Pd²⁺Later, the overall UV-visible absorption peak shifts upward and the intensity increases, most notably at 350nm, probably due to Pd²⁺Form a fairly stable complex with the nanoprobe, resulting in a change in the intensity of the absorption peak. The fluorescence lifetime was measured on the samples (Table 2), by adding 200. mu.M Pd²⁺The fluorescence lifetime is basically unchanged, which indicates that the fluorescence quenching may be caused by the nanoprobe and Pd²⁺Caused by specific interactions between them. In summary, Pd²⁺In the nano probe solutionThe mechanism of fluorescence quenching in liquid is static quenching.

TABLE 2 Presence or absence of quenching agent Pd²⁺Fluorescence lifetime of nanoprobe

In addition, in order to study the application of the prepared nanoprobe in cell imaging, the applicant also studied cytotoxicity and cell imaging by using the nanoprobe.

1. Cytotoxicity

Li-7 cells were seeded in 96-well plates using a special medium and cultured at 37 ℃ for 24 hours. Then, the medium was replaced with nanoprobe solutions of different concentrations (0, 1, 10, 100, 200, 400, 600, 800mg/L) and cultured for 24 hours. DMSO (150. mu.L) was then added to each well and the shaker was shaken at 100rpm for 10 minutes at room temperature. MTT solution (20. mu.L, 5mg/mL) was added to each well and incubation was continued for 4 hours. Absorbance at 490nm was measured using a microplate reader. The MTT method was used to assess the cytotoxicity of nanoprobes according to ISO 10993-5.

2. Cellular imaging

Li-7 cells were seeded in a laser confocal special culture dish and cultured for 24 hours. And taking out the culture dish with the cells growing well attached to the wall, sucking away the cell culture solution, and gently washing off dead cells and cell fragments on the surface by using a PBS (phosphate buffered saline) buffer solution. 2mL of the dedicated Li-7-containing cell culture solution was added to the blank group, NIR-CDs was added to the NIR-CDs group (2mL, 0.05mg/mL), and a nanoprobe culture solution (2mL, 0.55mg/mL, with 9% NIR-CDs, w/w) was added to each of the other 3 experimental groups, which were placed in a cell incubator for further incubation for 30 minutes. The culture medium was aspirated, gently washed 3 times with PBS buffer, and the residual NIR-CDs and nanoprobes were washed away. Then blank group and NIR-CDs group were added with special culture solution (2mL), and the other 2 experimental groups were separately treated with different concentrations (80, 160 μm/L) of Pd²⁺And (4) a culture solution. Incubating in incubatorAfter incubation for 30 minutes, the residual substrate was washed away by pipetting the PBS buffer several times along the edges. And finally, performing imaging work under a laser confocal microscope, setting a microscope excitation wavelength of 405nm, and selecting a bright field and a red channel (lambda em is 630-720 nm).

FIG. 16 is a graph of cell viability of Li-7 cells after 24 hours incubation at varying concentrations of nanoprobe. As can be seen, the cell viability of the cell was greater than 81% even though the concentration of the nanoprobe was increased to 800mg/L using the Li-7 cell for cytotoxicity studies, indicating that the nanoprobe had low cytotoxicity. In this regard, further cellular imaging was performed, as shown in fig. 17, and in the absence of nanoprobes (fig. 17a), it was observed that Li-7 cells in the blank did not fluoresce; when the nanoprobe is added (fig. 17b), bright red fluorescence appears in Li-7 cells, which indicates that the nanoprobe can effectively enter the cells and has excellent biocompatibility; in exogenous Pd²⁺With the addition (FIGS. 17c and 17d), the fluorescence intensity can be effectively reduced. In conclusion, the nano probe has good biological application potential.

In addition, the applicant also selects three types of water, namely Kangshifu mineral water, tap water in the Siyuan lake school zone of Guangxi national university and Siyuan lake water of Guangxi national university, to carry out actual water sample detection. In the absence of Pd²⁺In the case of addition, no Pd was detected²⁺Is present. By adding Pd with different concentrations into an actual water sample²⁺As shown in table 3, the spiking recovery rate of the nanoprobe is found to be 95.54% to 107.14%, and the relative standard deviation is less than 4.6%, which indicates that the nanoprobe can be used for detecting an actual water sample and has good detection performance.

TABLE 3 Pd in actual water samples²⁺Results of recovery rate measurement (number of parallel experiments n ═ 3)

In conclusion, the invention prepares NIR-CDs by adopting a solvothermal method and performs functional modification on the NIR-CDs to obtain the NIR-CDsA water-soluble near-infrared fluorescent nano probe capable of identifying palladium ions is widely applied, bright red luminescence (the luminescence center is 678 +/-2 nm), the half-peak width is narrow (25nm) is observed, and Pd is detected by the nano probe²⁺The fluorescence response of (2) was analyzed and found to be when Pd was present²⁺When present, the red fluorescence is quenched, and the fluorescence intensity ratio is F/F₀A significant reduction. Remove Pd²⁺Besides, after other metal ions are added, the nano probe solution is hardly changed, which shows that the nano probe has good ion selectivity. In Pd²⁺When existing, adding other metal ions, and obtaining a fluorescence intensity ratio F/F₀Basically, the change does not occur, and the nano probe has good ion interference resistance. The Pd pair of the nano probe is further researched²⁺And Pd at different concentrations²⁺Next, the fluorescence intensity of the nanoprobe changes. It was found that when Pd was added²⁺Fluorescence intensity ratio F/F₀The decrease starts, and after 30 minutes, the fluorescence intensity ratio starts to stabilize. At the same time, Pd at different concentrations²⁺Then, the fluorescence intensity of the nanoprobe gradually decreases as the concentration increases. In addition, also for Pd²⁺The fluorescence quenching mechanism of the nano probe is researched. Therefore, the near-infrared fluorescent nano probe has high selectivity, narrower half-peak width, larger Stokes shift, excellent stability and low cytotoxicity, and can be simply and conveniently used for Pd²⁺And (4) identifying and analyzing. More importantly, the cell imaging and the analysis of an actual water sample can be carried out through verification, and the method has a good application prospect.

The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.

Claims

1. A preparation method of a near-infrared fluorescent nano probe capable of identifying palladium ions is characterized by comprising the following steps:

2. The method for preparing a near-infrared fluorescent nanoprobe capable of identifying palladium ions according to claim 1, wherein the organic solvent in the step (2) is acetone, absolute ethyl alcohol or methanol.

3. The method for preparing a near-infrared fluorescent nanoprobe capable of identifying palladium ions as claimed in claim 1, wherein the mass-to-volume ratio of the powder to the organic solvent in step (2) is 1 g: 10-30 mL.

4. The method for preparing a near-infrared fluorescent nanoprobe capable of identifying palladium ions according to claim 1, wherein the filter membrane in the step (4) is a polyethersulfone membrane with the thickness of 0.22 μm.

5. The method for preparing a near-infrared fluorescent nanoprobe capable of identifying palladium ions as claimed in claim 1, wherein the volume ratio of the petroleum ether to the ethyl acetate in the petroleum ether and ethyl acetate eluent in the step (5) is 3: 1-1: 1.

6. The method for preparing a near-infrared fluorescent nanoprobe capable of identifying palladium ions according to claim 1, wherein the nonionic surfactant in the step (6) is Pluronic F-127, and the concentration of the nonionic surfactant is 40 μmol-L^-1。

7. The method for preparing a near-infrared fluorescent nanoprobe capable of identifying palladium ions according to claim 1, wherein the mass ratio of the near-infrared carbon dots to the nonionic surfactant in the step (6) is 1: 8-11.

8. A near-infrared fluorescent nanoprobe capable of identifying palladium ions, obtained by the preparation method according to any one of claims 1 to 7.

9. The use of the near-infrared fluorescent nanoprobe prepared by the method for preparing a near-infrared fluorescent nanoprobe capable of recognizing palladium ions as claimed in any one of claims 1 to 7 in palladium ion detection, actual water sample detection and cell imaging.

10. The use according to claim 9, wherein the concentration of the near-infrared fluorescent nanoprobe during the palladium ion detection is 0.5 mg-mL^-1。