CN112111266A - Nanocrystalline material for detecting temperature in organism, preparation method thereof and detection kit - Google Patents

Abstract

The application relates to the field of biological detection, in particular to a nanocrystalline material for detecting temperature in a living body, a preparation method thereof and a detection kit. A rare earth fluoride nanocrystalline material with a core-shell structure is disclosed, and the nanocrystalline formula of the nanocrystalline material is as follows: KBi₃F₁₀:Ce/Tb@KBi₃F₁₀Or KBi₃F₁₀:Ce/Eu@KBi₃F₁₀. The surface of the nanocrystal is coated with a layer of inert shellAnd good water solubility and biocompatibility are obtained through citric acid ligand modification. Due to rare earth ions Tb³⁺Or Eu³⁺The luminescence intensity of the nano crystal has a certain relation with the temperature change, the nano crystal can be used for preparing a temperature detection sensor in a living body, and meanwhile, the core-shell structure greatly enhances the fluorescence intensity and the luminescence efficiency of the nano crystal, so that the temperature detection is more sensitive and accurate, and the core-shell nano crystal can be well applied to the detection application of the fluorescence temperature in the living body.

Description

Nanocrystalline material for detecting temperature in organism, preparation method thereof and detection kit

Technical Field

The application relates to the field of biological detection, in particular to a nanocrystalline material for detecting temperature in a living body, a preparation method thereof and a detection kit.

Background

With the progress of science, people find a new hot spot in the field of temperature measurement: and (4) detecting the temperature of the biological fluorescence. Temperature is a very important factor for organisms, environmental temperature directly or indirectly affects the growth, development, living state, reproduction and distribution of organisms, and temperature is of great significance for understanding the action and behavior of organisms, which is important for early diagnosis and treatment of various diseases. Therefore, the method has important significance for detecting the temperature in the organism.

At present, the temperature measurement method mainly comprises a contact type (for example, temperature measurement of a thermocouple and a thermal resistor) and a non-contact type (for example, an infrared thermal imager). In strong electromagnetic field or acid-base chemistry experiments, the contact temperature measurement method cannot be used for temperature measurement. The application range of the infrared thermal imager non-contact temperature measurement method is wide, but the infrared thermal imager non-contact temperature measurement method has two limitations: firstly, temperature testing

The accuracy degree is related to the property of the material to be detected; secondly, near infrared light emitted by the detected temperature is easily absorbed by biological tissues, which is not beneficial to temperature test in the field of biological medical treatment.

Rare earth ion (Ln)³⁺) The doped micro-nano up-conversion luminescent material is commonly used for temperature sensing research due to the advantages of stable physicochemical properties, wide light emitting range, long fluorescence lifetime, low toxicity, deep tissue penetration depth, low background fluorescence and the like. Although many studies on principle applications have been reported, the low luminous efficiency limits the application and development of rare earth doped nanomaterials. The luminescence of the rare earth doped nano material can be enhanced by regulating and controlling the doped matrix, the doped elements and the doping mode (the traditional luminescence center single doping is replaced by the codoping of the luminescence center and the sensitized ions). In addition, core-shell cladding is also a means to effectively enhance the luminous efficiency. The homogeneous shell is epitaxially coated on the surface of the bare-core nano particle, so that defects and solution can be effectively isolatedQuenching of the agent or surface ligand to the luminescent center avoids loss of excitation energy, thereby enhancing luminescence.

Disclosure of Invention

In order to solve the technical problem, the application provides a rare earth fluoride nanocrystalline material with a core-shell structure, namely KBi₃F₁₀Ce/Tb or KBi₃F₁₀The surface of the Ce/Eu nanocrystal is coated with an inert shell layer, so that the fluorescence intensity and the luminous efficiency of the nanocrystal are enhanced, good water solubility and biocompatibility are obtained through citric acid ligand modification, the Ce/Eu nanocrystal can be used for preparing a temperature detection sensor in a living body, and the Ce/Eu nanocrystal has the advantages of non-contact and high sensitivity.

In order to achieve the above purpose, the present application adopts the following technical solutions:

a rare earth fluoride nanocrystalline material with a core-shell structure is disclosed, and the nanocrystalline formula of the nanocrystalline material is as follows: KBi₃F₁₀:Ce/Tb@KBi₃F₁₀Or KBi₃F₁₀:Ce/Eu@KBi₃F₁₀。

Preferably, the nanocrystalline material is a water-soluble nanocrystalline material, and the KBi is coated by a citric acid ligand₃F₁₀:Ce/Tb@KBi₃F₁₀Or KBi₃F₁₀:Ce/Eu@KBi₃F₁₀And (3) nanocrystal composition.

Preferably, KBi₃F₁₀:Ce/Tb@KBi₃F₁₀The nanocrystal is KBi₃F₁₀:20Ce/10Tb@KBi₃F₁₀，KBi₃F₁₀:Ce/Eu@KBi₃F₁₀The nanocrystal is KBi₃F₁₀:20Ce/6Eu@KBi₃F₁₀。

Further, the application provides a preparation method of the rare earth fluoride nanocrystalline material with the core-shell structure, and the method comprises the following steps:

1) 1 millimole potassium nitrate KNO₃2.1 mmoles of bismuth nitrate Bi (NO)₃)₃∙5H₂O, 0.6 mmol of cerium nitrate Ce (NO)₃)₃∙5H₂O, 0.3 mmole Terbium nitrate Tb (NO)₃)₃∙5H₂O or 0.18 mmole europium nitrate Eu (NO)₃)₃∙5H₂O and 4 mmol of citric acid were dissolved in 6 ml of deionized water, and after stirring for 20 minutes, 8 mmol of ammonium fluoride (NH) was added₄F) And 40 ml of polyethylene glycol;

2) stirring for 1 hour, heating for 12 hours at the temperature of 120 ℃ in an oil bath kettle, and supplementing deionized water in time in the heating process; 3) washing the product with the mixed solution of ethanol and deionized water for 3-5 times;

4) then 1 millimole of potassium nitrate KNO₃2.1 mmoles of bismuth nitrate Bi (NO)₃)₃∙5H₂O and 4 mmol of citric acid were dissolved in 6 ml of deionized water, and after stirring for 20 minutes, 8 mmol of ammonium fluoride (NH) was added₄F) And 40 ml of polyethylene glycol, heating to 150 ℃ for reaction for 1 hour, cooling to room temperature, then adding the product obtained in the step 3), stirring for 1 hour, heating for 12 hours at the temperature of 120 ℃ in an oil bath pan, and supplementing deionized water in time in the heating process;

5) and after the solution is cooled to room temperature, washing for 3-5 times by using a mixed solution of ethanol and deionized water to obtain the core-shell nanocrystal.

Further, the application provides that the nanocrystalline material is used for in vivo temperature detection application.

Further, the present application provides an in vivo temperature detection sensor comprising the nanocrystalline material according to claim 1 or 2 or 3.

The application is realized by providing KBi₃F₁₀Ce/Tb or KBi₃F₁₀Coating an inert shell layer on the surface of the Ce/Eu nanocrystal to obtain the core-shell structure nanocrystal, and modifying the core-shell structure nanocrystal through a citric acid ligand to obtain good water solubility and biocompatibility. According to rare earth ion Tb³⁺Or Eu³⁺The luminescence intensity of a specific energy level in the nano-crystalline can change along with the temperature, the nano-crystalline can be used for preparing a temperature detection sensor in a living body, and meanwhile, the core-shell structure greatly enhances the fluorescence intensity and the luminescence efficiency of the nano-crystalline, so that the temperature detection is more sensitive and accurate, and the core-shell nano-crystalline can be well applied to the raw materialsThe detection of fluorescence temperature in an object.

Drawings

FIGS. 1 (a) and (b) are respectively an XRD spectrum and a transmission electron micrograph of the product, and (c) is a transmission electron micrograph.

FIG. 2 (a) KBi₃F₁₀Tb in 20Ce/10Tb nanocrystal³⁺Excitation spectrum of ion in ultraviolet region, (b) KBi₃F₁₀Emission spectrum of 20Ce/10Tb nanocrystal under ultraviolet excitation condition, and luminous intensity of product under ultraviolet excitation condition along with sensitized ion Ce³⁺(c) With activating ion Tb³⁺(d) The variation of (2).

FIG. 3 shows fluorescence spectra of the bare core (a) and the core/inert shell (b) and corresponding photographs of luminescence under UV lamp excitation.

FIG. 4 HepG2 cells and nanocrystals at 5% CO₂Cell viability after 24 h of co-culture at 37 ℃.

FIG. 5 KBi₃F₁₀: 20Ce/10Tb@KBi₃F₁₀An emission spectrum in a temperature range of 98-573K under excitation of a wavelength of 258 nm;

FIG. 6 (a) KBi₃F₁₀Emission spectrum of 20Ce/6Eu nano crystal under ultraviolet excitation condition, (b) KBi₃F₁₀: 20Ce/6Eu@KBi₃F₁₀An emission spectrum in a temperature range of 98-573K under excitation of a wavelength of 258 nm; (c) tb³⁺Is/are as follows⁵D₄→⁷F₅And Eu³⁺Is/are as follows⁵D₀→⁷F₂And (3) a trend graph of emission intensity (normalization) with temperature.

Detailed Description

1 experimental part

1.1 Main instruments and reagents:

potassium nitrate (99.0%), bismuth nitrate (98.0%), cerium nitrate (99.9%), terbium nitrate (99.9%), europium nitrate (99.9%), citric acid (99.5%), ammonium fluoride (99.99%), ethylene glycol (99.8%), MTT and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, and anhydrous ethanol was purchased from national pharmaceutical group chemical agents, ltd; HepG2 was purchased from Shanghai ai research Biotech Co., Ltd.

1.2 KBi₃F₁₀:Ce/Tb@KBi₃F₁₀Preparation of nanocrystals

With KBi₃F₁₀: 20Ce/10Tb@KBi₃F₁₀For example, 1 millimole of potassium nitrate (KNO)₃) 1.1 mmole bismuth nitrate (Bi (NO)₃)₃∙5H₂O), 0.6 mmol of cerium nitrate (Ce (NO)₃)₃∙5H₂O), 0.3 mmol terbium nitrate (Tb (NO)₃)₃∙5H₂O) and 4 mmol citric acid were dissolved in 6 ml deionized water, stirred for 20 minutes, and 8 mmol ammonium fluoride (NH) was added₄F) And 40 ml of polyethylene glycol, stirring for 1 hour, heating for 12 hours at the temperature of 120 ℃ in an oil bath kettle, and supplementing deionized water in time during the heating process. The product is washed 3-5 times with a mixture of ethanol and deionized water.

Then 1 millimole of potassium nitrate KNO₃2.1 mmoles of bismuth nitrate Bi (NO)₃)₃∙5H₂O and 4 mmol of citric acid were dissolved in 6 ml of deionized water, and after stirring for 20 minutes, 8 mmol of ammonium fluoride (NH) was added₄F) And 40 ml of polyethylene glycol, heating to 150 ℃ for reaction for 1 hour, cooling to room temperature, then adding the product obtained in the step, stirring for 1 hour, heating for 12 hours at 120 ℃ in an oil bath pan, and supplementing deionized water in time in the heating process; and after the solution is cooled to room temperature, washing for 3-5 times by using a mixed solution of ethanol and deionized water to obtain the core-shell nanocrystal.

The ion-doped samples with different concentrations or Eu ion-doped samples are realized by changing the corresponding ion concentration or species in the precursor solution.

1.3 characterization Instrument

X-ray diffraction patterns (Bruker D8 Advance, Cu-K α (λ =1.5405 a)), transmission electron microscope (TEM, FEI Tecnai G2F 20), spectrometer (FLUROHUB-B, HORIBA JOBIN YVON), ultraviolet lamp power 50W, inductively coupled plasma atomic emission instrument (PerkinElmer Optima 3300 DV).

Preparation of X-ray diffraction samples: paving the dried nanocrystalline in the groove of the sample support;

preparation of transmission electron microscope samples: dissolving all the nanocrystals synthesized in each time in 4 ml of ethanol solution, and dropping 3-6 drops of liquid on the ultrathin carbon film after ultrasonic treatment for 5 minutes.

1.4 nanocrystalline morphology, size contrast of core-shell structure

KBi₃F₁₀The X-ray diffraction pattern of the 20Ce/10Tb product is shown in figure 1a, all diffraction peaks correspond to JCPDS No. 41-0842 of standard PDF card one by one, and no redundant diffraction peaks exist, which indicates that the product obtained by the method is pure cubic phase KBi₃F₁₀. The analysis result of the transmission electron microscope shows that the product is flaky and has good dispersibility. As shown in FIG. 1c, is a core-shell KBi₃F₁₀: 20Ce/10Tb@KBi₃F₁₀The transmission electron microscope photo of the nanocrystalline shows that the grain size of the nanocrystalline with the core-shell structure is more uniform and the dispersity is good, and the average grain size is increased to 42nm, which also shows that the nanocrystalline core surface is successfully coated with the shell structure.

1.5 core-shell structure nanocrystalline fluorescence enhancement analysis

As shown in FIG. 2a, the excitation spectrum is typical of Ce³⁺: 4f → 5d broadband transition peak, the optimal excitation wavelength is about 350 nm. Under ultraviolet excitation, the product showed bright down-transferred luminescence in the green region, corresponding to Tb³⁺F → f transition of ion (FIG. 2 b). With sensitizing ion Ce³⁺When the ion concentration is increased from 10 to 20 mol%, the absorption capacity of the nanocrystal is increased, and therefore, the luminous intensity is remarkably enhanced, when Ce is added³⁺At ion concentrations above 20 mol%, the probability of radiationless cross relaxation between the sensitizing and activating ions increases, leading to a decrease in the luminescence intensity (fig. 2 c). Similarly, Tb³⁺The optimum doping concentration of the ions is about 10 mol% (fig. 2 d). KBi₃F₁₀The fluorescent quantum efficiency of 20Ce/10Tb nanocrystal is as high as 56%.

In FIG. 3, it can be seen that the core-shell structure KBi₃F₁₀: 20Ce/10Tb@KBi₃F₁₀The fluorescence intensity of the nanocrystal is KBi₃F₁₀About 11 times of 20Ce/10Tb nanocrystalThe inert shell layer coating structure can greatly enhance the fluorescence intensity of the nanocrystalline. In addition, the shapes of the fluorescence spectra of the two types of nanocrystals are approximately the same, but the fluorescence intensity is obviously changed, and the main reason of the enhancement is that after the shell layer is coated, luminescent ions on the surface are transferred into the interior, so that the surface defects are greatly reduced. The insets are fluorescent digital photographs of two nanocrystals, with the fluorescence being significantly enhanced by the naked eye.

1.6 cytotoxicity assay

The method for detecting the cytotoxicity of MTT (3- (4, 5-dimethylthiazole-2-methyl) -2, 5-diphenyltetrazole ammonium bromide) can be used for reflecting the biocompatibility of the core-shell nanocrystal. First, cell culture was performed, and HepG2 cells were seeded in a 96-well plate at 37 ℃ with 5% CO₂Incubate for 24 hours in ambient. Solutions of different concentrations were then added to the cell culture dish and incubated in the same environment for 24 hours. Then, the MTT powder was further dissolved in a phosphate buffer solution, and 100. mu.L of the MTT (0.5 mg/mL) solution was added to a petri dish and cultured at 37 ℃ for 4 hours. Finally, dimethyl sulfoxide is added, and detection is carried out under a microplate reader. As shown in FIG. 4, the survival rate of the HepG2 cells is high under different sample concentrations, and even when the sample solution concentration is as high as 100 mug/mL, the survival rate of the cells is still maintained at about 88%.

1.7 testing of temperature Change Properties

Temperature adjustment an Oxford Instruments liquid nitrogen thermostat (Oxford Instruments, Optistat DN) and a thermostat (Oxford Instruments, ITC502S) were used³⁺The luminous intensity of the light source gradually decreases with increasing temperature.

To verify the versatility of this experimental protocol, water-soluble KBi was further prepared in this application₃F₁₀: 20Ce/6Eu@KBi₃F₁₀And (4) nanocrystals. As shown in FIG. 6a, the product shows Eu under the condition of ultraviolet excitation³⁺Characteristic emission peak. As shown in FIG. 6b, Eu³⁺The luminous intensity of the light source gradually decreases with increasing temperature. Shown as Tb in FIG. 6c³⁺⁵D₄→⁷F₅And Eu³⁺Is/are as follows⁵D₀→⁷F₂The variation trend of the emission intensity (normalization) along with the temperature shows that whether the emission intensity is Tb³⁺Is also Eu^{3 +}The luminescence intensity of the light source gradually decreases with increasing temperature, and the phenomenon is caused by non-radiative decay under the condition of thermal activation as reported in other documents.

2. Conclusion

The application prepares the water-soluble KBi by a solvothermal method₃F₁₀Ce/Tb and KBi₃F₁₀Ce/Eu nanocrystals of which KBi₃F₁₀The fluorescent quantum efficiency of 20Ce/10Tb nanocrystal is as high as 56%. An inert shell layer is coated on the surface of the nano-crystalline material to obtain the nano-crystalline material with a core-shell structure, and the nano-crystalline material is modified by a citric acid ligand to obtain good water solubility and biocompatibility. Due to rare earth ions Tb³⁺Or Eu³⁺The nano-crystal can be used for preparing a temperature detection sensor in a living body, and meanwhile, the core-shell structure greatly enhances the fluorescence intensity and the luminous efficiency of the nano-crystal and has the advantages of quick response and high spatial resolution, so that the core-shell nano-crystal has a good application prospect in the detection of the fluorescence temperature in the living body.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present disclosure, including any person skilled in the art, having the benefit of the present disclosure. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (7)

1. A rare earth fluoride nanocrystalline material of a core-shell structure, characterized in that the nanocrystalline material has a nanocrystalline molecular formula such asThe following: KBi₃F₁₀:Ce/Tb@KBi₃F₁₀Or KBi₃F₁₀:Ce/Eu@KBi₃F₁₀。

2. The rare earth fluoride nanocrystalline material with core-shell structure according to claim 1, characterized in that the nanocrystalline material is a water-soluble nanocrystalline material coated with KBi ligand by citric acid₃F₁₀:Ce/Tb@KBi₃F₁₀Or KBi₃F₁₀:Ce/Eu@KBi₃F₁₀And (3) nanocrystal composition.

3. The rare earth fluoride nanocrystalline material of core-shell structure according to claim 1 or 2, characterized in that KBi₃F₁₀:Ce/Tb@KBi₃F₁₀The nanocrystal is KBi₃F₁₀:20Ce/10Tb@KBi₃F₁₀，KBi₃F₁₀:Ce/Eu@KBi₃F₁₀The nanocrystal is KBi₃F₁₀:20Ce/6Eu@KBi₃F₁₀。

4. The preparation method of the rare earth fluoride nanocrystalline material with the core-shell structure, which is characterized by comprising the following steps:

1) 1 millimole potassium nitrate KNO₃2.1 mmoles of bismuth nitrate Bi (NO)₃)₃∙5H₂O, 0.6 mmol of cerium nitrate Ce (NO)₃)₃∙5H₂O, 0.3 mmole Terbium nitrate Tb (NO)₃)₃∙5H₂O or 0.18 mmole europium nitrate Eu (NO)₃)₃∙5H₂O and 4 mmol of citric acid were dissolved in 6 ml of deionized water, and after stirring for 20 minutes, 8 mmol of ammonium fluoride (NH) was added₄F) And 40 ml of polyethylene glycol;

2) stirring for 1 hour, heating for 12 hours at the temperature of 120 ℃ in an oil bath kettle, and supplementing deionized water in time in the heating process; 3) washing the product with the mixed solution of ethanol and deionized water for 3-5 times;

4) then 1 millimole of nitric acidPotassium KNO₃2.1 mmoles of bismuth nitrate Bi (NO)₃)₃∙5H₂O and 4 mmol of citric acid were dissolved in 6 ml of deionized water, and after stirring for 20 minutes, 8 mmol of ammonium fluoride (NH) was added₄F) And 40 ml of polyethylene glycol, heating to 150 ℃ for reaction for 1 hour, cooling to room temperature, then adding the product obtained in the step 3), stirring for 1 hour, heating for 12 hours at the temperature of 120 ℃ in an oil bath pan, and supplementing deionized water in time in the heating process;

5) and after the solution is cooled to room temperature, washing for 3-5 times by using a mixed solution of ethanol and deionized water to obtain the core-shell nanocrystal.

5. Use of a nanocrystalline material according to any one of claims 1-3 for in vivo temperature sensing applications.

6. An in vivo temperature detection sensor comprising the nanocrystalline material of claim 1, 2 or 3.

7. An in vivo temperature detection kit comprising the detection sensor according to claim 6.

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