CN107607499A - Composite Nano oxygen sensor material with high efficiency energy transmission, preparation method and applications - Google Patents

Abstract

The invention discloses a composite nano-oxygen sensing material with energy transfer phenomenon, a preparation method and its application, and belongs to the technical field of biological dissolved oxygen fluorescence detection materials. The invention prepares amphiphilic silane-coated loaded platinum (II) MESO-tetrakis(pentafluorobenzene) porphine (PtTFPP), coumarin 6 (C6), iron ferric oxide nanoparticles (Fe ₃ O ₄ ) composite nanomaterials, due to the loading of coumarin 6 (C6), Resonance-based energy transfer improves the red light emission intensity of the oxygen probe PtTFPP, and improves the oxygen sensitivity compared with coumarin-free 6 (C6); at the same time, it can use long-wavelength 458nm light excitation, reducing the large noise of ultraviolet excitation , biological tissue damage and other shortcomings, and because the emission peak of C6 at 500nm does not change with the oxygen content, while the emission peak of PtTFPP at 652nm decreases with the increase of oxygen content, the ratiometric fluorescence method can be used to improve the accuracy of oxygen monitoring. In addition, the loading of ferric oxide (Fe ₃ O ₄ ) in magnetic nanoparticles increases the functions of magnetic navigation and magnetic enrichment.

Description

Composite nano oxygen sensing material with high efficiency energy transfer, preparation method and its application

technical field

The invention belongs to the technical field of fluorescence detection materials for biological dissolved oxygen, and in particular relates to a composite nanometer oxygen sensing material with energy transfer phenomenon, a preparation method and an application thereof.

Background technique

Oxygen content at the body fluid and cell levels is an important basis for understanding the metabolism of cells and tissues, and is of great significance in the field of biomedicine. The amount of dissolved oxygen in the body usually fluctuates within a certain range. If the oxygen content in the body is too low for a long time, it will lead to the occurrence of some chronic diseases, such as cancer. Therefore, the level of oxygen in human cells is regarded as an information sign of whether the physiological process and metabolism of the human body are normal or not, and its quantitative detection is of great significance for the prevention and timely detection of cancer.

The use of fluorescent nanosensors doped with oxygen probe molecules to detect the amount of dissolved oxygen in cells has been recognized by researchers in recent years. Fluorescent oxygen probes generally use transition metal complexes, whose metal-ligand charge-transfer fluorescence properties are highly sensitive to oxygen content. Except for special cases, both fluorescence intensity and fluorescence lifetime decrease with the increase of oxygen concentration. Oxygen probe molecules are coated with oxygen-permeable nanoparticles to prepare a nanosensor, so that the probe enters the cytoplasm through endocytosis, and the detection of dissolved oxygen in the cell can be realized. But at present, its shortcomings in application are weak detection of fluorescence and low sensitivity, so the development of high-intensity fluorescent oxygen sensors is the direction of most scientific researchers in recent years.

Contents of the invention

In order to solve the above problems, the object of the present invention is to provide a composite nano-oxygen sensing material with high-efficiency energy transfer characteristics utilizing long-wavelength excitation and long-wavelength emission: Coumarin 6 (C6) with fluorescent labeling function can Platinum(II) MESO-tetrakis(pentafluorophenyl)porphin (PtTFPP) for oxygen sensing function undergoes efficient energy transfer. Amphiphilic silane-coated platinum(Ⅱ) MESO-tetrakis(pentafluorophenyl)porphine (PtTFPP), coumarin 6 (C6), ferric iron tetroxide nanoparticles (Fe ₃ O ₄ ) prepared based on this method Composite nanomaterials have good oxygen sensitivity and multifunctional biological applications.

The object of the present invention is to provide a composite nano-oxygen sensing material with high-efficiency energy transfer and a preparation method thereof.

A composite nano-oxygen sensing material with high-efficiency energy transfer, loaded with platinum(II) MESO-tetrakis(pentafluorophenyl)porphine (PtTFPP), coumarin 6(C6) and trioxide tetroxide coated with amphiphilic silane The iron nanoparticles (Fe ₃ O ₄ ) are composed according to the mass ratio of 75:2-8:1-10:20.

Preferably, the mass of the amphiphilic silane, platinum (II) MESO-tetrakis(pentafluorobenzene) porphine (PtTFPP), coumarin 6 (C6), and iron ferric oxide nanoparticles (Fe ₃ O ₄ ) The ratio is 75:5:6:20.

A method for preparing a composite nano-oxygen sensing material with high-efficiency energy transfer, the specific steps are as follows:

(1) Prepare amphiphilic silane, platinum(II) MESO-tetrakis(pentafluorophenyl)porphine (PtTFPP), coumarin 6 (C6), and ferric iron tetroxide (Fe ₃ O ₄ ) at a concentration of 1 ～10mg/mL tetrahydrofuran solution, and then mix the four solutions according to the volume ratio of 10:2-8:1-10:4; then add the tetrahydrofuran solution containing functional oily molecules to the mixed solution; among them, the amphiphile The volume ratio of functional silane to tetrahydrofuran solution containing functional oily molecules is 1:6.8-8.3.

(2) Ultrasonicate 1-5 mL of the mixed solution obtained in step (1) for 5-30 minutes to make it evenly mixed, then inject it into 5-20 mL of ammonia solution with pH=8-10, and perform hydrolysis reaction for 1-5 hours;

(3) Put the mixed solution obtained in step (2) into a dialysis bag with a molecular weight cut-off of 8000-14000, and dialyze for 24-48 hours to remove tetrahydrofuran, thereby obtaining a water-soluble composite nanomaterial coated with amphiphilic silane.

The application in the present invention is based on the efficient energy transfer of coumarin 6 (C6) to platinum (II) MESO-tetrakis(pentafluorobenzene) porphine (PtTFPP), which improves the oxygen sensing sensitivity. The above characteristics make this core-shell nanoparticle have great application value in biological oxygen sensing.

Another object of the present invention is to provide an application of a composite nano-oxygen sensing material with high-efficiency energy transfer in biological oxygen sensing. The composite nano-oxygen sensing material Fe ₃ O ₄ @PtTFPP/C6@silane, which is a composite nano-oxygen sensing material with efficient energy transfer from coumarin 6 (C6) to platinum (II) MESO-tetrakis(pentafluorobenzene) porphine (PtTFPP), The oxygen sensing sensitivity is improved, the oxygen quenching efficiency is as high as 94%, and the Stern-Volmer curve shows a good linear relationship. Compared with the sensing material Fe ₃ O ₄ @PtTFPP@silane without adding coumarin 6 (C6), Stern -Volmer curve slope increased by 19%, oxygen sensitivity increased. At the same time, 458nm long-wavelength excitation replaces ultraviolet excitation, which avoids the disadvantages of high noise and damage to biological tissues. The addition of magnetic iron ferric oxide nanoparticles increases the magnetic navigation and magnetic enrichment functions of the material. The combination of magnetic, optical, and oxygen-sensing properties makes this composite nano-oxygen sensing material promising for biological applications.

In the present invention, the oxygen probe molecule platinum (II) MESO-tetrakis(pentafluorobenzene) porphine (PtTFPP) is used as the oxygen sensor molecule, and the energy transfer occurs between the oxygen and the PtTFPP molecule through collision or other ways, so that the luminescence of the PtTFPP luminescent molecule is weakened , to detect the oxygen concentration based on this oxygen quenching effect. Platinum (Ⅱ) MESO-tetrakis(pentafluorobenzene) porphine (PtTFPP) has corresponding absorption peaks at wavelengths of 400nm, 506nm and 540nm. At the same time, its excitation position can excite 652nm red light emission within the range of 350nm to 460nm. In view of its spectral characteristics, small molecules that can overlap with the excitation wavelength of platinum (Ⅱ) MESO-tetrakis(pentafluorophenyl) porphine (PtTFPP) were searched for. Coumarin 6 (C6) has an excellent fluorescent labeling function, and its excitation wavelength can excite 500nm green light emission within the range of 370nm to 480nm. Therefore, 458nm blue light is selected as the common excitation wavelength of the two, which can realize platinum (II) MESO-tetra(pentafluorobenzene) porphine (PtTFPP) emitting red light at 652nm, and coumarin 6 (C6) emitting green light at 500nm. , coumarin 6 (C6) to platinum (Ⅱ) MESO-tetrakis (pentafluorobenzene) porphine (PtTFPP) efficient energy transfer, platinum (Ⅱ) MESO-tetrakis (pentafluorobenzene) porphine (PtTFPP) at 652nm The emission intensity at the wavelength position increases greatly, and the emission intensity at the 500nm wavelength position of coumarin 6 (C6) decreases obviously. Since the occurrence of energy transfer requires a small distance between two small molecules, the effect is not obvious in the solution dispersion state, so the amphiphilic silane coating is used to make the distance close enough for efficient energy transfer. In addition, coumarin 6 (C6) does not have the function of oxygen sensing, and its green light does not change with the change of oxygen concentration. Therefore, the introduction of coumarin 6 (C6) can also use the ratiometric fluorescence monitoring method to increase the concentration of oxygen. Sensing accuracy. Platinum(II) MESO-tetrakis(pentafluorophenyl)porphine (PtTFPP) has an efficient excitation site in the ultraviolet region, which will cause disadvantages such as large noise and damage to organisms. The addition of coumarin 6 (C6) will generate higher intensity red light excitation at the 458nm long-wavelength excitation position. It can be seen that the introduction of coumarin 6 (C6) has brought many performance improvements, which is of great significance. In order to make the material have the functions of magnetic navigation and magnetic enrichment, ferroferric oxide nanoparticles (Fe ₃ O ₄ ), a magnetic material, are added. The present invention uses Fe ₃ O ₄ as the core, amphiphilic silane coating and loading platinum (II) MESO-tetrakis (pentafluorobenzene) porphine (PtTFPP), coumarin 6 (C6) nanocomposite material in a wide range It has a magnetic response within a certain magnetic field range, and can use magnetic characteristics to achieve cell separation. The material emits bright red light, and the luminous intensity is sensitive to oxygen. Compared with pure platinum(II) MESO-tetrakis(pentafluorophenyl)porphine (PtTFPP), it exhibits good luminescent thermodynamic stability. The combination of magnetic, optical, and oxygen sensing properties makes this nanocomposite promising for biological applications.

Description of drawings

Figure 1: Transmission electron micrograph of the composite nano-oxygen sensing material (Fe ₃ O ₄ @PtTFPP/C6@silane); where (a) is 200nm, (b) is 50nm;

Figure 2: (a) The emission spectrum of Fe ₃ O ₄ @PtTFPP@silane prepared by loading different concentrations of PtTFPP, with an excitation wavelength of 458nm; (b) Fe ₃ O prepared by loading different concentrations of coumarin 6 (C6) ₄ The emission spectrum of @PtTFPP/C6@silane, the excitation wavelength is 458nm;

Figure 3: (a) Four kinds of nanoparticles: Fe ₃ O ₄ @PtTFPP@silane, Fe ₃ O ₄ @C6@silane, Fe ₃ O ₄ @PtTFPP/C6@silane and pure PtTFPP excitation spectra; (b) four Kinds of nanoparticles: Fe ₃ O ₄ @PtTFPP@silane, Fe ₃ O ₄ @C6@silane, Fe ₃ O ₄ @PtTFPP/C6@silane and pure PtTFPP emission spectra;

Figure 4: Ultrafast kinetic testing of four nanoparticles: coumarin 6 in Fe ₃ O ₄ @C6@silane, Fe ₃ O ₄ @PtTFPP/C6@silane (PtTFPP 1 mg/mL, 60, 80, 100 μL) (C6) lifetime;

Figure 5: The emission spectrum of the water-soluble magnetic composite nanomaterial (Fe ₃ O ₄ @PtTFPP/C6@silane) prepared in Example 1 under the excitation of 458nm excitation light; the intensity value of the emission spectrum at 652nm will vary with Decrease with the increase of oxygen content, while the intensity value at 500nm remains unchanged with the increase of oxygen content;

Figure 6: Stern-Volmer curves, indicating that the magnetic composite nanomaterials (Fe ₃ O ₄ @PtTFPP@silane, Fe ₃ O ₄ @PtTFPP/C6@silane) have good oxygen sensing performance, and the addition of C6 improves the energy transfer Oxygen sensitivity;

Figure 7: Verification of reversible oxygen sensing of composite nanomaterials (Fe ₃ O ₄ @PtTFPP/C6@silane);

Figure 8: Oxygen sensing in MCF-7 cells before and after adding glucose oxidase; the inset is a confocal image.

detailed description

In order to better understand the present invention, the solution of the present invention will be further described through specific examples below, and the protection scope of the present invention should include the entire content of the claims, but is not limited thereto.

Example 1

Preparation of Composite Nano Oxygen Sensing Material (Fe ₃ O ₄ @PtTFPP/C6@silane)

(1) Iron ferric oxide nanoparticles were synthesized according to the co-precipitation method. In order to increase its stability, the surface was coated with oleic acid (Wang, J., et al. Remote manipulation of micronomachines containing magnetic nanoparticles. Optics Letters 34,581 -583 (2009)).

(2) The prepared oily iron ferric oxide nanoparticles are formulated into 5 mg/mL tetrahydrofuran solution, n-octyltrimethoxysilane (purchased from sigma company) is formulated into 7.5 mg/mL tetrahydrofuran solution, platinum ( Ⅱ) MESO-tetrakis(pentafluorobenzene) porphine (PtTFPP) (purchased from sigma company), coumarin 6 (C6) (purchased from sigma company) were formulated into 1mg/mL tetrahydrofuran solution respectively; Platinum (II) MESO-tetrakis(pentafluorobenzene) porphine (PtTFPP, 1mg/mL), coumarin 6 (C6, 1mg/mL), and amphiphilic silane (7.5mg/mL) were placed on a magnetic stirrer and stirred For 5 minutes, place ferric oxide nanoparticles (5 mg/mL) in an ultrasonic cleaner for 5 minutes;

(3) Take 60 μL of platinum (II) MESO-tetrakis(pentafluorobenzene) porphine (PtTFPP, 1 mg/mL), 50 μL of coumarin 6 (C6, 1 mg/mL), and 100 μL of amphiphilic silane (7.5 mg/mL) , 40 μL of iron ferric oxide nanoparticles (5 mg/mL) were mixed, and then 750 μL of a tetrahydrofuran solution (THF) of functional oily molecules was added to the mixed solution to prepare a solution with a total amount of 1 ml.

(4) Ultrasound the mixed solution obtained in step (3) for 5 to 30 minutes to mix it evenly, then pour it into 5 mL of ammonia solution (pH=9), and hydrolyze it for 3 hours at room temperature;

(5) Put the mixed solution obtained in step (4) into a dialysis bag with a molecular weight cut-off of 8000 to 14000, and dialyze for 24 hours to remove tetrahydrofuran, thereby obtaining the composite nano-oxygen sensor material with energy transfer phenomenon of the present invention . The product concentration was 0.177 mg/mL.

Embodiment 2 comparative example

Preparation of Composite Nano Oxygen Sensing Material (Fe ₃ O ₄ @PtTFPP@silane)

Repeat Example 1, the difference is that the functional oily molecule loaded this time does not contain coumarin 6 (C6) with fluorescent labeling function (purchased from sigma company). All the other steps are the same as in Example 1. The resulting product concentration was 0.168 mg/mL.

As shown in Figure 1(a)(b): Fe ₃ O ₄ @PtTFPP/C6@silane NPs are well dispersed, and the outer layer has a thin transparent coating layer. The single nanoparticle is similar to a spherical shape, with a diameter of about 100 nm. It can be seen that it is composed of many small ferric oxide nanoparticles. This is because ferric oxide nanoparticles will attract each other and agglomerate.

As shown in Figure 2, according to the preparation method in Example 1, first determine the optimal concentration of PtTFPP, based on four different concentrations of Fe ₃ O ₄ @PtTFPP@silane (PtTFPP, 1mg/mL, 20, 40, 60, 80μL), The emission spectrum test was carried out with an excitation wavelength of 458nm. As shown in figure (a), when the amount of PtTFPP was 60 μL, the corresponding emission peak intensity was the largest, so 60 μL was determined to be the most suitable amount of PtTFPP (1 mg/mL). On the basis of establishing the most suitable amount of PtTFPP, based on four different concentrations of Fe ₃ O ₄ @PtTFPP/C6@silane (C6, 1mg/mL, 10, 20, 50, 100μL), using 458nm excitation wavelength for emission spectrum test , as shown in Figure (b), when the amount of C6 is 50 μL, the corresponding emission peak intensity is the largest, so 50 μL is determined to be the most suitable amount of C6 (1 mg/mL).

As shown in Figure 3, it can be concluded from the excitation spectrum in (a) that there is an overlapping area between the excitation positions of C6 and PtTFPP, and the characteristic emission of PtTFPP and C6 can be simultaneously excited by excitation with 458nm light. (b) The emission spectrum shows that at 652nm, the luminous intensity of Fe ₃ O ₄ @PtTFPP/C6@silane is significantly stronger than that of Fe ₃ O ₄ @PtTFPP@silane, and at 500nm Fe ₃ O ₄ @PtTFPP/C6@silane Weaker than Fe ₃ O ₄ @C6@silane, C6 has a fluorescence resonance energy transfer effect with PtTFPP, which increases the luminous intensity at 652nm and decreases at 500nm.

As shown in Figure 4, in order to study the ultrafast dynamics in the excited state in detail, ultrafast spectroscopy is used to analyze the lifetime change. Based on four kinds of nanoparticles: Fe ₃ O ₄ @C6@silane, Fe ₃ O ₄ @PtTFPP/C6@silane (PtTFPP 1mg/mL, 60, 80, 100μL), using 458nm wavelength excitation, the lifetime test results are shown in Figure 4 Show. For PtTFPP (Fe ₃ O ₄ @C6@silane) without adding PtTFPP, the lifetime of C6 is 1.9ns. However, with the increasing amount of PtTFPP (60, 80, 100 μL PtTFPP), the lifetime of C6 in Fe ₃ O ₄ @PtTFPP/C6@silane decreased, which were 1.51, 1.48, and 1.10 ns. Due to the energy transfer from C6 to PtTFPP, the time for C6 to stay in the excited state is shortened, thus its lifetime is shortened. Ultrafast spectroscopic tests fully demonstrate the resonant energy transfer from C6 to PtTFPP.

As shown in Figure 5 (a), the emission peak intensity of Fe ₃ O ₄ @PtTFPP/C6@silane at 652nm decreases continuously with the increase of oxygen content, but the emission peak intensity at 500nm remains unchanged. Therefore, the Fe ₃ O ₄ @PtTFPP/C6@silane material has a good ratiometric fluorescence effect. Define R as the ratio of the emission intensities of PtTFPP and C6, and the oxygen sensitivity of Fe ₃ O ₄ @PtTFPP/C6@silane nanoparticles as Q

Q＝(R _N2 -R _O2 )/R _N2

Among them, R _N2 and R _O2 represent the emission intensity ratios in pure nitrogen environment and pure oxygen environment, respectively, so the oxygen sensitivity of Fe ₃ O ₄ @PtTFPP/C6@silane nanoparticles is calculated to be higher than 94%.

As shown in Figure 6, the corresponding Stern-Volmer curves are drawn for Fe ₃ O ₄ @PtTFPP/C6@silane(a) and Fe ₃ O ₄ @PtTFPP@silane(b), compared to Fe ₃ O ₄ @PtTFPP/C6@silane, After adding C6, the slope of Stern-Volmer curve fitting increased from 0.7658 to 0.9059. The results show that Fe ₃ O ₄ @PtTFPP/C6@silane has better oxygen sensitivity and a good linear relationship after adding C6.

Example 3

Oxygen sensing test of Fe ₃ O ₄ @PtTFPP/C6@silane and Fe ₃ O ₄ @PtTFPP@silane

When using 458nm excitation light to excite Fe ₃ O ₄ @PtTFPP/C6@silane composite nanomaterials, because it contains platinum(Ⅱ) MESO-tetra(pentafluorobenzene) porphine, it will emit red light, and its peak position is at 652nm, And the intensity value of the emitted light changes with the change of the surrounding oxygen content. However, coumarin 6 (C6) emits green light, and its peak position is at 500nm, and the intensity of the emitted light does not change with the change of the surrounding oxygen content. When using a gas mixer to configure mixed gases with different oxygen contents (volume fractions of oxygen are 0, 20, 40, 60, 80, 100% respectively), pass it into the sealed cuvette, each time Continue for 20 minutes, so that the gas in the cuvette reaches an equilibrium state. Finally, the composite nanomaterial in the cuvette is tested for fluorescence emission spectrum. Under the excitation of 458nm excitation light, the intensity value of its emission spectrum at 652nm will gradually decrease with the increase of oxygen content. However, the intensity at 500 nm remains constant with the increase of oxygen content. The same test procedure was used for Fe ₃ O ₄ @PtTFPP@silane.

As shown in Figure 7, the reversibility test was carried out on Fe ₃ O ₄ @PtTFPP/C6@silane. By alternately feeding pure oxygen and pure nitrogen, it shows that Fe ₃ O ₄ @PtTFPP/C6@silane has good reversibility and can be reused. works well.

As shown in Figure 8, glucose (8mM, 100μL) was added to the cell culture dish, monitored after 5 minutes, glucose oxidase (1.4mg/mL, 50μL) was added quickly, and then the fluorescence change was monitored over time. After adding oxidase, 7 min showed that the marked region 1 had the greatest red fluorescence intensity. According to the numerical change of the red fluorescence intensity, the change curve of the red fluorescence intensity in area 1 is obtained, as shown in figure (a), as time goes on, the red fluorescence becomes stronger before 7 minutes, and then slowly decreases. The illustration in (a) shows the enhanced red fluorescence more intuitively. Because the material has good ratio fluorescence characteristics, red/green is defined as the ratio of red fluorescence intensity to green fluorescence intensity. The red/green change in area 1 is shown in Figure (b). In the first 7 minutes, red/green continued to rise , and then decreased slightly and stabilized. The inset shows that the green fluorescence is stable and the red fluorescence is significantly enhanced.

The above-mentioned embodiments of the present invention are examples for clearly illustrating the present invention, rather than limiting the implementation of the present invention. For those of ordinary skill in the art, some changes can be made on the basis of the above description. can achieve a certain effect. However, those related to the technical solutions of the present invention are still within the scope of protection of the present invention.

Claims (4)

1. the composite Nano oxygen sensor material with high efficiency energy transmission, it is characterised in that the load coated by amphiphilic silane Enter (phenyl-pentafluoride) porphines (PtTFPP) of platinum (II) MESO- tetra-, coumarin 6 (C6) and ferriferrous oxide nano-particle (Fe₃O₄) according to Mass ratio is 75:2-8:1-10:20 compositions.

2. there is the composite Nano oxygen sensor material of high efficiency energy transmission as claimed in claim 1, it is characterised in that described Amphiphilic silane, (phenyl-pentafluoride) porphines (PtTFPP) of platinum (II) MESO- tetra-, coumarin 6 (C6), ferriferrous oxide nano-particle (Fe₃O₄) mass ratio be 75:5:6:20.

3. the preparation method of the composite Nano oxygen sensor material with high efficiency energy transmission as claimed in claim 1, it is special Sign is, comprises the following steps that：

(1) by amphiphilic silane, (phenyl-pentafluoride) porphines (PtTFPP) of platinum (II) MESO- tetra-, coumarin 6 (C6), ferroso-ferric oxide (Fe₃O₄) tetrahydrofuran solution that concentration is 1~10mg/mL is each configured to, four kinds of solution are pressed 10 afterwards：2-8：1-10：4 Volume ratio mixed；The tetrahydrofuran solution containing feature oiliness molecule is added into the mixed solution again；Wherein, The volume ratio of amphiphilic silane and the tetrahydrofuran solution containing feature oiliness molecule is 1:6.8-8.3.

(2) make within 5~30 minutes it well mixed the mixed solution ultrasound that 1~5mL steps (1) obtain, it is then injected into 5~ In 20mL, pH=8~10 ammonia spirit, hydrolysis 1~5 hour；

(3) mixed solution that step (2) obtains is fitted into the bag filter that molecular cut off is 8000~14000, dialysis 24~ 48 hours to remove tetrahydrofuran, so as to obtain the Water Soluble Compound nano material of amphiphilic silane cladding.

4. the composite Nano oxygen sensor material with high efficiency energy transmission is in bio tank/oxygen sensing side as claimed in claim 1 The application in face.