Surface confinement long-afterglow luminescent probe as well as preparation method and application thereof

Abstract

The invention discloses a surface confinement long afterglow luminescent probe, and a preparation method and application thereof, and the preparation method comprises the following preparation steps: mixing zinc nitrate, gallium nitrate and sodium germanate, dissolving in deionized water, stirring, reacting, centrifugally washing, and collecting a product; dissolving the product, zinc nitrate, sodium germanate, gallium nitrate and chromic nitrate in deionized water; reacting to obtain a reaction product; performing centrifugal washing, and collecting a product; dispersing the product into dimethylformamide, dropwise adding 3-aminopropyltriethoxysilane, and condensing and refluxing to obtain a product; dispersing the product into dimethylformamide, adding succinic anhydride and 4-dimethylaminopyridine, and stirring for reaction to obtain a reaction product; adding the reaction product into 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, carrying out stirring reaction, centrifuging the solution, and dispersing the solution into PBS (Phosphate Buffer Solution); adding BHQ-3-NH2 into the solution, reacting, and centrifuging to obtain a probe; the probe provided by the invention has better FRET efficiency.

Description

Technical Field

[0001] The invention belongs to the field of inorganic materials, and in particular relates to a surface confined long afterglow luminescence probe, a preparation method and an application thereof. Background Art

[0002] The nonradiative dipole-dipole coupling between two adjacent molecules Resonance energy transfer is crucial for the application of nanomaterials in remote sensing, cell imaging, single-molecule spectroscopy and optoelectronics. However, the resonance energy transfer process between conventional nanomaterials and specific molecules is usually difficult to control, mainly because the energy transfer process is heavily dependent on the distance between the donor and the acceptor. At the FRET critical distance, the energy transfer rate is equal to the internal decay rate of the donor. When the distance between the donor and the acceptor is greater than the FRET critical distance, the FRET efficiency is usually lower than the expected efficiency, which also severely limits the application of long-afterglow luminescent materials based on FRET, especially in high-sensitivity detection; therefore, achieving efficient long-range FRET efficiency, increasing the signal intensity change of the long-afterglow probe, and thus improving the sensitivity of enzyme activity detection is a research-worthy issue. Summary of the invention

[0003] In view of the deficiencies in the prior art, the present invention proposes a surface confined long afterglow luminescence probe, a preparation method and applications thereof.

[0004] A method for preparing a surface confined long afterglow luminescence probe comprises the following steps:

[0005] (1) mixing and dissolving zinc nitrate, gallium nitrate and sodium germanate in deionized water, and adjusting the pH of the mixture;

[0006] (2) stirring the above-mentioned mixed solution and reacting it to obtain a reaction product A;

[0007] (3) centrifuging and washing the above reaction product to collect the product ZGGO nanoparticles;

[0008] (4) dissolving the ZGGO nanoparticles, zinc nitrate, sodium germanate, gallium nitrate and chromium nitrate in deionized water, and adjusting the pH of the mixture;

[0009] (5) stirring the mixed solution and reacting it to obtain a reaction product B;

[0010] (6) The above reaction product is centrifuged and washed, and the collected product is ZGGO@ZGGO:Cr nanoparticles;

[0011] (7) dispersing the above ZGGO@ZGGO:Cr nanoparticles in dimethylformamide, adding 3-aminopropyltriethoxysilane dropwise, condensing and refluxing, and washing with dimethylformamide after the reflux to obtain ZGGO@ZGGO:Cr-NH2 nanoparticles;

[0012] (8) dispersing the above ZGGO@ZGGO:Cr-NH2 nanoparticles in dimethylformamide, adding succinic anhydride and 4-dimethylaminopyridine to stir the reaction, and washing with a mixture of ethanol and water to obtain a reaction product C;

[0013] (9) adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the above reaction product, stirring the reaction, centrifuging the solution and dispersing it into PBS;

[0014] (10) BHQ-3-NH2 was added to the above solution, reacted, centrifuged and the product was washed with deionized water to obtain ZGGO@ZGGO:Cr-BHQ-3.

[0015] Preferably, the pH value of the mixed solution in step (1) and step (4) is 8-9.

[0016] Preferably, the reaction temperature in step (2) and step (5) is 220° C., and the reaction time is 10 h.

[0017] Preferably, the condensation reflux temperature is 70-100°C.

[0018] Preferably, the reaction temperature in step (8) is 37°C.

[0019] A surface confined long afterglow luminescence probe comprises ZGGO@ZGGO:Cr nanoparticles with surface modified BHQ-3 organic molecules.

[0020] The probe prepared by the above preparation method or the application of the above probe in sensitive detection of enzyme activity.

[0021] The beneficial effects of the present invention are as follows:

[0022] Improve the FRET efficiency of long afterglow luminescence probes and effectively construct enzyme activity long afterglow luminescence probes. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] 1. Figure 1Transmission electron microscopy images of ZGGO@ZGGO:Cr nanoparticles with different shell thicknesses. The scale bar is 100 nm.

[0025] 2. Figure 2 Particle size distribution diagram of ZGGO@ZGGO:Cr nanoparticles with different shell thicknesses.

[0026] 3. Figure 3 Phosphorescence intensity of ZGGO@ZGGO:Cr nanoparticles with different shell thicknesses before and after adding BHQ-3.

[0027] 4. Figure 4 This is the linear curve of the long afterglow luminescence probe for detecting different concentrations of azoreductase.

[0028] 5. Figure 5 FRET efficiency of ZGGO@ZGGO:Cr nanoparticles with different shell thicknesses after adding different BHQ-3.

[0029] 6. Figure 6 The change in fluorescence lifetime of ZGGO:Cr and ZGGO@ZGGO:Cr nanoparticles with different shell thicknesses before and after adding BHQ-3. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1

[0032] Azoreductase (AzoR): purchased from Sigma

[0033] Nicotinamide adenine dinucleotide phosphate (NADPH): purchased from China Bio-Technology Co., Ltd.

[0034] BHQ-3-NH2: Customized by the company, purity 95%

[0035] Except for the above contents, unless otherwise specified, the methods and reagents used in this application are commonly used in the art or well known to those skilled in the art.

[0036] A method for preparing a surface confined long afterglow luminescence probe, the method comprising the following steps:

[0037] The precursors include zinc nitrate, gallium nitrate and sodium germanate. After the precursors are mixed in a certain ratio, the pH value of the mixed solution is adjusted to about 8.5 with concentrated ammonia water.

[0038] The reaction solution was placed in a high temperature and high pressure resistant reactor and reacted at 220° C. for 10 hours.

[0039] The reaction product was centrifuged and washed with water to collect the product Zn 1.4 Ga 1.2 Ge 0.4 O4 nanoparticles.

[0040] The above Zn 1.4 Ga 1.2 Ge 0.4 O4 nanoparticles are mixed with zinc nitrate, gallium nitrate, sodium germanate and cadmium nitrate in a beaker, and the pH value of the mixture is adjusted to about 8.5 with concentrated ammonia water.

[0041] The reaction solution was placed in a high temperature and high pressure resistant reactor and reacted at 220° C. for 10 hours.

[0042] The reaction product was centrifuged and washed with water to collect the product Zn 1.4 Ga 1.2 Ge 0.4 O4@Zn 1.4 Ga 1.2 Ge 0.4 O4:Cr nanoparticles.

[0043] The long afterglow nanoparticles prepared above are surface modified.

[0044] The prepared Zn 1.4 Ga 1.2 Ge 0.4 O4@Zn 1.4 Ga 1.2 Ge 0.4 O4:Cr nanoparticles were dispersed in N,N-dimethylformamide, 3-aminopropyltriethoxysilane was added dropwise, and condensed and refluxed at 80°C for 10 hours to obtain particles ZGGO@ZGGO:Cr-NH2 with amino groups modified on the surface.

[0045] The reaction solution was washed with N,N-dimethylformamide, dispersed in N,N-dimethylformamide, and succinic anhydride and 4-dimethylaminopyridine were added to react at room temperature for 6 hours to obtain particles ZGGO@ZGGO:Cr-COOH with succinimide ester modified on the surface.

[0046] The reaction solution was washed with ethanol and water, and a certain amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added, and stirred at 37° C. for 30 minutes. The solution was quickly centrifuged and dispersed in a certain amount of PBS.

[0047] A certain amount of BHQ-3-NH2 was added to the above solution and reacted in outdoor for 4 to 8 h. The product was centrifuged and washed with deionized water to obtain ZGGO@ZGGO:Cr-BHQ-3.

[0048] Comparative Example 1

[0049] Azoreductase (AzoR): purchased from Sigma

[0050] Nicotinamide adenine dinucleotide phosphate (NADPH): purchased from China Bio-Technology Co., Ltd.

[0051] Except for the above contents, unless otherwise specified, the methods and reagents used in this application are commonly used in the art or well known to those skilled in the art.

[0052] The precursors include zinc nitrate, gallium nitrate and sodium germanate. After the precursors are mixed in a certain ratio, the pH value of the mixed solution is adjusted to about 8.5 with concentrated ammonia water.

[0053] The reaction solution was placed in a high temperature and high pressure resistant reactor and reacted at 220° C. for 10 hours.

[0054] The reaction product was centrifuged and washed with water to collect the product Zn 1.4 Ga 1.2 Ge 0.4 O4 nanoparticles.

[0055] The above Zn 1.4 Ga 1.2 Ge 0.4 O4 nanoparticles are mixed with zinc nitrate, gallium nitrate, sodium germanate and cadmium nitrate in a beaker, and the pH value of the mixture is adjusted to about 8.5 with concentrated ammonia water.

[0056] The reaction solution was placed in a high temperature and high pressure resistant reactor and reacted at 220° C. for 10 hours.

[0057] The reaction product was centrifuged and washed with water to collect the product Zn 1.4 Ga 1.2 Ge 0.4 O4@Zn 1.4 Ga 1.2 Ge 0.4 O4:Cr nanoparticles.

[0058] The long afterglow nanoparticles are not surface modified.

[0059] The modified long afterglow nanoparticles prepared in Example 1 and Comparative Example 1 were tested for phosphorescence intensity.

[0060] 1 mg / mL ZGGO@ZGGO:Cr-BHQ-3 and

[0061] Zn 1.4 Ga 1.2 Ge 0.4 O4@Zn 1.4 Ga 1.2 Ge 0.4 O4:Cr nanoparticles were placed in a fluorescence spectrometer and the emission phosphorescence spectrum was scanned using 260 nm as the excitation wavelength. Figure 3 , the data of different concentrations of BHQ-3 were processed to obtain the FRET efficiency Figure 5 ,From the data, it can be seen that after the surface of long afterglow nanoparticles are ,modified with BHQ-3, the probe obtains better FRET efficiency.

[0062] Linear curve of the long afterglow luminescence probe for detecting different concentrations of azoreductase.

[0063] Experimental materials: AzoR, NADPH, ZGGO@ZGGO:Cr-BHQ-3.

[0064] Experimental reagents: phosphate buffered saline.

[0065] Experimental equipment: constant temperature culture oscillator, centrifuge, double row tubes, nitrogen, oil pump, centrifuge tubes.

[0066] Experimental conditions: 30℃.

[0067] Experimental steps: ① Dissolve the prepared probe in a certain amount of PBS solution, and perform three cycles of freeze-vacuum-thaw process in a nitrogen environment to remove oxygen in the system. ② Take an appropriate amount of the solution in 1) and add a certain amount of azoreductase and NADPH, pass nitrogen to remove oxygen in the system, and react at 37°C for 24 hours. ③ Use a fluorescence instrument to detect the intensity before and after the reaction.

[0068] Experimental conclusion: When using azoreductase and NADPH, the afterglow intensity of ZGGO@ZGGO:Cr-BHQ-3 is restored. When using different concentrations of azoreductase, the afterglow intensity of the particles is restored as shown in the figure. Figure 4 .

[0069] The reaction solution was washed with phosphate buffer and dispersed into a 1 mg / mL solution for detection.

[0070] Fluorescence lifetime test of long afterglow luminescence probe.

[0071] Experimental materials: ZGGO@ZGGO: Cr-COOH, BHQ-3-NH2, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[0072] Experimental reagents: phosphate buffered saline (PBS).

[0073] Experimental equipment: constant temperature oscillator, centrifuge, fluorescence meter (can measure fluorescence lifetime)

[0074] Experimental conditions: 37℃

[0075] Experimental steps: First, ZGGO@ZGGO:Cr-COOH was added with a certain amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) and stirred at 37°C for 30 minutes. The solution was quickly centrifuged and dispersed into a certain amount of PBS.

[0076] A certain amount of BHQ-3-NH2 was added to the above solution (1 / 3 / 5 / 7 nmol BHQ-3-NH2 for 1 mg of nanoparticles), and the reaction was carried out outdoors for 4 to 8 h. The product was centrifuged and washed with deionized water to obtain ZGGO@ZGGO:Cr-BHQ-3 connected with different concentrations of BHQ-3.

[0077] The reaction solution was washed with PBS and dispersed into a 1 mg / mL solution. A fluorescence instrument was used to perform a lifespan test and the lifespan was calculated.

[0078] At the same time, the unmodified ZGGO@ZGGO:Cr was used as the control group and the fluorescence lifetime was also tested. The results are as follows Figure 6 shown.

[0079] The luminescence effect of zinc gallium germanate material is enhanced by preparing different core-shell thicknesses, and then the detection of azoreductase is realized by connecting a quencher. The long afterglow nanomaterial with nanometer size, low cost, low toxicity and mass production is synthesized by hydrothermal method, and the long afterglow nanomaterial prepared by the present invention has a longer electron lifetime. The nanomaterial is further used with azoreductase that cuts azo bonds to monitor azoreductase in bacteria.

[0080] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0081] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A method for preparing a surface confined long afterglow luminescence probe, characterized in that: The following steps are involved: (1) mixing and dissolving zinc nitrate, gallium nitrate and sodium germanate in deionized water, and adjusting the pH of the mixture; (2) stirring the above-mentioned mixed solution and reacting it to obtain a reaction product A; (3) centrifuging and washing the above reaction product to collect the product ZGGO nanoparticles; (4) dissolving the ZGGO nanoparticles, zinc nitrate, sodium germanate, gallium nitrate and chromium nitrate in deionized water, and adjusting the pH of the mixture; (5) stirring the mixed solution and reacting it to obtain a reaction product B; (6) The above reaction product is centrifuged and washed, and the collected product is ZGGO@ZGGO:Cr nanoparticles; (7) dispersing the above ZGGO@ZGGO:Cr nanoparticles in dimethylformamide, adding 3-aminopropyltriethoxysilane dropwise, condensing and refluxing, and washing with dimethylformamide after the reflux to obtain ZGGO@ZGGO:Cr-NH2 nanoparticles; (8) dispersing the above ZGGO@ZGGO:Cr-NH2 nanoparticles in dimethylformamide, adding succinic anhydride and 4-dimethylaminopyridine to stir the reaction, and washing with a mixture of ethanol and water to obtain a reaction product C; (9) adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the above reaction product, stirring the reaction, centrifuging the solution and dispersing it into PBS; (10) BHQ-3-NH2 was added to the above solution, reacted, centrifuged and the product was washed with deionized water to obtain ZGGO@ZGGO:Cr-BHQ-3.

2. The method for preparing the surface confined long afterglow luminescence probe according to claim 1, characterized in that: The pH value of the mixed solution in step (1) and step (4) is 8-9.

3. The method for preparing the surface confined long afterglow luminescence probe according to claim 1, characterized in that: The reaction temperature in step (2) and step (5) is 220° C., and the reaction time is 10 h.

4. The method for preparing the surface confined long afterglow luminescence probe according to claim 1, characterized in that: The condensation reflux temperature is 70-100°C.

5. The method for preparing the surface confined long afterglow luminescence probe according to claim 1, characterized in that: The reaction temperature in step (8) is 37°C.

6. A surface confined long afterglow luminescence probe, characterized in that: Includes ZGGO@ZGGO:Cr nanoparticles with surface modified with BHQ-3 organic molecules.

7. Use of the probe prepared by any one of the preparation methods of claims 1 to 5 or the probe of claim 6 in sensitive detection of enzyme activity.

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