Fluorescence-colorimetric bimodal nanoprobe for amikacin sulfate detection and biosensor

Abstract

The invention discloses a fluorescence-colorimetric bimodal nanoprobe and a biosensor for amikacin sulfate detection, and relates to the technical field of drug safety and environmental hazard detection, the fluorescence-colorimetric bimodal nanoprobe comprises up-conversion nanoparticles and Congo red; the UCNPs is prepared by taking rare earth chloride, oleic acid, 1-octadecene, ammonium fluoride and sodium hydroxide as raw materials through a solvothermal method. According to the invention, a fluorescence method and a colorimetric method are innovatively combined, and the fluorescence-colorimetric bimodal nano probe is designed and is used for real-time analysis and visual detection of AMK; the near-infrared excitation characteristic and the stable chemical property of UCNPs are utilized, background fluorescence interference is reduced, the AMK detection sensitivity, accuracy and anti-interference capacity are remarkably improved, and the method can be applied to AMK detection in actual samples.

Description

Technical Field

[0001] The present invention relates to the technical field of drug safety and environmental hazard detection, and in particular to a fluorescence-colorimetry dual-mode nanoprobe and biosensor for detecting amikacin sulfate. Background Art

[0002] Amikacin sulfate (AMK), as an aminoglycoside broad-spectrum antibiotic, has significant effects in treating a variety of bacterial infections, but its overdose may cause side effects such as nephrotoxicity and tinnitus. In addition, when AMK is excreted from the body, the unmetabolized part may enter the environment through urine and feces, causing pollution and posing potential risks to the environment and human health. Therefore, the development of a rapid and accurate AMK detection method is crucial to assess its impact on the human body and the natural environment.

[0003] Existing AMK detection technologies include spectrophotometry, ion chromatography, Rayleigh scattering and HPLC, which are often limited in practical applications due to high material consumption, cumbersome operation, high cost and long time consumption. Fluorescent probes have shown great potential in life sciences, environmental sciences and other fields due to their advantages such as high sensitivity, high accuracy and low cost. The dual-mode detection system combines fluorescence and colorimetry to provide more reliable AMK quantitative analysis through dual signals. However, most fluorescent probes use short-wavelength excitation, which is easy to produce background fluorescence and reduce accuracy. Therefore, the development of fluorescence-colorimetric dual-mode probes with low background fluorescence interference is of great significance to improve the accuracy of AMK detection.

[0004] Lanthanide-doped upconversion nanoparticles (UCNPs) show great potential in the field of sensing due to their unique photophysical properties. UCNPs can convert low-energy near-infrared light into high-energy ultraviolet / visible light, which not only enhances the penetration depth of light in biological tissues, but also significantly reduces the interference of autofluorescence. The high chemical stability, low toxicity and high photostability of UCNPs make them have broad application prospects in the field of environmental and drug detection, including drug safety and environmental monitoring. At present, there are no reports on the detection of AMK based on ratiometric fluorescence and colorimetry of UCNPs. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a fluorescence-colorimetric dual-modal nanoprobe based on UCNPs, which can be used for amikacin sulfate detection and has the advantages of high accuracy, high anti-interference, high sensitivity and visual detection. Specifically, the present invention has developed a ratiometric fluorescent probe, which uses red fluorescence as a reference signal to improve the accuracy of detection. Under 980 nm excitation, UCNPs mainly show two main emission peaks (540 nm and 655 nm). After the introduction of Congo red (CR), it produces an absorption peak at 540 nm, which overlaps with the green emission peak of up-conversion luminescence, resulting in quenching of green emission, while red emission is not affected. Therefore, the fluorescence-colorimetric dual-modal nanoprobe described in the present invention has the advantages of visualization and high sensitivity, and can achieve more accurate AMK detection.

[0006] The technical problem to be solved by the present invention is achieved by adopting the following technical solutions: One of the objects of the present invention is to provide a fluorescence-colorimetry dual-modality nanoprobe, comprising UCNPs and CR.

[0007] A second object of the present invention is to provide a biosensor comprising the fluorescence-colorimetric dual-modal nanoprobe.

[0008] The third object of the present invention is to provide the use of the fluorescence-colorimetry dual-modal nanoprobe or the biosensor in the detection of amikacin sulfate.

[0009] The present invention firstly prepares hydrophobic UCNPs by a solvothermal method, then prepares hydrophilic UCNPs by an acid treatment method, and then combines the UCNPs with CR to prepare a fluorescence-colorimetric dual-modal nanosensor, and utilizes the sensor to realize visual detection of AMK.

[0010] The present invention uses a 980 nm laser to excite UCNPs, generating emission peaks at 540 nm and 655 nm. When CR is combined with UCNPs, the absorption peak of CR overlaps with the 540 nm green emission peak of UCNPs, resulting in quenching of the green emission, while the red emission of 655 nm remains unchanged. As the concentration of AMK increases, the absorption peak of CR decreases, causing the green color of the solution to gradually deepen, thereby achieving colorimetric and visual detection of AMK. At the same time, by monitoring the change in the ratio of green to red emission intensities, fluorescent modal detection of AMK is achieved. Therefore, the dual-modal nanoprobe of the present invention combines the intuitiveness of colorimetric detection with the high sensitivity of fluorescent detection, providing a new method for the detection of AMK.

[0011] The beneficial effects of the present invention are: 1. The present invention applies UCNPs to the detection of AMK, and uses upconversion fluorescence excited by near-infrared light as the detection signal of AMK, which effectively reduces the background fluorescence, thereby significantly improving the sensitivity and stability of AMK detection.

[0012] 2. The present invention innovatively combines fluorescence and colorimetry to design a fluorescence-colorimetry dual-modal nanoprobe for real-time analysis and visual detection of AMK, thereby improving the sensitivity, accuracy and anti-interference ability of AMK detection and can be used for the detection of AMK in actual samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a schematic diagram of the principle of using the nanoprobe of the present invention for AMK detection; Figure 2 The up-conversion luminescence spectrum of the nanoprobe of the present invention used for AMK detection; Figure 3 (A) is a low-resolution transmission electron microscopy image of NaYF4:Yb,Er upconversion nanoparticles prepared in Example 1; Figure 3 (B) Figure 3 (C) Figure 3 (D) are low-resolution transmission electron microscopy images, high-resolution transmission electron microscopy images, and X-ray diffraction patterns of the NaYF4:Yb,Er@NaYF4 upconversion nanoparticles prepared in Example 1, respectively; Figure 4 The responsiveness of the nanoprobe prepared in Example 1 to different concentrations of AMK; (A) The effect of adding different concentrations of AMK (0-35 μM) on the upconversion luminescence spectrum of the nanoprobe; (B) I 540 / I 655 Linear fitting curve of CR absorbance and AMK concentration; (C) Effect of adding different concentrations of AMK (0~35 µM) on the absorption spectrum of the nanoprobe; (D) Linear fitting curve of CR absorbance and AMK concentration; Figure 5 The selectivity and anti-interference test results of the nanoprobe prepared in Example 1 in the presence of relevant interferents; (A) selectivity and anti-interference test results in fluorescence mode; (B) selectivity and anti-interference test results in colorimetric mode; where 1-blank; 2-AMK; 3-Ca 2+ ; 4-Mg 2+ ; 5.Na + 6.CO3 2- ; 7-PO4 2- ; 8-SO4 2- ; 9-glucose; 10-ascorbic acid; 11-citric acid; 12-glutathione. DETAILED DESCRIPTION

[0014] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments and diagrams.

[0015] The invention provides a fluorescence-colorimetry dual-mode nanoprobe, comprising UCNPs and CR.

[0016] Furthermore, the concentration of UCNPs in the nanoprobe is 0.1-2 mg / mL, and the concentration of CR is 9.5-10.5 mmol / L.

[0017] Furthermore, the nanoprobe further comprises an acetate buffer solution. Preferably, the pH value of the acetate buffer solution is 4-5.

[0018] Furthermore, the UCNPs are prepared by a solvothermal method using rare earth chloride, oleic acid (OA), 1-octadecene (ODE), ammonium fluoride (NH4F) and sodium hydroxide (NaOH) as raw materials. Specifically, the rare earth chloride includes but is not limited to yttrium, ytterbium and erbium chlorides.

[0019] Furthermore, the reaction temperature of the solvothermal method is 100-300°C, and the reaction time is 30-60 min. Rare earth chloride is used as a precursor, OA is used as a ligand, OA and ODE are used as a mixed solvent, and react with NH4F and NaOH under high temperature conditions.

[0020] As a specific embodiment, the preparation method of UCNPs comprises the following steps: (1) NaYF4:Yb,Er upconversion nanoparticles were prepared by reacting yttrium chloride hexahydrate (YCl3·6H2O), ytterbium chloride hexahydrate (YbCl3·6H2O), erbium chloride hexahydrate (ErCl3·6H2O), OA, ODE, NH4F and NaOH; (2) The core-shell structured NaYF4:Yb,Er@NaYF4 upconversion nanoparticles were prepared by reacting NaYF4:Yb,Er upconversion nanoparticles, YCl3·6H2O, OA, ODE, NH4F and NaOH.

[0021] Preferably, the preparation process of the UCNPs is in a protective atmosphere, such as argon.

[0022] Preferably, the UCNPs are treated with acid to remove surface OA to obtain hydrophilic UCNPs. As a specific embodiment, the acid treatment uses dilute hydrochloric acid with a pH of 0.8 to 1.2.

[0023] The present invention provides a biosensor, comprising the fluorescence-colorimetry dual-modality nanoprobe.

[0024] The present invention also provides application of the fluorescence-colorimetry dual-mode nanoprobe or the biosensor in the detection of amikacin sulfate.

[0025] Specifically, the detection of amikacin sulfate is carried out by fluorescence detection at an excitation wavelength of 980 nm and an emission wavelength of 500-700 nm; and colorimetric analysis is carried out at an absorption wavelength of 400-700 nm.

[0026] Example 1 (1) Preparation of NaYF4:Yb,Er upconversion nanoparticles Weigh 3.9 mmol YCl3·6H2O, 1 mmol YbCl3·6H2O, 0.1 mmol ErCl3·6H2O and mix with 30 mLOA and 75 mL ODE, heat to 160℃ and stir for 1 h, cool to room temperature, add 50 mL methanol solution containing 0.74 g NH4F and 0.5 g NaOH, heat to 40℃ and stir for 50 min, then heat to 100℃ in vacuum and keep warm for 20 min, then heat to 300℃ in argon atmosphere and keep warm for 1 h, cool to room temperature, take 5 mL of the reaction mixture, wash it three times with ethanol and cyclohexane to obtain NaYF4:Yb,Er upconversion nanoparticles for later use.

[0027] (2) Preparation of core-shell NaYF4:Yb,Er@NaYF4 upconversion nanoparticles 1 mmol YCl3·6H2O was mixed with 6 mL OA and 15 mL ODE, and heated to 160°C in an argon-filled environment with stirring until the solution became transparent, and then cooled to room temperature; the NaYF4:Yb,Er upconversion nanoparticles prepared in step (1) were added under stirring, and 15 mL of a methanol solution containing 0.148 g NH4F and 0.1 g NaOH was added dropwise, and the mixture was heated to 50°C and stirred for 40 min, and then heated to 100°C in a vacuum and kept warm for 20 min, and then heated to 300°C in an argon atmosphere and kept warm for 1 h, and then cooled to room temperature, and the product was precipitated with ethanol, and the product was washed with ethanol three times to obtain NaYF4:Yb,Er@NaYF4 upconversion nanoparticles for later use.

[0028] (3) Acid treatment of NaYF4:Yb,Er@NaYF4 upconversion nanoparticles 200 mg of NaYF4:Yb,Er@NaYF4 upconversion nanoparticles prepared in step (2) were dispersed in 60 mL of dilute hydrochloric acid (pH = 1), ultrasonically treated for 1 h to accelerate the protonation and dissociation of the ligand OA, centrifuged, and washed twice with ultrapure water; the acid-treated UCNPs were redispersed in 20 mL of ultrapure water to obtain a hydrophilic UCNPs solution for later use.

[0029] (4) Preparation of nanoprobes: 200 μL of the hydrophilic UCNPs solution prepared in step (3) was mixed evenly with 100 μL of 10 mMCR solution in a test tube, and the final volume of the solution was fixed to 2 mL with acetate buffer (pH = 4.5) to obtain nanoprobes.

[0030] from Figure 3 It can be observed that the UCNPs synthesized in this example exhibit a spherical morphology with uniform particle size distribution; after being encapsulated by the NaYF4 shell, the average diameter of the UCNPs increased from 26.03 nm ( Figure 3 A) increases to 28.06 nm ( Figure 3 B); NaYF4:Yb,Er@NaYF4 upconversion nanoparticles have uniformly distributed lattice fringes ( Figure 3 C); The XRD characteristic peaks of NaYF4:Yb,Er upconversion nanoparticles and NaYF4:Yb,Er@NaYF4 upconversion nanoparticles match well with the standard PDF card (powder diffraction file, JCPDS16-0334) ( Figure 3 D), this hexagonal crystal structure facilitates energy transfer between rare earth ions, thereby significantly improving the efficiency of upconversion luminescence (UCL).

[0031] Example 2 The preparation method of the nanoprobe is the same as that of Example 1, except that the solvothermal reaction conditions for preparing NaYF4:Yb,Er upconversion nanoparticles are first heated to 100°C in vacuum for 30 min and then heated to 250°C in argon atmosphere for 1 h.

[0032] Example 3 The preparation method of the nanoprobe is the same as that of Example 1, except that the solvothermal reaction conditions for preparing NaYF4:Yb,Er@NaYF4 upconversion nanoparticles are first heated to 150°C in vacuum for 20 min and then heated to 300°C in argon atmosphere for 1 h.

[0033] Example 4 The preparation method of the nanoprobe is the same as that of Example 1, except that dilute hydrochloric acid with a pH of 0.8 is used in the acid treatment of the NaYF4:Yb,Er@NaYF4 upconversion nanoparticles.

[0034] Example 5 The preparation method of the nanoprobe is the same as that of Example 1, except that an acetate buffer solution with a pH of 4 is used in the preparation of the nanoprobe.

[0035] Example 6 AMK aqueous solution was added to the nanoprobe prepared in Example 1 to make the final concentration of AMK 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 30, 35 μM, respectively. The mixture was evenly mixed and incubated for 10 min. The upconversion fluorescence spectrum of the solution in the range of 500-700 nm was recorded with a 980 nm laser to complete the fluorescence detection of AMK ( Figure 4 A and 4B); at the same time, the absorbance of the solution was measured in the range of 400~700nm to achieve colorimetric detection of AMK ( Figure 4 C and 4D).

[0036] from Figure 4 As can be seen in A, the green UCL intensity at 540 nm increases when the AMK concentration increases from 0 μM to 35 μM. At the same time, the red UCL at 655 nm remains unchanged, and the inset shows the color change intuitively. 540 / I 655 As a detection signal, the signal showed a good linear relationship with the AMK concentration in the AMK concentration range of 0~35 μM ( Figure 3 B). The limit of detection (LOD) was calculated according to the formula LOD = 3*δ / S, where δ is the standard deviation based on ten blank experiments and S is the slope of the linear regression equation. According to this formula, the LOD in fluorescence mode is 0.35 nM. In addition, as the AMK concentration increases from 0 μM to 35 μM, the absorbance of the probe gradually decreases ( Figure 3 C). The inset shows the color transition of the probe solution from dark red to light red, providing an intuitive visual detection of AMK. Further analysis showed that the absorbance at 497 nm had a good linear relationship with the AMK concentration, with an LOD of 0.86 nM and a correlation coefficient R² of 0.998 ( Figure 3 D). Therefore, the nanoprobe designed in the present invention exhibits high sensitivity for the detection of AMK in both fluorescence and colorimetric modes.

[0037] Example 7 Take 200 μL of lake water sample and add it to 1.8 mL of the nanoprobe prepared in Example 1. After mixing evenly, measure the I 540 / I 655 and absorbance, into Figure 4 B and Figure 4The AMK concentration was calculated by the linear equation in D. Subsequently, 10 μL of AMK aqueous solution with different concentrations was added to 2 mL of the nanoprobe prepared in Example 1 for spike recovery measurement. The concentration of AMK in the water sample was calculated according to the calibration curve. All corresponding detection processes were performed independently, and the results are shown in Table 1.

[0038] Recovery rate = (AB) / C × 100% Among them, A is the concentration measured after adding standard AMK to the sample, B is the concentration of AMK in the sample, and C is the spiked concentration.

[0039] Table 1

[0040] As can be seen from Table 1, when the spiked concentrations were 0, 10, and 20 µM, the recoveries of AMK in lake water samples were 96.8–106.8%, and the standard deviations (RSDs) were 1.5–3.7%, which were relatively low.

[0041] The detection performance of the nanoprobe prepared in Example 1 was compared with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) technology to verify the accuracy of the nanoprobe in measuring AMK.

[0042] Table 2

[0043] As can be seen from Table 2, the data obtained by the two detection methods are highly consistent, which provides strong evidence for the high sensitivity and accurate detection performance of the nanoprobe described in the present invention in complex samples and demonstrates its stability and sensitivity under variable environmental conditions.

[0044] Example 8 Selectivity and anti-interference test: 35 μM AMK aqueous solution and 500 μM interfering substances (interfering substances are Ca 2+ Mg 2+ 、Na + 、CO3 2- PO4 2- 、SO4 2- , glucose, ascorbic acid, citric acid, glutathione aqueous solution) was added to the nanoprobe prepared in Example 1, mixed evenly, and incubated for 10 min, and then the up-conversion fluorescence spectrum of the solution in the range of 500-700 nm was recorded with a 980 nm laser to complete the fluorescence detection of AMK ( Figure 5 At the same time, the absorbance of the solution was measured in the range of 400-700 nm to achieve colorimetric detection of AMK ( Figure 5 red bars in B).

[0045] After adding the above-mentioned interfering substances to the nanoprobe prepared in Example 1, 35 μM AMK aqueous solution was further added and mixed evenly. After incubation for 10 min, the upconversion fluorescence spectrum of the solution in the range of 500-700 nm was recorded using a 980 nm laser to complete the fluorescence detection of AMK ( Figure 5 The blue bar graph in A); at the same time, the absorbance of the solution was measured in the range of 400-700 nm to achieve colorimetric detection of AMK ( Figure 5 blue bars in B).

[0046] from Figure 5 It can be seen that in the fluorescence mode, the addition of interfering substances did not affect the fluorescence intensity of UCNPs, while the addition of AMK significantly changed the I 540 / I 655 Emission ratio ( Figure 5 A). In the colorimetric mode, the addition of the same concentration of AMK resulted in a significant decrease in absorbance, demonstrating the selectivity of the probe ( Figure 5 B). Therefore, the nanoprobe designed in the present invention shows good selectivity and anti-interference ability for AMK detection, and is suitable for quantitative analysis of AMK in complex samples.

[0047] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. 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, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A fluorescence-colorimetric dual-modal nanoprobe, characterized in that: The fluorescence-colorimetric dual-modality nanoprobe includes UCNPs and CR.

2. The fluorescence-colorimetric dual-modality nanoprobe according to claim 1, characterized in that: The concentration of UCNPs in the nanoprobe is 0.1-2 mg / mL, and the concentration of CR is 9.5-10.5 mmol / L.

3. The fluorescence-colorimetry dual-modality nanoprobe according to claim 1, characterized in that: The nanoprobe also includes an acetate buffer solution; the pH value of the acetate buffer solution is 4-5.

4. The fluorescence-colorimetry dual-modality nanoprobe according to claim 1, characterized in that: The UCNPs are prepared by a solvothermal method using rare earth chloride, oleic acid, 1-octadecene, ammonium fluoride and sodium hydroxide as raw materials; the rare earth chloride includes chlorides of yttrium, ytterbium and erbium; the reaction temperature of the solvothermal method is 100-300°C and the reaction time is 30-60 min.

5. The fluorescence-colorimetry dual-modality nanoprobe according to claim 4, characterized in that: The preparation method of the UCNPs comprises the following steps: (1) NaYF4:Yb,Er upconversion nanoparticles were prepared by reaction of YCl3·6H2O, YbCl3·6H2O, ErCl3·6H2O, OA, ODE, NH4F and NaOH; (2) The core-shell structured NaYF4:Yb,Er@NaYF4 upconversion nanoparticles were prepared by reacting NaYF4:Yb,Er upconversion nanoparticles, YCl3·6H2O, OA, ODE, NH4F and NaOH.

6. The fluorescence-colorimetry dual-modality nanoprobe according to claim 5, characterized in that: The preparation process of the UCNPs is carried out in a protective atmosphere.

7. The fluorescence-colorimetry dual-modality nanoprobe according to claim 5, characterized in that: The UCNPs are treated with acid to remove surface OA, thereby obtaining hydrophilic UCNPs; the acid treatment uses dilute hydrochloric acid with a pH of 0.8 to 1.

2.

8. A biosensor comprising the fluorescence-colorimetric dual-modal nanoprobe according to any one of claims 1 to 7.

9. Use of the fluorescent-colorimetric dual-modal nanoprobe according to any one of claims 1 to 7 or the biosensor according to claim 8 in the detection of amikacin sulfate.

10. The use according to claim 9, characterized in that: The amikacin sulfate detection is performed by fluorescence detection at an excitation wavelength of 980 nm and an emission wavelength of 500-700 nm; and colorimetric analysis is performed at an absorption wavelength of 400-700 nm.

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