Fluorescence-colorimetric bimodal nanoprobe and biosensor for CAT detection

Abstract

The invention discloses a fluorescence-colorimetric bimodal nanoprobe and a biosensor for CAT detection, and relates to the technical field of nano biological materials, the fluorescence-colorimetric bimodal nanoprobe comprises UCNPs, hydrogen peroxide, ferrous ions and neutral red; the preparation method comprises the following steps: firstly, preparing UCNPs through a specific method, and then constructing the fluorescent-colorimetric bimodal nanoprobe by utilizing interaction of hydrogen peroxide, ferrous ions, neutral red and the UCNPs; in a fluorescence mode, the activity of CAT is reflected by measuring the quenching degree of an up-conversion fluorescence signal; in the colorimetric mode, the CAT activity is detected by observing the color fading degree of neutral red; the bimodal detection method not only improves the accuracy and sensitivity of detection, but also realizes visual rapid detection of CAT activity.

Description

Technical Field

[0001] The invention relates to the technical field of nano-biomaterials, and in particular to a fluorescence-colorimetry dual-mode nano-probe and a biosensor for CAT detection. Background Art

[0002] Catalase (CAT) is a terminal oxidase widely distributed in animals, plants and microorganisms in nature. CAT decomposes hydrogen peroxide into water and oxygen through enzymatic reactions, effectively inhibiting the conversion of anion free radicals into hydroxyl free radicals in organisms, and then participates in various biological processes such as cell metabolism and signal transduction. It also plays a key role in the biological defense system and protects cells from oxidative stress damage. However, abnormal increases in CAT concentrations are also closely related to a series of diseases, including diabetes, cancer, cardiovascular disease, Alzheimer's disease, and foodborne diseases caused by bacterial contamination, posing a threat to the health of organisms. Therefore, there is an urgent need for a method that can accurately and sensitively detect CAT content.

[0003] At present, the methods for determining CAT include chemiluminescence, fluorescence analysis, spectrophotometry, and electrochemical methods, but these methods have problems such as long measurement cycle, complex operation, and insufficient sensitivity. Fluorescence colorimetric signal output analysis has shown significant advantages in CAT detection. This method combines the high sensitivity, high signal-to-noise ratio, and rapid response characteristics of fluorescence with the intuitiveness and convenience of colorimetry, thereby achieving accurate, rapid, and intuitive detection of CAT. During the day, the tester can preliminarily judge the presence of CAT by the visible color change of the solution; at night or in a dimly lit environment, the activity of CAT in the sample to be tested can be further accurately confirmed by the change in fluorescence intensity with the auxiliary irradiation of ultraviolet light. This dual-modal detection method not only improves the accuracy of detection, but also greatly expands its application scenarios, making environmental detection more efficient and convenient. However, traditional fluorescence analysis usually uses short-wavelength light to excite fluorescent substances, which often produces strong background fluorescence, interfering with the detection of target signals, thereby affecting the accuracy and sensitivity of detection.

[0004] As a new type of rare earth luminescent material, upconversion nanoparticles (UCNPs) have the advantages of narrow emission band, low toxicity, good chemical stability and photostability, and show broad application prospects in the fields of bioimaging, drug delivery and release, and detection. In CAT detection, UCNPs have significant advantages. UCNPs can convert long-wavelength near-infrared light into short-wavelength ultraviolet or visible light, avoiding the background fluorescence interference caused by short-wavelength excitation in traditional fluorescence analysis, thereby improving the accuracy and sensitivity of detection. At present, there is no report on the use of fluorescence-colorimetry synergistic signal output based on UCNPs for the detection of CAT activity. 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, and apply the probe to the detection of CAT activity in body fluids, which has the characteristics of high signal-to-noise ratio, good selectivity, high sensitivity, and rapid visualization detection.

[0006] The present invention first prepares UCNPs by a specific method, and then utilizes the interaction between hydrogen peroxide, ferrous ions, neutral red and UCNPs to construct a fluorescence-colorimetric dual-mode nanoprobe. Among them, UCNPs, as a luminophore, can convert long-wavelength near-infrared light into short-wavelength visible light, avoiding the interference of background fluorescence in traditional fluorescence analysis. The Fenton reaction involving hydrogen peroxide and ferrous ions will cause the neutral red to fade, while restoring the up-conversion fluorescence signal, and CAT can decompose hydrogen peroxide, thereby preventing the occurrence of this reaction, so that the neutral red absorbance and the up-conversion fluorescence signal remain unchanged. In the fluorescence mode, the activity of CAT is reflected by measuring the quenching degree of the up-conversion fluorescence signal; in the colorimetric mode, the CAT activity is detected by observing the fading degree of neutral red. This dual-mode detection method not only improves the accuracy and sensitivity of the detection, but also realizes the visual rapid detection of CAT activity.

[0007] The technical problem to be solved by the present invention is achieved by adopting the following technical solutions: The first object of the present invention is to provide a fluorescence-colorimetric dual-modality nanoprobe, which comprises UCNPs, hydrogen peroxide, ferrous ions and neutral red.

[0008] In the present invention, hydrogen peroxide participates in the Fenton reaction, resulting in the fading of neutral red and the recovery of the fluorescence signal of UCNPs, which provides an intuitive signal change for the detection of CAT activity; ferrous ions act as a catalyst for the Fenton reaction, promoting the reaction between hydrogen peroxide and neutral red, thereby realizing sensitive detection of CAT activity. The concentration and existence state of ferrous ions have an important influence on the reaction rate and signal intensity; neutral red, as an azo dye, has characteristic absorption in the green visible light region, overlapping with the green emission light of UCNPs, resulting in quenching of green luminescence. However, under the action of the Fenton reaction, neutral red fades, eliminating the quenching effect on the green emission light, allowing the green luminescence to be restored. This process not only provides a signal change for the detection of CAT activity in the fluorescence mode, but also realizes the visual detection of CAT activity through the colorimetric mode.

[0009] The second object of the present invention is to provide a biosensor comprising the fluorescence-colorimetry dual-modality nanoprobe.

[0010] The third object of the present invention is to provide the use of the fluorescence-colorimetry dual-modality nanoprobe or biosensor in CAT detection.

[0011] like Figure 1 As shown in the figure, under the excitation of 980 nm laser, UCNPs exhibit emission peaks at 540 nm and 655 nm. Neutral red is an azo dye with characteristic absorption in the green visible light region, which overlaps with the green emission light of UCNPs, resulting in quenching of green luminescence, while the luminescence at 655 nm remains unaffected. Therefore, the overall red UCL is presented. In the presence of divalent ferrous ions, hydrogen peroxide undergoes a Fenton reaction with it, causing neutral red to fade, thereby eliminating the quenching of green emission light and allowing green luminescence to recover. If CAT is introduced into the Fenton reaction system, hydrogen peroxide will be decomposed, preventing the occurrence of the Fenton reaction and maintaining the quenched state of green luminescence. Therefore, a luminescence modality can be constructed to detect CAT activity by up-converting the intensity of green emission. At the same time, the fading reaction of the neutral red dye is conducive to detecting CAT activity through a colorimetric modality, thereby forming a luminescence-colorimetric dual signal output response to CAT activity. This dual response not only improves the sensitivity and accuracy of the detection, but also makes the detection process more intuitive and convenient.

[0012] The present invention uses red fluorescence as a reference signal to prepare a ratiometric fluorescent probe, and the detection result is more reliable. Figure 2 A explains the luminescence mechanism of UCNPs described in the present invention. The upconversion luminescence process originates from Yb 3+ and Er 3+ The energy transfer upconversion process between ions. The 540 nm and 655 nm emission peaks shown in the spectrum correspond to their excited states ( 4 S 3 / 2 and 4 F 9 / 2 energy level) to the ground state ( 4 I 15 / 2 energy transition of energy levels. Figure 2B shows the rationality of the nanoprobe described in the present invention for CAT activity detection from a spectroscopy perspective. Neutral red has characteristic absorption at 525 nm, while the upconversion green emission is located at 540 nm. Therefore, the absorption spectrum of neutral red and the upconversion green emission spectrum have good overlap, which easily leads to upconversion emission quenching. Since neutral red is not specially modified on the surface of UCNPs, this quenching mechanism should be attributed to the inner filter effect. Under the Fenton reaction, neutral red fades, while green upconversion emission is not affected. After adding CAT, hydrogen peroxide is decomposed, causing neutral red to maintain the quenching effect on green upconversion. In this process, the red upconversion emission peak at 655 nm remains unchanged. Accordingly, the green UCL is selected as the luminescent signal for detection, while the red emission serves as a reference signal, together forming a ratio probe. Therefore, the ratio optical probe prepared by the present invention has the advantages of self-calibration and anti-interference, and can achieve more accurate and reliable CAT activity detection.

[0013] The beneficial effects of the present invention are: 1. The present invention applies UCNPs to the detection of CAT. With its unique upconversion luminescence property, i.e., the ability to convert long-wavelength near-infrared light into short-wavelength visible light, it effectively avoids the interference of biological background fluorescence and significantly improves the sensitivity and accuracy of CAT detection. This application not only expands the scope of use of UCNPs in the field of biological detection, but also provides a new technical path for achieving efficient and specific CAT detection.

[0014] 2. The present invention creatively designs a dual-modal detection probe that combines fluorescence and colorimetry. The probe realizes visualization and quantitative detection of CAT concentration through Fenton reaction and enzymatic hydrolysis of CAT. In fluorescence mode, the detection limit is as low as 0.051 mU / mL, and in colorimetric mode, its detection limit reaches 1.32 mU / mL, showing extremely high detection sensitivity. In addition, the use of dual-modal detection methods further enhances the reliability and anti-interference ability of detection, opening up new possibilities for on-site rapid visualization detection of biomarkers. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a schematic diagram of the principle of using the nanoprobe of the present invention for CAT activity detection; Figure 2 (A) is a schematic diagram of energy transfer upconversion between green and red emissions in UCNPs of the present invention; Figure 2 (B) UV-visible absorption spectra of the probe solution containing CAT (red solid line) and without CAT (red dashed line), and upconversion luminescence spectra of the probe solution containing CAT (blue dashed line) and without CAT (red solid line); Figure 3(A) is a low-resolution transmission electron microscopy image of NaYF4:Yb,Er core nanoparticles prepared in Example 1; Figure 3 (B) is a low-resolution transmission electron microscopy image of NaYF4:Yb,Er@NaYF4 upconversion nanoparticles prepared in Example 1; Figure 3 (C) is a high-resolution transmission electron microscopy image of NaYF4:Yb,Er@NaYF4 upconversion nanoparticles prepared in Example 1; Figure 3 (D) is the X-ray diffraction pattern of NaYF4:Yb,Er@NaYF4 upconversion nanoparticles prepared in Example 1; Figure 4 The responsiveness of the nanoprobe prepared in Example 1 to different active CATs; (A) Relationship between the upconversion luminescence spectrum change trend and CAT concentration; (B) I 540 / I 655 Ratio-CAT concentration linear fitting analysis; (C) Relationship between UV-visible absorption spectrum evolution and CAT concentration; (D) Absorbance (525 nm)-CAT concentration linear fitting analysis; Figure 5 The selectivity and anti-interference test results of the nanoprobe prepared in Example 1 in the presence of 500 µM concentration of relevant interferents; (A) selectivity and anti-interference test results in fluorescence mode; (B) selectivity and anti-interference test results in colorimetric mode; wherein, 1-blank, 2-CAT, 3-urease, 4-acetylcholinesterase, 5-cellulase, 6-lysozyme, 7-insulin, 8-L-lysine, 9-L-aspartic acid, 10-uric acid. DETAILED DESCRIPTION

[0016] 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.

[0017] The invention provides a fluorescence-colorimetry dual-mode nanoprobe, which comprises UCNPs, hydrogen peroxide, ferrous ions and neutral red.

[0018] Furthermore, the concentration of UCNPs in the nanoprobe is 0.1~1 mg.mL -1 The concentration of hydrogen peroxide is 0.5~2 mmol L -1 The concentration of ferrous ions is 1-3 mmol L -1 The concentration of neutral red is 0.1~0.3 mmol L -1 .

[0019] The present invention finely regulates the concentrations of UCNPs, hydrogen peroxide, ferrous ions and neutral red, with the aim of ensuring that the prepared nanoprobe has high sensitivity and accuracy in both fluorescence and colorimetric modes.

[0020] Furthermore, the UCNPs are prepared by a solvothermal method using rare earth chloride, oleic acid, 1-octadecene, ammonium fluoride and sodium hydroxide as raw materials.

[0021] Furthermore, the rare earth chlorides include but are not limited to chlorides of yttrium, ytterbium and erbium.

[0022] Furthermore, the reaction temperature of the solvothermal method is 280-320°C, and the reaction time is 30-90 min. Rare earth chloride is used as a precursor, oleic acid (OA) is used as a ligand, OA and 1-octadecene (ODE) are used as a mixed solvent, and react with ammonium fluoride (NH4F) and sodium hydroxide (NaOH) under high temperature conditions.

[0023] As a specific embodiment, the preparation method of UCNPs comprises the following steps: (1) NaYF4:Yb,Er core 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) NaYF4:Yb,Er@NaYF4 upconversion nanoparticles were prepared by reacting NaYF4:Yb,Er core nanoparticles, YCl3·6H2O, OA, ODE, NH4F and NaOH; (3) The NaYF4:Yb,Er@NaYF4 upconversion nanoparticles were treated with acid to remove the surface oleic acid and obtain hydrophilic UCNPs.

[0024] Furthermore, the acid treatment uses dilute hydrochloric acid with a pH of 0.1-2.

[0025] The present invention provides a biosensor, comprising the fluorescence-colorimetry dual-modality nanoprobe.

[0026] The present invention provides application of the fluorescence-colorimetry dual-mode nanoprobe or biosensor in CAT detection.

[0027] The fluorescence-colorimetric dual-mode nanoprobe performs fluorescence detection within the range of an excitation wavelength of 980 nm and an emission wavelength of 500-700 nm; and performs colorimetric analysis within the range of an absorption wavelength of 400-700 nm.

[0028] Preparation Example 1 (1) Preparation of NaYF4:Yb,Er core nanoparticles YCl3·6H2O, YbCl3·6H2O, ErCl3·6H2O (the molar amounts of the substances used are 3.9 mmol, 1 mmol, and 0.1 mmol, respectively) are mixed with 30 mL OA and 75 mL ODE, heated to 160°C and stirred for 1 h, and cooled to room temperature; then 50 mL of a methanol solution containing 0.74 g NH4F and 0.5 g NaOH is added dropwise, heated to 50°C and stirred for 40 min, 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, cooled to room temperature, 5 mL of the reaction mixture is taken, and washed three times with ethanol and cyclohexane to obtain NaYF4:Yb,Er core nanoparticles for later use.

[0029] (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, heated to 160°C and stirred until the solution became transparent, and then cooled to room temperature; the prepared NaYF4:Yb,Er core nanoparticles were added under stirring, and 15 mL of a methanol solution containing 0.148 g NH4F and 0.1 g NaOH was added dropwise, heated to 50°C and stirred for 40 min, 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, cooled to room temperature, precipitated the product with ethanol, and washed the product with ethanol three times to obtain NaYF4:Yb,Er@NaYF4 upconversion nanoparticles for later use.

[0030] (3) Acid treatment of NaYF4:Yb,Er@NaYF4 upconversion nanoparticles 200 mg of the NaYF4:Yb,Er@NaYF4 upconversion nanoparticles prepared above 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, washed twice with ultrapure water, and the acid-treated UCNPs were redispersed in 20 mL of ultrapure water to obtain a hydrophilic UCNPs solution for later use.

[0031] from Figure 3It can be seen that the UCNPs synthesized above are spherical nanoparticles with uniform diameters, where the average diameter of the core of the UCNPs is 25.98 nm. After a layer of NaYF4 inert shell is coated on the core, the average diameter of the UCNPs increases to 27.86 nm. High-resolution transmission electron microscopy imaging shows that the UCNPs have uniformly distributed and clearly visible lattice fringes with a crystal plane spacing of 0.51 nm, corresponding to the (110) crystal plane of hexagonal NaYF4, which confirms the successful preparation of hexagonal UCNPs. The crystal structure of the core-shell UCNPs was analyzed by X-ray powder diffraction, further confirming the formation of a standard hexagonal structure. This hexagonal phase crystal structure promotes efficient energy transfer between rare earth ions, thereby enhancing the upconversion luminescence (UCL) performance.

[0032] Preparation Example 2 The preparation method of the nanoprobe is the same as that of Example 1, except that when preparing NaYF4:Yb,Er core nanoparticles, the preparation raw materials used are 3.85 mmol, 1 mmol, 0.15 mmol of YCl3·6H2O, YbCl3·6H2O, ErCl3·6H2O with molar amounts of 30 mLOA and 75 mL ODE, respectively.

[0033] Preparation Example 3 The preparation method of the nanoprobe is the same as that of Example 1, except that when preparing NaYF4:Yb,Er core nanoparticles, the solvothermal reaction is heated to 280°C in an argon atmosphere and kept warm for 90 min.

[0034] Preparation Example 4 The preparation method of the nanoprobe is the same as that of Example 1, except that when preparing NaYF4:Yb,Er core nanoparticles, the solvothermal reaction is heated to 320°C in an argon atmosphere and kept warm for 30 min.

[0035] Preparation Example 5 The preparation method of the nanoprobe is the same as that of Example 1, except that 100 mL of dilute hydrochloric acid (pH=0.5) is used to ultrasonically treat the NaYF4:Yb,Er@NaYF4 upconversion nanoparticles for 1 h to remove the oleic acid on the surface.

[0036] Application Example 1 Fluorescence detection of CAT: 300 μL of hydrogen peroxide solution (10 mM) and 100 μL of CAT solution of different concentrations (different masses of CAT powder (200000 U / g) were dispersed in 10 mM HEPES solution to obtain CAT solutions of different concentrations) were added to a 5 mL centrifuge tube, and the solution was calibrated to 1 mL with HEPES buffer (10 mM). After the enzyme catalyzed reaction was carried out at 25°C for 12 min, an acidic ferrous chloride solution (6 mM, 1 mL, pH=1) was immediately added to terminate the enzymatic reaction, and then a UCNPs-neutral red composite solution (150 μL of the hydrophilic UCNPs solution prepared in Example 1 and 100 μL of neutral red solution (4.05 mM)) was added, and the solution was fixed to 3 mL of probe solution with a pH of 3, and the solution was shaken and then subjected to a fading reaction for 30 min. Under 980 nm laser excitation, the up-conversion fluorescence spectrum of the probe solution in the range of 500-700 nm was recorded, and the luminescence mode was constructed by the intensity change of the green emission light to detect the CAT activity.

[0037] Colorimetric detection of CAT: 300 μL of hydrogen peroxide solution (10 mM) and 100 μL of CAT solution of different concentrations (different masses of CAT powder (200000 U / g) were dispersed in 10 mM HEPES solution to obtain CAT solutions of different concentrations) were added to a 5 mL centrifuge tube, and the solution was calibrated to 1 mL with HEPES buffer (10 mM). After the enzyme catalytic reaction was carried out at 25°C for 12 min, an acidic ferrous chloride solution (6 mM, 1 mL, pH=1) was immediately added to terminate the enzymatic reaction, and then a UCNPs-neutral red composite solution (150 μL of the hydrophilic UCNPs solution prepared in Example 1 and 100 μL of neutral red solution (4.05 mM)) was added, and the solution was fixed to 3 mL of probe solution with a pH of 3, and the solution was shaken and then subjected to a fading reaction for 30 min. The color change of the probe solution was observed and recorded under visible light, and the absorbance test was performed on it. The colorimetric detection of CAT was achieved by the fading degree of neutral red and the absorbance change.

[0038] from Figure 4It can be seen that when the CAT concentration increases from 0 mU / mL to 160 mU / mL, the quenching efficiency of neutral red (NR) on UCL gradually increases, resulting in a decrease in the green emission intensity, while the red UCL intensity remains unchanged. Therefore, a ratiometric fluorescent probe was constructed using the red emission at 655 nm as the reference signal and the green emission at 540 nm as the detection signal. This ratiometric fluorescent probe can eliminate the interference of environmental factors such as pH changes, fluorescence self-quenching, and background signals, and achieve more accurate, reliable, and anti-interference visual detection. As the green UCL is gradually quenched, the overall UCL color changes from green to red. The results show that within the CAT concentration range of 0 to 160 mU / mL, I 540 / I 655 The UCL signal showed a significant linear relationship ( Figure 3 B). The limit of detection (LOD) was calculated using the formula LOD = 3σ / S, where σ is the standard deviation of the background noise obtained from 10 blank experiments and S is the slope of the linear regression equation obtained from the sensitivity test. According to this formula, the LOD in the luminescence mode was determined to be 0.051 mU / mL. In addition, Figure 3 C shows the colorimetric response of the probe solution to CAT. As the concentration of CAT gradually increases, the absorbance of NR at 525 nm also increases accordingly. The picture in the inset clearly shows the transition of the probe solution from light red to dark red, indicating that the presence of CAT can be visually judged by the naked eye. Figure 3 As shown in Figure D, within the concentration range of 0 to 160 mU / mL, there is a significant linear relationship between the absorbance at 525 nm and the CAT concentration, with a LOD of 1.32 mU / mL (R²=0.998). Therefore, the nanoprobe described in the present invention exhibits high sensitivity to CAT detection in both luminescence and colorimetric modes.

[0039] Application Example 2 Selectivity and anti-interference test of nanoprobes: Solutions containing potential interfering substances (including urease, acetylcholinesterase, cellulase, lysozyme, insulin, L-lysine, L-aspartic acid, and uric acid) were added to the probe solution prepared in Example 1 and mixed evenly. The fluorescence spectrum was recorded under 980 nm laser excitation, and the color change was observed under visible light to evaluate the selectivity and anti-interference ability of the probe.

[0040] from Figure 5 It can be seen that after adding 500 μM interfering substances, the UCL and absorption spectrum of the probe remain unchanged. When 160 mU / mL CAT solution is added to the probe, I 540 / I 655The intensity ratio of the probe solution changed significantly, and the UCL changed from green to red. Further, CAT was added to the solution with interfering substances to test the selectivity and anti-interference ability. It was found that the absorbance of the probe solution increased, and the I 540 / I 655 The intensity ratio was significantly reduced. These results fully demonstrated the high selectivity and strong anti-interference ability of the nanoprobe for CAT detection, indicating that it is suitable for accurate detection in complex sample environments.

[0041] Application Example 3 Nanoprobe detection of CAT in serum: Take a human serum sample and dilute it with deionized water to a certain multiple. Take the diluted serum sample and add the probe solution prepared in Example 1, mix well, and perform CAT detection in fluorescence and colorimetric modes respectively. Calculate the concentration of CAT in serum based on the known standard curve.

[0042] In the experiments of adding CAT at different concentrations (0, 30, 60 mU / mL), the recovery of CAT ranged from 97.98% to 104.97%, while the relative standard deviation (RSD) value was low, ranging from 1.3% to 5.3% (see Table 1). From the data in Table 1, it can be inferred that the nanoprobe of the present invention can sensitively and accurately detect the CAT content in human serum.

[0043] Table 1

[0044] 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-colorimetry dual-mode nanoprobe includes UCNPs, hydrogen peroxide, ferrous ions and neutral red.

2. The fluorescence-colorimetric dual-modality nanoprobe according to claim 1, characterized in that: The concentration of UCNPs in the nanoprobe is 0.1~1 mg.mL -1 The concentration of hydrogen peroxide is 0.5~2 mmol L -1 The concentration of ferrous ions is 1~3mmol L -1 The concentration of neutral red is 0.1~0.3 mmol L -1 .

3. 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, NH4F and NaOH as raw materials.

4. The fluorescence-colorimetric dual-modal nanoprobe according to claim 3, characterized in that: The rare earth chlorides are chlorides of yttrium, ytterbium and erbium.

5. The fluorescence-colorimetric dual-modality nanoprobe according to claim 3, characterized in that: The reaction temperature of the solvent thermal method is 280-320° C., and the reaction time is 30-90 min.

6. The fluorescence-colorimetry dual-modality nanoprobe according to claim 3, characterized in that: The preparation method of the UCNPs comprises the following steps: (1) NaYF4:Yb,Er core nanoparticles were prepared by reaction of YCl3·6H2O, YbCl3·6H2O, ErCl3·6H2O, oleic acid, 1-octadecene, NH4F and NaOH; (2) NaYF4:Yb,Er@NaYF4 upconversion nanoparticles were prepared by reacting the above NaYF4:Yb,Er core nanoparticles, YCl3·6H2O, oleic acid, 1-octadecene, NH4F and NaOH; (3) The above-mentioned NaYF4:Yb,Er@NaYF4 upconversion nanoparticles were treated with acid to obtain hydrophilic UCNPs.

7. The fluorescence-colorimetry dual-modality nanoprobe according to claim 6, characterized in that: The acid treatment uses dilute hydrochloric acid with a pH of 0.1-2.

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

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

10. The use according to claim 9, characterized in that: The fluorescence-colorimetric dual-mode nanoprobe performs fluorescence detection within the range of an excitation wavelength of 980 nm and an emission wavelength of 500-700 nm; and performs colorimetric analysis within the range of an absorption wavelength of 400-700 nm.

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