Upconversion nanoprobe for dual signal output detection of tyrosinase and its application

Abstract

The invention discloses an upconversion nanoprobe for detecting tyrosinase with dual signal output and application thereof, and relates to the technical field of nanobiomaterials. The upconversion nanoprobe comprises UCNPs, tyramine and iron ions; the UCNPs has a core-shell structure, with β-NaYF4:Yb,Er as the core and NaYF4 as the shell; the upconversion nanoprobe of the invention forms a dual-signal self-calibration capability by integrating the advantages of fluorescence determination method and colorimetry; the method not only utilizes the UCL signal excited by near-infrared light, greatly improving the detection accuracy, but also provides an intuitive and convenient method for the detection of tyrosinase through colorimetric reaction; the dual-signal output detection mode effectively alleviates signal fluctuations, thereby ensuring the accuracy and reliability of tyrosinase analysis.

Description

Technical Field

[0001] The invention relates to the technical field of nano-biomaterials, and in particular to an up-conversion nano-probe for detecting tyrosinase with dual signal output and application thereof. Background Art

[0002] As the core enzyme in the process of melanin synthesis, tyrosinase plays a vital role in the body. Its activity level is not only closely related to various physiological functions of the body, but also often used as an important diagnostic indicator in the medical field, especially in the early screening of skin cancer and melanoma. However, the existing tyrosinase detection methods such as spectrophotometry, fluorescence analysis, electrochemical methods and immunoassay technology, although each has its own advantages, still have limitations. For example, although the fluorescence assay has high sensitivity and high selectivity, it is easily interfered by background fluorescence; and although the colorimetric method is cost-effective and intuitive, its detection accuracy needs to be improved.

[0003] In order to overcome the shortcomings of existing technologies, researchers have been exploring more accurate, efficient and low-cost methods for tyrosinase detection. In recent years, lanthanide-doped upconversion nanoparticles (UCNPs) have shown great potential in the field of biomarker detection due to their unique optical properties. UCNPs have an excellent ability to convert low-energy near-infrared (NIR) light into high-energy visible light, which can significantly enhance the penetration of light in biological tissues while effectively reducing the autofluorescence noise caused by short-wavelength excitation. These properties make UCNPs an ideal choice for detecting tyrosinase.

[0004] Although UCNPs have shown great potential in biomarker detection, there are still challenges in constructing highly sensitive upconversion nanoprobes for tyrosinase detection. In particular, there is a lack of suitable energy acceptors. Therefore, the development of a new, highly sensitive, background-free dual-mode probe is crucial to improve the accuracy of tyrosinase detection. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an upconversion nanoprobe for detecting tyrosinase with dual signal output, which utilizes the unique optical properties of UCNPs and combines the specific catalytic activity of tyrosinase to achieve background-free and highly sensitive detection of tyrosinase.

[0006] The technical problem to be solved by the present invention is achieved by adopting the following technical solutions:

[0007] One of the purposes of the present invention is to provide an upconversion nanoprobe for detecting tyrosinase with dual signal output, wherein the upconversion nanoprobe comprises UCNPs, tyramine and iron ions.

[0008] The present invention combines UCNPs with tyramine and iron ions to form a label-free sensing system. Tyramine is used as a substrate of tyrosinase. Tyrosinase specifically oxidizes tyrosamine to generate dopamine. Dopamine reacts with iron ions to generate a purple product. The purple product acts as an energy acceptor to quench the upconversion luminescence (UCL) of UCNPs. At the same time, the generation of the purple product is monitored by a colorimetric reaction to achieve dual-signal output detection of tyrosinase.

[0009] The second object of the present invention is to provide the use of the up-conversion nanoprobe in the preparation of a tyrosinase detection reagent.

[0010] The third object of the present invention is to provide the application of the up-conversion nanoprobe in constructing an intelligent sensor for tyrosinase detection.

[0011] A fourth object of the present invention is to provide an intelligent sensor, comprising the upconversion nanoprobe, a 980 nm laser, a cuvette, a smart phone and a 3D printing accessory.

[0012] The beneficial effects of the present invention are:

[0013] 1. Improve detection sensitivity and accuracy. The present invention constructs a label-free sensing system by combining UCNPs with specific tyrosinase recognition molecules, thereby achieving background-free detection of tyrosinase. The near-infrared excitation, high photostability, and low spontaneous fluorescence of UCNPs, as well as the specific reaction of tyrosinase with tyrosine, improve the detection sensitivity and accuracy. The detection limit in the luminescence mode can be as low as 0.001 U mL -1 , providing a powerful tool for the accurate detection of tyrosinase.

[0014] 2. Realize dual-signal self-calibration and enhance reliability. The present invention provides a method for detecting tyrosinase by luminescence-colorimetry dual-signal output, which integrates the advantages of fluorescence determination and colorimetry to form a dual-signal self-calibration capability; this method not only utilizes the UCL signal excited by near-infrared light, greatly improving the detection accuracy, but also provides an intuitive and convenient method for the detection of tyrosinase through colorimetric reaction; the dual-signal output detection method effectively alleviates signal fluctuations, thereby ensuring the accuracy and reliability of tyrosinase analysis.

[0015] 3. Easy operation and low cost. Compared with the traditional tyrosinase detection method, the nanoprobe provided by the present invention is easier to operate and does not require complex instruments and equipment and cumbersome experimental steps; at the same time, UCNPs do not require complex and time-consuming surface modification when constructing upconversion nanoprobes, which is conducive to the rapid and low-cost preparation of upconversion nanoprobes and reduces the detection cost.

[0016] 4. It has broad application prospects. The nanoprobe provided by the present invention can not only be used for early screening and diagnosis of diseases such as skin cancer and melanoma in the medical field, but can also be expanded to the detection of other diseases related to tyrosinase activity; in addition, through the rational design and use of other new substrates, the nanoprobe is expected to become a multifunctional tool for detecting several other enzymes, and has broad application prospects in the fields of biomedical research, clinical diagnosis and environmental monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of the principle of using the upconversion nanoprobe of the present invention for tyrosinase detection;

[0018] Figure 2 The UV-visible absorption spectrum (red) and UCL spectrum (blue) of the upconversion nanoprobe used for tyrosinase detection according to the present invention;

[0019] Figure 3 Structural characterization diagrams of NaYF4:Yb,Er upconversion nanoparticles and NaYF4:Yb,Er@NaYF4 upconversion nanoparticles prepared in Example 1; (A) Low-resolution transmission electron microscopy (TEM) image of NaYF4:Yb,Er upconversion nanoparticles; (B) Low-resolution TEM image of NaYF4:Yb,Er@NaYF4 upconversion nanoparticles; (C) High-resolution TEM image of NaYF4:Yb,Er@NaYF4 upconversion nanoparticles; (D) X-ray diffraction (XRD) pattern of NaYF4:Yb,Er@NaYF4 upconversion nanoparticles;

[0020] Figure 4 The upconversion nanoprobe prepared in Example 1 was used to detect different concentrations of tyrosinase (0-5.5 U·mL -1 ) responsiveness; (A) UCL spectrum in the range of 500-600 nm, the inset shows the change of UCL color with the increase of tyrosinase concentration; (B) Linear fitting curve between UCL intensity at 540 nm and tyrosinase concentration; (C) UV-visible absorption spectrum in the range of 400-800 nm, the inset shows the change of probe solution color with the increase of tyrosinase concentration; (D) Linear fitting curve between absorbance at 536 nm and tyrosinase concentration;

[0021] Figure 5The selectivity and anti-interference test results of the upconversion nanoprobe prepared in Example 1; (A) in luminescence mode; (B) in colorimetric mode; where 1-blank, 2-tyrosinase, 3-bovine serum albumin (BSA), 4-human serum albumin (HSA), 5-alkaline phosphatase (ALP), 6-acetylcholinesterase (AchE), 7-glucose oxidase (GOX), 8-Zn 2+ , 9-Cl - , 10-SO4 2- , 11-ascorbic acid, 12-L-cysteine. DETAILED DESCRIPTION

[0022] 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.

[0023] The invention provides an upconversion nanoprobe for detecting tyrosinase with dual signal outputs. The upconversion nanoprobe comprises UCNPs, tyramine and iron ions.

[0024] Furthermore, the iron ions are derived from at least one of ammonium ferric sulfate and ferric chloride, including but not limited to these.

[0025] Furthermore, the concentration of UCNPs in the upconversion nanoprobe is 0.1-1 mg / mL, the concentration of tyramine is 0.5-2 mmol / L, and the concentration of iron ions is 0.3-0.7 mmol / L, based on 2 mL of the upconversion nanoprobe solution. The present invention ensures that the prepared upconversion nanoprobe has high sensitivity and accuracy in both fluorescence and colorimetric modes by regulating the concentrations of UCNPs, tyramine, and iron ions, thereby achieving efficient detection of tyrosinase.

[0026] 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. The rare earth chloride is used as a precursor, OA is used as a ligand, and OA and ODE are used as a mixed solvent. The rare earth chloride includes but is not limited to yttrium, ytterbium, and erbium chlorides. The reaction temperature of the solvothermal method is 280-320°C, and the reaction time is 30-90 min.

[0027] As a preferred technical solution, the UCNPs have a core-shell structure, with β-NaYF4:Yb,Er as the core and NaYF4 as the shell.

[0028] As a specific embodiment, the preparation method of UCNPs comprises the following steps:

[0029] (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;

[0030] (2) NaYF4:Yb,Er@NaYF4 upconversion nanoparticles with core-shell structure were prepared by reacting NaYF4:Yb,Er upconversion nanoparticles, YCl3·6H2O, OA, ODE, NH4F and NaOH;

[0031] (3) The NaYF4:Yb,Er@NaYF4 upconversion nanoparticles were treated with acid to obtain hydrophilic UCNPs.

[0032] In order to improve the water solubility and biocompatibility of UCNPs, the present invention uses acid treatment to convert the synthesized hydrophobic UCNPs into hydrophilic UCNPs. Specifically, the acid treatment uses dilute hydrochloric acid with a pH of 0.1-2.

[0033] In order to avoid the influence of oxidation reaction on the nanoparticles, the preparation process of the UCNPs is carried out in a protective atmosphere (such as argon).

[0034] In the present invention, the upconversion nanoprobe performs fluorescence detection at an excitation wavelength of 980 nm and an emission wavelength of 540-655 nm, and performs colorimetric analysis at an absorption wavelength of 400-700 nm. The intensity changes of green emission at 540 nm and red emission at 655 nm are used to evaluate the activity of tyrosinase.

[0035] The present invention provides application of the up-conversion nanoprobe in preparing a tyrosinase detection reagent.

[0036] The present invention provides application of the up-conversion nanoprobe in constructing an intelligent sensor for tyrosinase detection.

[0037] The present invention provides an intelligent sensor, comprising the upconversion nanoprobe, a 980 nm laser, a cuvette, a smart phone and 3D printing accessories. Through the camera of the smart phone and its built-in color analysis software, the light signal emitted by the upconversion nanoprobe can be accurately captured and analyzed, thereby realizing the rapid and accurate determination of the tyrosinase concentration in the sample.

[0038] The ammonium ferric sulfate solution in the present invention is an ammonium ferric sulfate aqueous solution; the tyramine solution is a tyramine aqueous solution; and the ferric chloride solution is a ferric chloride aqueous solution.

[0039] Example 1: Preparation of UCNPs

[0040] (1) Preparation of NaYF4:Yb,Er upconversion nanoparticles

[0041] 3.9 mmol YCl3·6H2O, 1 mmol YbCl3·6H2O, and 0.1 mmol ErCl3·6H2O were weighed and mixed with 30 mLOA and 75 mL ODE, heated to 160℃ and stirred for 1 h, and cooled to room temperature; then 50 mL of methanol solution containing 20 mmol NH4F and 12.5 mmol NaOH was added dropwise, heated to 50℃ and stirred for 30 min, then heated to 100℃ in vacuum and kept warm for 20 min, and then heated to 300℃ in argon atmosphere and kept warm for 1 h. After the reaction, the solution was cooled to room temperature and washed three times with ethanol and cyclohexane to obtain NaYF4:Yb,Er upconversion nanoparticles.

[0042] (2) Preparation of core-shell NaYF4:Yb,Er@NaYF4 upconversion nanoparticles

[0043] 1 mmol YCl3·6H2O was mixed with 6 mL OA and 15 mL ODE, heated to 160°C in an argon-filled environment and stirred 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 10 mL of a methanol solution containing 4 mmol NH4F and 2.5 mmol NaOH was added dropwise, heated to 50°C and stirred for 30 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. After the reaction was completed, the solution was precipitated with ethanol and washed three times to obtain NaYF4:Yb,Er@NaYF4 upconversion nanoparticles.

[0044] (3) Acid treatment of NaYF4:Yb,Er@NaYF4 upconversion nanoparticles

[0045] Weigh 200 mg of the NaYF4:Yb,Er@NaYF4 upconversion nanoparticles prepared in step (2) and disperse them in 60 mL of dilute hydrochloric acid (pH = 1), ultrasonically treat for 1 h to promote the protonation and dissociation of OA ligands, centrifuge, wash twice with ultrapure water to remove residual acid and unreacted OA ligands, and redisperse the acid-treated upconversion nanoparticles in 2 mL of ultrapure water to obtain a hydrophilic UCNPs solution.

[0046] from Figure 1It can be seen that under 980 nm laser excitation, UCNPs exhibit characteristic emissions at 540 nm and 655 nm, and the overall UCL appears green. After the addition of tyrosinase, tyramine is oxidized to dopamine. In order to construct an energy receptor suitable for upconversion sensing system, the present invention introduces iron ions into the sensing system. The catechol part in dopamine reacts with Fe 3+ Ion coordination, followed by oxidation to form o-semiquinone, the oxidation product appears purple, and its absorption peak in the visible light region overlaps with the emission peaks of UCNPs at 540 nm and 655 nm, resulting in quenching of the emission at these two wavelengths. Due to the maximum overlap between the absorption peak of the purple product and the green UCL, the quenching of the green UCL is more significant than that of the red UCL. Therefore, the entire upconversion probe emits a weak red light. Through this colorimetric reaction, the present invention successfully constructs a suitable upconversion energy receptor for tyrosinase detection, and realizes dual-signal output detection of tyrosinase without background interference.

[0047] from Figure 2 It can be seen that in the absence of tyrosinase, the absorption of tyramine in the visible light range is weak, so the UCL intensity of UCNPs is not affected. After the addition of tyrosinase, tyramine is converted into dopamine under the catalysis of tyrosinase, and dopamine then reacts with Fe 3+ The reaction triggered a colorimetric reaction, forming a strong absorption peak at 536 nm. This absorption peak overlapped with both the 540 nm green emission and the 655 nm red emission of UCNPs, resulting in the quenching of these two emission peaks. Therefore, a luminescent and colorimetric nanoprobe with green UCL emission as the detection signal was developed. It is worth noting that the surface of UCNPs is exposed after acid treatment. Therefore, the distance between UCNPs and the energy acceptor is not sufficient to realize the luminescence resonance energy transfer process. The quenching behavior of UCL should be attributed to the inner filter effect (IFE). In addition, the formation of a purple product enhances the intuitiveness of this detection method.

[0048] from Figure 3 It can be seen that the average particle size of the core UCNPs is 26 nm, while the particle size of the core-shell UCNPs increases to 28 nm. Through high-resolution TEM observation, UCNPs exhibit lattice fringes consistent with the hexagonal phase (110) crystal plane, proving that they have excellent crystallinity. In addition, the XRD spectrum shows sharp diffraction peaks that are completely consistent with the standard pattern (JCPDS 16-0334), further confirming the high crystallinity and hexagonal structure of UCNPs. This structure is conducive to energy transfer between rare earth ions and is crucial to improving UCL efficiency.

[0049] Example 2: Preparation of UCNPs

[0050] The preparation method is the same as that of Example 1, except that 3.8 mmolYCl3·6H2O, 1.1 mmolYbCl3·6H2O, 0.2 mmolErCl3·6H2O, 40 mLOA and 60 mL ODE are used in the preparation of NaYF4:Yb,Er upconversion nanoparticles.

[0051] Example 3: Preparation of UCNPs

[0052] The preparation method 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 100°C in a vacuum for 30 min and then heated to 320°C in an argon atmosphere for 1 h.

[0053] Example 4: Preparation of UCNPs

[0054] The preparation method is the same as that of Example 1, except that when the NaYF4:Yb,Er@NaYF4 upconversion nanoparticles are subjected to acid treatment, 120 mL of dilute hydrochloric acid (pH=1.5) is used for ultrasonic treatment for 2 h.

[0055] Example 5: Preparation of upconversion nanoprobes

[0056] Take 100 μL of ammonium ferric sulfate solution (10 mmol / L), 100 μL of the hydrophilic UCNPs solution (10 mg / mL) prepared in Example 1, 200 μL of tyramine solution (10 mmol / L) and 1 mL of PBS solution (pH = 7.5, 10 mmol / L), mix them evenly, and dilute the solution volume to 2 mL with deionized water to obtain an upconversion nanoprobe solution.

[0057] Example 6: Preparation of upconversion nanoprobes

[0058] Take 100 μL of ferric chloride solution (10 mmol / L), 200 μL of the hydrophilic UCNPs solution (10 mg / mL) prepared in Example 1, 200 μL of tyramine solution (15 mmol / L) and 1 mL of PBS solution (pH = 7.5, 10 mmol / L), mix them evenly, and dilute the solution volume to 2 mL with deionized water to obtain an upconversion nanoprobe solution.

[0059] Example 7: Luminescence and colorimetric responsiveness testing of upconversion nanoprobes

[0060] 100 μL of tyrosinase of different concentrations was added to the upconversion nanoprobe solution prepared in Example 5, and diluted to 2 mL with PBS solution (pH = 7.5, 10 mmol / L) to make the tyrosinase concentration range from 0 to 5.5 U·mL-1 The solution was allowed to stand at 37°C for 90 min to ensure sufficient reaction, and then the UCL spectrum and UV-visible absorption spectrum of the solution were measured.

[0061] Depend on Figure 4 It can be seen that as the tyrosinase concentration ranges from 0 to 5.5 U·mL -1 As the wavelength range increases, the green UCL intensity of the nanoprobe at 540 nm continues to decrease, and the color of the probe changes from green to light red (see Figure 4 A). The change of green UCL showed a good linear relationship with the concentration of tyrosinase. Correlation analysis showed that it was in the range of 0~5.5 U·mL -1 The linear correlation coefficient (R 2 ) is close to 1( Figure 4 B). The limit of detection (LOD) was calculated using the formula LOD = 3*δ / S, where δ is the standard deviation of 10 blank experiments, representing the background noise level, and S is the slope of the linear regression equation. In this luminescence mode, the LOD is 0.0010 U mL -1 . Figure 3 C shows that as the concentration of tyrosinase increases, the absorption spectrum response of the probe solution increases, which is consistent with the process in which tyrosinase catalyzes the oxidation of tyrosine to produce colorless dopamine, which then reacts with iron ions to produce a purple product. The appearance of the purple product intuitively indicates the presence of tyrosinase ( Figure 4 C illustration). Further, Figure 4 D shows that the absorbance of the probe solution at 536 nm also shows a linear relationship with the tyrosinase concentration, with a very high correlation. 2 =0.999, the minimum detection limit reaches 0.0025 U·mL -1 In summary, the upconversion nanoprobe developed in the present invention can respond to the presence of tyrosinase through dual signals of luminescence and absorption, thereby realizing sensitive detection of tyrosinase activity.

[0062] Example 8: Selectivity and anti-interference test of upconversion nanoprobe

[0063] Tyrosinase or different types of interfering substances (interfering substances include BSA, HSA, ALP, AchE, GOX, Zn 2+ , Cl - 、SO4 2-, ascorbic acid, L-cysteine), mixed well to ensure full reaction, and recorded the UCL spectrum and UV-visible absorption spectrum of the solution under the excitation of 980 nm laser. The color change of the solution was carefully observed under visible light to comprehensively evaluate the selectivity and anti-interference ability of the upconversion nanoprobe.

[0064] from Figure 5 It can be seen that the UCL intensity and absorbance of the probe solution did not change significantly after the addition of interfering substances, which proves that the probe has excellent selectivity; while the addition of tyrosinase will cause the UCL intensity of the solution to decrease significantly and the absorbance to increase significantly ( Figure 5 A and Figure 5 B). In particular, at a wavelength of 540 nm, the significant decrease in UCL intensity and the increase in absorbance confirmed the high sensitivity of the probe to tyrosinase. In the presence of tyrosinase, the probe solution was purple, showing an intuitive dual-mode response of luminescence and colorimetry. The results show that the upconversion nanoprobe of the present invention can also accurately detect tyrosinase in complex samples, showing excellent anti-interference performance, and is suitable for clinical diagnosis and biological detection.

[0065] Example 9: Detection of tyrosinase in serum using upconversion nanoprobes

[0066] Human serum was taken as a sample, the collected serum sample was diluted, and the upconversion nanoprobe solution prepared in Example 5 was added, and after being mixed evenly, the activity of tyrosinase was detected in the serum sample with the probe added using fluorescence and colorimetry modes. The actual concentration of tyrosinase in the serum sample was calculated by comparing with the pre-established standard curve, and the results are shown in Table 1.

[0067] Table 1

[0068]

[0069] As shown in Table 1, when the concentration of tyrosinase added was 0, 1, and 2 U·mL -1 When the luminescence method was used, the recovery rate was 99.2-100.8%, and the relative standard deviation was 2.5-3.9%; the recovery rate of the colorimetric method was 98.6-101.0%, and the relative standard deviation was 3.9-4.8%. This shows that the upconversion nanoprobe of the present invention can sensitively and accurately determine tyrosinase in human serum, and can provide an effective analytical tool for clinical diagnosis and biomedical research.

[0070] 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 dual-signal output upconversion nanoprobe for detecting tyrosinase, characterized in that: The upconversion nanoprobe includes UCNPs, tyramine and iron ions; The UCNPs are prepared by a solvothermal method using rare earth chloride, OA, ODE, NH4F and NaOH as raw materials; The reaction temperature of the solvothermal method is 280-320°C, and the reaction time is 30-90 min; The preparation method of the UCNPs comprises the following steps: (1) NaYF4:Yb,Er upconversion nanoparticles were prepared by reacting YCl3·6H2O, YbCl3·6H2O, ErCl3·6H2O, OA, ODE, NH4F and NaOH; (2) NaYF4:Yb,Er@NaYF4 upconversion nanoparticles were prepared by reacting NaYF4:Yb,Er upconversion nanoparticles, YCl3·6H2O, OA, ODE, NH4F and NaOH; (3) The NaYF4:Yb,Er@NaYF4 upconversion nanoparticles were treated with acid to obtain hydrophilic UCNPs.

2. The dual-signal output upconversion nanoprobe for detecting tyrosinase according to claim 1, characterized in that: The iron ions are derived from at least one of ammonium ferric sulfate and ferric chloride.

3. The upconversion nanoprobe for detecting tyrosinase with dual signal output according to claim 1, characterized in that: The preparation process of the UCNPs is carried out in a protective atmosphere.

4. The upconversion nanoprobe for detecting tyrosinase with dual signal output according to any one of claims 1 to 3, characterized in that: The up-conversion nanoprobe performs fluorescence detection within the range of an excitation wavelength of 980 nm and an emission wavelength of 540-655 nm, and performs colorimetric analysis within the range of an absorption wavelength of 400-700 nm.

5. Use of the upconversion nanoprobe according to any one of claims 1 to 3 in the preparation of a tyrosinase detection reagent.

6. Use of the upconversion nanoprobe according to any one of claims 1 to 3 in constructing an intelligent sensor for tyrosinase detection.

7. A smart sensor comprising the upconversion nanoprobe according to any one of claims 1 to 3, a 980 nm laser, a cuvette, a smart phone and a 3D printing accessory.

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