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CN-121978072-A - Fluorescent reagent composition for detecting hyaluronidase and application thereof

CN121978072ACN 121978072 ACN121978072 ACN 121978072ACN-121978072-A

Abstract

The invention discloses a fluorescent reagent composition for detecting hyaluronidase and application thereof, which are characterized by comprising sodium hyaluronate or a hyaluronic acid solution, a magnetic nano material Fe 3 O 4 NPs solution and a fluorescent nano material UCNPs solution, wherein the detection of the hyaluronidase comprises the following steps of adding a urine sample to be detected containing the hyaluronidase into the sodium hyaluronate solution to degrade the sodium hyaluronate, adding the fluorescent nano material UCNPs solution into a reaction product to enable the reaction product to be subjected to electrostatic adsorption with unhydrolyzed sodium hyaluronate, outputting a fluorescent signal F 0 , adding the magnetic nano material Fe 3 O 4 NPs solution to perform magnetic separation, taking supernatant to measure the fluorescent signal F, and taking F/F 0 as an output signal to calculate the concentration of the hyaluronidase in the urine sample to be detected.

Inventors

  • YAN ZEJUN
  • YANG BING
  • HUANG YOUJU
  • CHEN JING
  • DING CAIPING
  • LI MING
  • ZHANG DONG
  • LIU YADONG

Assignees

  • 宁波大学附属第一医院(宁波市第一医院)

Dates

Publication Date
20260505
Application Date
20260408

Claims (9)

  1. 1. A fluorescent reagent composition for detecting hyaluronidase is characterized by comprising sodium hyaluronate or a hyaluronic acid solution, a magnetic nano material Fe 3 O 4 NPs solution and a fluorescent nano material UCNPs solution, wherein the concentration of the sodium hyaluronate or the hyaluronic acid solution is 1-2 mg/mL, the concentration of the magnetic nano material Fe 3 O 4 NPs solution is 0.5-2 mg/mL, and the concentration of the fluorescent nano material UCNPs solution is 0.5-2 mg/mL.
  2. 2. The fluorescent reagent composition for detecting hyaluronidase according to claim 1 is characterized in that the preparation method of the magnetic nanomaterial Fe 3 O 4 NPs is characterized in that ferric chloride hexahydrate, sodium acetate and ethylene glycol are used as raw materials, and the magnetic nanomaterial Fe 3 O 4 NPs is prepared by a hydrothermal synthesis method.
  3. 3. The fluorescent reagent composition for detecting hyaluronidase according to claim 2, wherein the preparation method of the magnetic nanomaterial Fe 3 O 4 NPs comprises the specific steps of dissolving 1-2 g ferric chloride hexahydrate in 30-50 mL glycol solution, completely dissolving the ferric chloride hexahydrate by ultrasonic and stirring, adding 3-4 g sodium acetate and 0-2 g sodium dodecyl sulfate, stirring to obtain a uniform solution by ultrasonic, after the solid is completely dissolved, loading the uniform solution into a reaction kettle, reacting at 190-220 ℃ for 5-15 h, waiting for the reaction to be completed, naturally cooling to room temperature, magnetically washing with absolute ethanol and deionized water, and finally drying in vacuum to obtain the magnetic nanomaterial Fe 3 O 4 NPs,Fe 3 O 4 NPs with a particle size of 150-350 nm.
  4. 4. The fluorescent reagent composition for detecting hyaluronidase according to claim 1, wherein the preparation method of the fluorescent nanomaterial UCNPs is characterized in that the fluorescent nanomaterial UCNPs is prepared by taking polyethyleneimine, sodium chloride, yttrium nitrate hexahydrate, ytterbium nitrate pentahydrate, erbium nitrate hexahydrate, ammonium fluoride and ethylene glycol as raw materials.
  5. 5. The fluorescent reagent composition for detecting hyaluronidase according to claim 4, wherein the preparation method of the fluorescent nanomaterial UCNPs comprises the following specific steps: (1) Sequentially dissolving 0.3-0.5 g polyethylenimine, 0.1-0.2 g sodium chloride, 0.5-1.5 mmol yttrium nitrate hexahydrate, 0.1-0.5 mmol ytterbium nitrate pentahydrate and 0.01-0.05 mmol erbium nitrate hexahydrate in 10-30 mL glycol to obtain a clear and transparent solution A; (2) Ultrasonically dissolving 0.2-0.5 g ammonium fluoride in 10-20 mL glycol to obtain solution B; (3) Slowly dripping the solution A into the solution B, keeping stable stirring in the whole process, transferring the mixed solution into a reaction kettle, reacting at 180-220 ℃ for 1-10 h, naturally cooling to room temperature after finishing, washing the reaction product with acetone, washing with ethanol and water alternately for multiple times, centrifuging, taking precipitate, and vacuum drying to obtain the fluorescent nano material UCNPs with the particle size of 40-80 nm.
  6. 6. The method of claim 1, wherein the fluorescent reagent composition is used for detecting the concentration of the hyaluronidase serving as a bladder cancer marker in urine.
  7. 7. The use according to claim 6, which is not for the purpose of disease diagnosis, wherein the detection of the concentration of the bladder cancer marker hyaluronidase in the urine comprises the steps of: Step 1, adding a urine sample to be tested containing hyaluronidase into sodium hyaluronate or hyaluronic acid solution to degrade the sodium hyaluronate or hyaluronic acid; step 2, adding fluorescent nano material UCNPs solution into the reaction product obtained in the step1, and measuring a solution fluorescence signal value F 0 ; Step 3, adding a magnetic nano material Fe 3 O 4 NPs solution into the solution obtained in the step 2, then performing magnetic separation to remove sediment, and taking supernatant to measure a fluorescence signal value F; And 4, taking the F/F 0 as an output signal, and calculating to obtain the concentration of the hyaluronidase in the urine sample to be detected according to the relation between the F/F 0 value and the hyaluronidase concentration.
  8. 8. The method according to claim 7, wherein in the step 1, the urine sample is centrifuged at a speed of 3000-5000 rpm/min for 5-10 min, the upper clear urine sample is taken as the urine sample to be tested, 10-50. Mu.L of the urine sample to be tested is added to 5-20. Mu.L of 1-2 mg/mL sodium hyaluronate or hyaluronic acid solution, and 50-200. Mu.L of phosphate buffer solution with pH=5-7 and concentration of 10-100 mM is added for reaction at 30-40 ℃ for 60-120 min.
  9. 9. The method according to claim 8, wherein in the step 2, 15-40. Mu.L of 0.5-2 mg/mL fluorescent nanomaterial UCNPs solution is added to the reaction product obtained in the step 1, the reaction product is incubated for 5-10 min to enable the reaction product to be subjected to electrostatic adsorption with undegraded sodium hyaluronate or hyaluronic acid, fluorescence intensity F 0 is measured by a 980 nm laser machine, in the step 3, 5-15. Mu.L of 0.5-2 mg/mL magnetic nanomaterial Fe 3 O 4 NPs solution is continuously added, after the reaction product is uniformly mixed, magnetic separation is carried out for 5-10 min, and supernatant fluid is taken to measure fluorescence intensity F.

Description

Fluorescent reagent composition for detecting hyaluronidase and application thereof Technical Field The invention belongs to the technical field of bladder cancer marker detection, and particularly relates to a fluorescent reagent composition for detecting hyaluronidase and application thereof. Background Bladder Cancer (BC) belongs to a malignant tumor of the urinary system, most of the bladder cancer is non-muscle invasive bladder cancer (NMIBC), the rest is Muscle Invasive Bladder Cancer (MIBC), the prognosis and the treatment scheme of the bladder cancer are obviously different, wherein the survival rate of NMIBC patients in 5 years can reach 95%, the treatment is mainly based on local operation combined perfusion treatment, and if the survival rate of the bladder cancer in 5 years after the bladder cancer is developed into MIBC is only 10%, the comprehensive treatment mainly based on radical operation is needed. The existing clinical diagnosis means have obvious defects that the detection sensitivity of cystoscopy, cytology examination and imaging detection on early bladder cancer is limited, missed detection is easy to occur, pathological sections are used as grading basis, the method belongs to invasive operation, and the patient acceptance is low. Therefore, for early diagnosis and accurate classification of bladder cancer, which is the core of improving patient survival rate and optimizing therapeutic strategies, development of low-invasive, low-cost diagnosis and classification methods is urgently needed. Hyaluronidase (HAase) is taken as an enzyme class which can hydrolyze hyaluronic acid, can enhance the tissue fluid permeability by regulating and controlling the metabolism of hyaluronic acid in extracellular matrix, participates in the process of remodelling extracellular matrix, and has biological behaviors closely related to cell migration and invasion. Particularly in the diagnosis of bladder cancer, the abnormal body fluid level of HAase is often related to the occurrence and progress of bladder tumor, and can assist in early diagnosis, progress monitoring and prognosis evaluation of bladder cancer. However, the existing HAase detection method has the inherent defects that the sensitivity of the traditional turbidity method is insufficient, the detection requirement of low content HAase in a bladder cancer sample is difficult to meet, the ELISA method can only detect the mass concentration of HAase instead of the enzyme activity, and the operation flow is complex and the cost is high. Fluorescence is a common detection means, and has the characteristics of high sensitivity, detection limit reduced by more than one order of magnitude compared with a turbidity method, high specificity, low cost, simplicity and the like. However, the existing kit for fluorescence detection HAase is rarely applied to bladder cancer detection, and is mainly because (1) the stability of fluorescent probe molecules is insufficient and is easily interfered by complex samples, and (2) the detection range is not matched with the differentiation range of bladder cancer, so that lower detection limit is needed for better diagnosis and classification of bladder cancer tumor. In view of the foregoing, there is a need to develop a simple, rapid, highly sensitive HAase detection reagent to meet the clinical demands of early screening and accurate classification of bladder cancer. Disclosure of Invention The invention aims to solve the technical problem of providing a fluorescent reagent composition for detecting hyaluronidase, which has high sensitivity, strong specificity, high accuracy and simple operation, and application thereof, and is used for ultrasensitive detection of bladder cancer biomarker HAase so as to meet clinical requirements of early screening and accurate grading of bladder cancer. The technical scheme includes that the fluorescent reagent composition for detecting hyaluronidase comprises sodium hyaluronate or hyaluronic acid solution, magnetic nano material Fe 3O4 NPs solution and fluorescent nano material UCNPs solution, wherein the concentration of the sodium hyaluronate or the hyaluronic acid solution is 1-2 mg/mL, the concentration of the magnetic nano material Fe 3O4 NPs solution is 0.5-2 mg/mL, and the concentration of the fluorescent nano material UCNPs solution is 0.5-2 mg/mL. The preparation method of the magnetic nano material Fe 3O4 NPs comprises the following steps of taking ferric chloride hexahydrate, sodium acetate and ethylene glycol as raw materials, and preparing the magnetic nano material Fe 3O4 NPs through a hydrothermal synthesis method. Further, the preparation method of the magnetic nano material Fe 3O4 NPs comprises the specific steps of dissolving 1-2 g ferric chloride hexahydrate in 30-50 mL glycol solution, completely dissolving the ferric chloride hexahydrate through ultrasonic and stirring, adding 3-4 g sodium acetate and 0-2 g sodium dodecyl sulfate, stirring into a uniform