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Preparation and application of a type I hyaluronidase fluorescent nanosensor

A hyaluronidase and nanosensor technology, which is applied in the fields of nanotechnology and analysis and detection to achieve the effects of simple operation, good social value and application prospects, and high specificity

Active Publication Date: 2022-06-21
CHANGSHA UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, this sensing system has not been reported in the detection of Hyal-1 in urine

Method used

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  • Preparation and application of a type I hyaluronidase fluorescent nanosensor
  • Preparation and application of a type I hyaluronidase fluorescent nanosensor
  • Preparation and application of a type I hyaluronidase fluorescent nanosensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Preparation and characterization of nanosensors

[0032] 1 mg of TPC3 dye and 2 mg of CHA polymer were added to 1 ml of DMSO, and 3 ml of ultrapure water was slowly added dropwise to the solution under magnetic stirring at 37 °C for 30 min, and white micelle particles gradually appeared in the solution. After the reaction, the unwrapped TPC3 dye molecules were removed from the reaction solution with a dialysis bag with a molecular weight cut-off of 3500 KD, and dialyzed for 3 days. During this process, the white micellar particles were slowly dispersed in the dialysis bag until they disappeared to obtain a uniform size TPC3@CHA micellar solution. The final product was stored at 4°C. Depend on figure 1 (A) The TEM image shows that the size of TPC3@CHA is about 160 nm, and there is no agglomeration phenomenon, and the uniformity is good.

Embodiment 2

[0033] Example 2: Titration curve of nanosensor response

[0034] The determination of the titration curve was carried out under the optimized conditions. figure 2 (A) shows that after different concentrations of Hyal-1 (0-12 μg / mL), the fluorescence intensity of the nanosensor increases significantly with the increase of Hyal-1 concentration. and from figure 1 (B) It can be seen that after the nanosensor responds to Hyal-1, the size of the nanosensor after being degraded by Hyal-1 is about 5 nm. figure 2 (B) shows that the signal-to-background ratio (S / B) of the nanosensor and the analyte Hyal-1 in the range of 0.25-6.0 μg / mL exhibits a good linear relationship, and the linear function formula is y=1.267x+ 2.1811;R 2 =0.9956. The probe can accurately detect Hyal-1 in urine.

Embodiment 3

[0035] Example 3: Selective investigation of nanosensors

[0036] The nanosensor was added to PB buffer at a concentration of 0.02mmol / LTPC3@0.3mg / mL CHA, and then different representative interference species (0.1μM / mL H) were added to it. 2 O 2 ,Urea,Uric acid,Glu,Gly,Cys,Glucose,VC,GSH,Cathepsin,Trypsin,Thrombin,Galactosidase,Ribnudease,Hyal-2(5μg / mL),Hyal-3(5μg / mL),Hyal-4( 5 μg / mL), PH-20 (5 μg / mL) and Hyal-1 (5 μg / mL)). At the same time, add a blank group, only add 2 μL PBS buffer solution, shake well, measure and record the fluorescence intensity value of each group solution at excitation wavelength = 375 nm, emission wavelength = 495 nm, and obtain the effect of various species on the nano-sensing fluorescence signal. influences. The signal-to-background ratio is the fluorescence value of the solution after adding various interfering species divided by the fluorescence value of the blank group. The experiment was repeated three times and the average value was taken. ...

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Abstract

The invention discloses a polymer nanometer sensor and its application for detecting type 1 hyaluronidase (Hyal-1). Based on the principle of differential degradation of hyaluronic polymer nanomicelles by different hyaluronidases, the invention encapsulates hydrogen bond-induced enhanced dye molecules in nanomicelles, and uses this stimulus-responsive nanomaterial to regulate the relationship between dyes and water. Intermolecular interaction, design of highly selective and sensitive Hyal-1 fluorescent nanosensor. The type-1 hyaluronidase detection kit includes a type-1 hyaluronidase fluorescent nanometer sensor and a solvent. The results show that the kit has the advantages of high specificity, good sensitivity, easy operation, economical and practical, and realizes the detection of Hyal‑1 in actual urine samples. Therefore, the inventive method has original innovation, good social value and application prospect, and is expected to be applied to the detection and analysis of Hyal-1 in the urine of bladder cancer patients.

Description

technical field [0001] The invention belongs to the fields of nanotechnology and analysis and detection, and relates to the construction of a type 1 hyaluronidase fluorescent nano sensor based on the principle of hydrogen bond induction and enhancement and a new method for type 1 hyaluronidase detection and analysis. Background technique [0002] Hyaluronidases (HAases) belonging to the endoglycosidase family are able to degrade hyaluronic acid (HA) into oligosaccharide fragments to maintain various physiological functions such as angiogenesis, fertilization process, embryonic development and cell motility. HAases have isoforms with similar gene sequences but different functions, among which type 1 hyaluronidase (Hyal-1) usually exists in lysosomes to catalyze the degradation of intracellular HA to maintain key physiological functions. When the body function is abnormal or diseased, the intracellular Hyal-1 will be overexpressed and spilled into the tissue and body fluids. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64B82Y15/00
CPCG01N21/6428B82Y15/00
Inventor 杨盛罗金秋杨荣华
Owner CHANGSHA UNIVERSITY OF SCIENCE AND TECHNOLOGY
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