Preparation and application of type I hyaluronidase fluorescent nano sensor
A hyaluronidase and nanosensor technology, applied in the fields of nanotechnology and analysis and detection, to achieve the effects of mild conditions, high specificity, good social value and application prospects
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Embodiment 1
[0031]Example 1: Preparation and characterization of nanosensors
[0032]Add 1 mg of TPC3 dye and 2 mg of CHA polymer to 1 ml of DMSO, slowly drop 3 ml of ultrapure water into the solution under magnetic stirring at 37°C, react for 30 minutes, and white micelle particles gradually appear in the solution. After the reaction, the reaction solution was used to remove the unwrapped TPC3 dye molecules with a molecular weight cut-off 3500KD dialysis bag, and dialyzed for 3 days. During this process, the white micellar particles were slowly dispersed in the dialysis bag until they disappeared to prepare a TPC3@CHA micellar solution of uniform size. The final product was stored at 4°C. byfigure 1 (A) The transmission electron microscope image shows that the size of TPC3@CHA is about 160nm, and there is no agglomeration phenomenon, and the uniformity is good.
Embodiment 2
[0033]Example 2: Titration curve of nano sensor response
[0034]The titration curve is determined under optimized conditions.figure 2 (A) shows that after different concentrations of Hyal-1 (0-12μg / mL), the fluorescence intensity of the nanosensor increases significantly with the increase of Hyal-1 concentration. And fromfigure 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 5nm.figure 2 (B) shows that the signal-to-background ratio (S / B) of the nanosensor and the analyte Hyal-1 in the range of 0.25-6.0μg / mL shows a good linear relationship, and the linear function is y = 1.267x + 2.1811;R2 = 0.9956. The probe can accurately detect Hyal-1 in urine.
Embodiment 3
[0035]Example 3: Selectivity investigation of nano sensor
[0036]Take the nanosensor and add it to the PB buffer at a concentration of 0.02 mmol / L TPC3@0.3mg / mL CHA, and then add various representative interference species (0.1 μM / mL H2O2, Urea, Uric acid, Glu, Gly, Cys,Glucose, VC, GSH, Cathepsin, Trypsin, Thrombin, Galactosidase, Ribnudease, Hyal-2 (5μg / mL), Hyal-3 (5μg / mL ), Hyal-4( 5μg / mL), PH-20 (5μg / mL) and Hyal-1(5μg / mL)). At the same time, add a blank group, add only 2 μL PBS buffer solution, shake well, measure and record the fluorescence intensity of each group of solutions at excitation wavelength = 375 nm and emission wavelength = 495 nm, to obtain various species of nano-sensing Influence of fluorescence signal. 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 is repeated three times, and the average value is taken.
[0037]Result analysis: su...
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