Application Of High Sensitivity Fluorescent Nano Probe Based On FRET System In Real-time Detection Of Tumor Marker Hyaluronidase

Cancer is one of the leading causes of human death worldwide.Cancer has a wide-ranging physical and emotional impact on the life of patients.The cost of treatment also presents various challenges to the healthcare system and patients.The latest global cancer burden report of the International Agency for Research on Cancer（IARC）in 2021 shows that there are 19.29 million new cancer cases and 9.96 million cancer deaths,and an average of 2,000 people are diagnosed with cancer every hour globally.Cancer incidence has gradually increased every year since 1975.As a serious non-communicable disease,cancer is the biggest obstacle to the health development of every country.Therefore,early detection of tumors is of key importance for tumor prevention.The current diagnostic techniques for cancer are cytological diagnosis and histopathological biopsy,which have high specificity and low sensitivity.Therefore,an efficient and sensitive early detection method is urgently needed.In this work,a novel FRET system fluorescent nanoprobe was designed and synthesized for the detection of tumor marker hyaluronidase（HAase）.The specific research results are as follows:（1）Poly（lactic acid）（PLA）was synthesized at 130°C by using water as the initiator and zinc lactate as the catalyst to initiate the ring-opening polymerization of D,L-lactide.Proton nuclear magnetic resonance（¹H-NMR）and Fourier transform infrared spectroscopy（FT-IR）were used to characterize the product,which proved that the product contained PLA structural units.The molecular weight of PLA was calculated to be 1735 from the peak area of ¹H-NMR.Gel permeation chromatography proves that the number-average molecular weight and weight-average molecular weight of PLA are 3683 and 3456 respectively,and the polydispersity index is 1.08.1-Ethyl-3-（3-dimethylaminopropyl）carbodiimide（EDC）and N-hydroxysuccinimide（NHS）were used as cross-linking agents to prepare a hyaluronic-acid-adipate dihydrazide-poly（lactic acid）（HA-ADH-PLA,HAP）copolymer containing hydrophilic and hydrophobic blocks.It was characterized by ¹H-NMR,FT-IR and thermogravimetric analysis（TGA）,which proved that a new amide bond was generated at the connection site of the two substances,thus confirming the successful synthesis of HAP.Fe Cl₂?4H₂O was used as the iron source,and FeSe₂ nanoparticles were synthesized by hydrothermal method with selenium powder as the selenium source,and were characterized by transmission electron microscopy（TEM）and nanosizer.The results showed that the particle size of FeSe₂ was 5.62±1.32 nm and uniform distributed.X-ray diffraction（XRD）showed that the peak shape position,peak size and intensity of FeSe₂ diffraction peaks are consistent with the standard card.X-ray photoelectron spectroscopy（XPS）confirmed the presence of Fe and Se elements in FeSe₂NPs.In the high-resolution Fe 2p spectrum,two characteristic peaks of FeSe₂ are observed at 707.2 and 720.1 e V.In the high-resolution Se 3d spectrum,two peaks are observed at 54.8 and 55.6 e V,corresponding to the characteristic peaks of Se 3d_5/2 and Se 3d_3/2,respectively.The peak at 56.5 e V is attributed to the Se-Se bond.These results demonstrate the successful synthesis of FeSe₂.Using FeSe₂ as a fluorescence quencher,HAP-coated FeSe₂ core-shell structure nanoparticles were prepared by ultrasonic double emulsion method.By activating the carboxyl group on the surface of nanoparticles,a large amount of positively charged polyethyleneimine（PEI）was attached to the nanoparticles surface with a dense structure.Fluorescein isothiocyanate（FITC）was then attached to nanoparticles as a fluorescent marker to detect tumor cells,thereby preparing a complete fluorescent nanoprobe HAPPF@FeSe₂NPs.Energy-dispersive X-ray spectroscopy（EDX）demonstrated the presence of C,N,O,S,Fe and Se elements,and Element mapping further verifies the uniform distribution of various elements.TEM and DLS showed that the particle size of HAPPF@FeSe₂NPs was 32.67±7.55 nm.FT-IR demonstrated that PEI and FITC were successfully attached to the surface of nanoparticles.The nanoprobes should have good dispersibility,so we measured the average particle sizes of the nanoprobes in distilled water,phosphate buffer saline（PBS）and DMEM medium measured using DLS as 32.7 nm,32.6 nm and 35.1 nm,and the polydispersity index（PDI）are 0.35,0.35 and 0.37,respectively.（2）Before in-cell testing,the sensitivity and selectivity of the nanoprobes need to be tested in vitro.We tested the effects of p H,temperature,salt concentration and light irradiation time on the stability of FITC and fluorescent probes.The results show that FITC and fluorescent probes have good stability at p H 7 and temperature 24-36°C,and the fluorescence intensity will not be affected by high salt concentration and light exposure.The detection limit of the nanoprobe was determined to be 0.08 U/m L under the optimal conditions,with good sensitivity.A series of potential interfering substances were selected to investigate the selectivity of the nanoprobe,and the results show that the nanoprobe is specific for the detection of HAase.（3）As a nanostructure,the probe needs to be evaluated for its biocompatibility.The biocompatibility of nanoprobes was studied in terms of hemocompatibility and cytocompatibility in vitro.All the results indicated that the nanoprobes present better cytocompatibility and hemocompatibility.（4）The probe has excellent tumor detection ability through cell imaging and fluorescent labeling studies,and can be used as a real-time and efficient tumor detection probe.The synthesized HAPPF@FeSe₂NPs were co-incubated in He La cells and L929 cells under optimal conditions to observe the fluorescent signal of nanoprobes in detecting tumor cells.The results showed that the fluorescence was recovered in He La cells due to the presence of HAase in tumor cells,but no significant fluorescence signal was observed in L929 cells.This proves that the nanoprobe can be used as a probe to detect hyaluronidase in tumor cells.