Construction And Application Of Hyaluronidase Biosensors

As an indispensable part of the extracellular matrix,hyaluronic acid（HA）with different molecular weights and tissue concentrations can mediate many cellular events in some physiological processes,such as embryo morphogenesis,cell regeneration and migration,angiogenesis and wound healing,and play different physiological roles.The molecular weight and tissue concentration of HA are firmly regulated by the synergistic activity of biosynthesis and degradation processes.Therefore,the synthesis and degradation of HA have a significant impact on various physiological and pathological processes.The degradation of HA is mainly controlled by hyaluronidase（HAase）.Studies have shown that HAase is a potential tumor marker,and it is overexpressed in of certain cancer patients,hence the development of simple and sensitive method for HAase detection is significant for the clinical diagnosis and treatment of cancer.This thesis focuses on the construction of novel biosensors based on HA and the application for HAase detection.The project can be outlined as follows:1.A simple electrochemiluminescence（ECL）biosensor has been designed for HAase detection based on ECL composite materials with adjustable molecular weight.Ru（bpy）₃²⁺is adsorbed on HA through electrostatic interaction to form HA-Ru（bpy）₃²⁺complex as the ECL composite materials.In the presence of HAase,the HA in HA-Ru（bpy）₃²⁺can be cleaved into fragments by enzymatic hydrolysis,and high molecular weight HA-Ru（bpy）₃²⁺complex can be transformed into the low molecular weight HA-Ru（bpy）₃²⁺complex fragment.Therefore,the low molecular weight HA-Ru（bpy）₃²⁺complex fragment can be separated through centrifugal filtration easily.The resulting ultrafiltrate containing low molecular weight HA-Ru（bpy）₃²⁺complex fragment can be used to characterize the concentration of HAase in the sample.The ECL intensity has a linear relationship with the concentration of HAase in the range of2.0～40 U/m L with a detection limit of 0.33 U/m L.The proposed ECL system has been applied to detect HAase in urine samples and HAase inhibitor with high sensitivity and selectivity.2.A sensitive biosensor constructed by the reliable controlled release system and the mature ECL analytical technique has been devised for the quantification of HAase with high efficiency and selectivity.Ru（bpy）₃²⁺doped Si O₂ nanoparticles（Ru@Si O₂NPs）,as ECL signal probes,were trapped in the hydrogel fabricated by HA and polyethylenimine（PEI）evenly and steadily.When HAase existed,the hydrogel was decomposed by HAase,and the Ru@Si O₂ NPs escaped from the hydrogel into the supernate.Then the ECL signal produced from the supernate can be detected and used to characterize HAase concentration.The result showed a good linear relationship between ECL intensity and HAase concentration ranged from 2.0 to 60 U/m L,and the limit of detection was 2.0 U/m L.In addition,since PEI and HA can be replaced with target-responsive probes（such as double strand amino-modified DNA）for hydrogel fabrication,the hydrogel shows outstanding universality and extendibility which can be widely used to design controlled release ECL biosensors for various biomarkers’detection.3.A novel ECL biosensor on the basis of charge controllable nanoparticles has been devised to quantify HAase concentration.The Ru@Si O₂ NPs have been synthesized firstly.And then amino groups have been modified on the surface of Ru@Si O₂ NPs through APTES to synthesize Ru@Si O₂-NH₂ NPs that containing a lot of positive charges.Finally,Ru@Si O₂-NH₂ NPs have been modified by HA which has a large amount of carboxyl groups through amide bond,and then the Ru@Si O₂-HA NPs with a lot of negative charges has have been synthesized as the charge controllable ECL indicator in this study.The electrostatic repulsion was occurred between the Ru@Si O₂-HA NPs with a lot of negative charges and the ITO electrode surface that also has a lot of negative charges,and leading to a low intensity ECL signal.In the presence of HAase,the HA on the surface of the Ru@Si O₂-HA NPs can be decomposed,and therefore the Ru@Si O₂-HA NPs with a lot of negative charges can be transformed into Ru@Si O₂-NH₂ NPs with a lot of positive charges.Ru@Si O₂-NH₂ NPs can be concentrated nearby the surface of the ITO electrode through electrostatic attraction,and result an enhanced ECL signal,which can be recorded and used for HAase activity characterization.The result showed a good linear relationship between ECL intensity and HAase concentration ranged from 2.0to 60 U/m L,and the limit of detection was 2.0 U/m L.Furthermore,the proposed novel strategy has tremendous application potential in the assay of nucleic acid,aptamer,enzyme and any other biomarkers.4.A novel biosensor coupled with controlled release system has been designed for HAase determination using commonly reached electronic balance as readout without complex analytical instruments and skilled technicians.Pt@Si O₂nanoparticles（NPs）,which can catalyze the breakdown of H₂O₂ into O₂ and H₂O,was embedded in the hydrogel constructed by PEI and HA.In the presence of HAase,the hydrogel was broken down as HAase can catalyze the degradation of HA and hence the Pt@Si O₂ NPs in the hydrogel was released.The released Pt@Si O₂ NPs mixed with H₂O₂ solution in a drainage device,and then O₂ was generated due to the decomposition of H₂O₂,resulting in an enhancement of pressure in the drainage device because of the low solubility of O₂.A certain amount of H₂O was overflowed from the drainage device because the difference of the pressure between the inner and outer of the drainage device.The overflowed H₂O was collected by a tube and its amount was measured by electronic balance easily.The weight of the H₂O has a linear relationship with the HAase concentration in the range of 1.0～60 U/m L（120 min enzymatic hydrolysis time）and 0.20～10 U/m L（240 min enzymatic hydrolysis time）.The developed biosensor has been applied to detect the activity of HAase in urine samples,and the result shows that no significant difference between ELISA kit and proposed method.