Research Of The Interaction Between Blood Proteins And Drugs Based On The Interference Effect Of Colloidal Crystal Films

The blood contains a variety of rich proteins,such as human serum albumin（HSA）,immunoglobulins（Ig）and fibrinogen（Fibrinogen,Fg）can maintain osmotic pressure and drug transportation,immune protection,blood coagulation and thrombolysis,respectively.Drugs will interact with proteins in a series of ways,surface plasmon resonance（SPR,Biacore）and bio-layer interferometry（BLI,Fortebio）are relatively mature techniques for biomolecule interaction analysis at present.Nonetheless,due to the limitation of the substrate,the above two methods are insufficient in the analysis of the interaction between blood proteins and drugs.Ordered porous layer interferometry（OPLI）is a label free technology for the kinetic monitoring of biochemical reaction process high throughput,low cost,simple and fast analysis through replacing the silica colloidal crystal film sensing substrate.In this thesis,a variety of the diameter of the three dimension silica colloidal crystal film were prepared;orderly porous layer interference measurement system was optimized;the adsorption behavior of three blood proteins was studied;the interaction characteristic between small molecular drugs and human serum albumin method were established and the kinetic was analysis;the application of thrombolysis detection was expanded based on silica colloidal crystal film as the thrombus model"microscaffold".The main work of this paper is as follows:1.A batch preparation of silica colloidal crystal film were realized by"vertical deposition method".Combining the microscope with the ordered porous layer interferometry system,a micro-ordered porous layer interferometry system was constructed.The photonic bandgap and interference peak were compared to calculate the detection sensitivity of the photonic crystal sensor from both theoretical and experimental aspects with the silica colloidal crystal film as an example.The theoretical calculation showed that optical thickness features had a lower detection limit in the detection sensitivity of photonic crystal sensor.The experimental results show that optical thickness had smaller relative error,better linear relationship and lower detection limit compared with the photonic bandgap with periodic characteristics of photonic crystals.2.The adsorption behavior which is protein corona formation process of three representative blood proteins on the surface.The results showed that the three proteins had different adsorption processes and optical parameters.The process of protein adsorption of HSA on Si O₂ surface with three different surface curvature was studied.The results showed that the larger the curvature,the faster the adsorption of protein,the greater the thickness.This method can be directly used to monitor the protein adsorption process of plasma and whole blood on the surface of nanoparticles from the results,which provided the basis for the subsequent study of the interaction with drug molecules on the adsorption characteristics of blood proteins.3.HSA is one of the most important transport carriers of small molecule drugs in vivo,and has a very important affinity relationship with drugs.In this thesis,a real-time method for detecting the affinity between HSA and indomethacin was established by ordered porous layer interferometry and silica colloidal crystal films.Firstly,HSA was modified on silica colloidal crystal films by covalent binding method.And the adsorption and desorption process of indomethoxin with HSA was evaluated by optical thickness change as a parameter in real time.Finally,the most suitable silica colloidal crystal film was selected,and the method was verified by different affinity drugs.The kinetics of the above drugs and HSA were analyzed by real-time adsorption and desorption.4.A new thrombolysis detection method in situ was developed by using silica colloidal crystal film as the framework,loading fibrin and preparing interference thrombus model.The process of thrombolysis was studied in real time by monitoring the optical thickness changes resulting from the dissolution of fibrin on the loaded film and providing kinetic data.The whole thrombolytic process was recorded and compared with the fibrin plate method from nattokinase,urokinase and lumbrokinase as thrombolytic drug models.This method has good sensitivity to nattokinase,urokinase and lumbrokinase,and can be used as a real-time,low-cost and rapid detection system for drug thrombolysis detection system.