Thin Film Interference Effect Of Ordered Nanoporous Structures And Their Applications On Biosensing

Biomolecular interactions happen throughout the whole life activities,and real time monitoring of these processes has a broad application prospect in the fields of life science research,medical diagnosis,new drug development,agriculture and food production,etc.Label-free biosensing is very suitable for monitoring of biomolecular interactions because it can be used for real-time and in-situ analysis.Reflectometric Interference Spectroscopy(RIfS)is a label-free sensing method based on white light interference of a thin film which is used for monitoring interactions between biomolecules by observing optical thickness(the product of the refractive index and film thickness)of the film.RIfS has developed rapidly in recent years for its ability to do high throughput,low cost,simple and rapid analysis.Construction of interference effective substrates is the key part of RIfS sensing method and lots of research work has been made.The interference substrates reported at present all have inherent defects and deficiencies and new interference effective substrates need to be developed.Colloidal crystals are three-dimensionally ordered arrays of monodispersed nanospheres which include two type,opals and inverse opals.Much research has focused on the construction of photonic crystals.A systematic investigation of RIfS properties of colloidal crystals is presented in this paper for the first time and a class of new interference effective substrates with excellent performance has been developed.A RIfS sensing system based on the interference effects of colloidal crystals was constructed and the detection of trace enzymes and the kinetics analysis of enzymatic reactions were realized.The ability of the RIfS system to analyze biomolecular interactions in complex biological fluids was also explored.The ordered porous films with larger pore size and porosity were obtained by further optimization of the substrates.The above works provide new methods and ideas for the dynamic monitoring of biomolecule interactions.The main work of this paper is as follows:1.A simple approach for simultaneously assembling multiple silica colloidal crystal(SCC)films with the same quality was presented by the vertical deposition method,with some modifications and improvements.A systematic investigation into the evolution of the thickness and optical properties of SCC films formed under the changeable particle volume fraction and particle size was presented.It was found that the thickness of the film was positively correlated with the concentration of colloidal suspension,which was determined together by solvent evaporation and particle sedimentation.In addition,it was found that the standard deviation of the diameter of the particles affects the optical properties of the films along the growth direction through systematic study of different regions and diffraction peaks of the film2.A RIfS sensing system was constructed by combining a microscope with the traditional RIfS and the acquisition and recording of microregion reflectometric interference spectrum is realized.Furthermore,a systematic investigation into the RIfS properties of SCC films with different sphere diameters and film thicknesses in this work was also done for the first time.The influence of silica sphere diameter and film thickness on the RIfS signals was studied.Results showed that the film thickness is the key factor of RIfS signals.The influence of film thickness on response to refractive index changes of the proposed system was also investigated.Thicker film is more sensitive in responding to changes in the index of refraction.Finally,the optimized thickness of SCC film as the interference substrate was obtained,which was 4?6 m,and the film with this thickness was used as the interference substrate for subsequent sensing research.3.An in-situ,real-time and label-free sensing system of monitoring the digestive process of gelatin with trypsin was also demonstrated in this work by combining gelatin-modified silica colloidal crystal films with RIfS.The functionalized method was optimized,and the quantitative analysis of trypsin concentration was realized.The system showed excellent sensitivity with a wide linear range.Furthermore,the chemical selectivity of the sensor system was verified.Kinetic parameters of enzymatic reaction were also obtained since the digestion curve could be recorded in real time.4.In this part,the RIfS system combined with functionalized SCC films as interference effective substrates was used for real-time monitoring of the interaction between immunoglobulin G from human serum(hIgG)and protein A from staphylococcus aureus(SPA)within whole blood samples.The influences of SPA concentration,p H value and buffer con-centration on system response during the SPA functionalized stage were studied for the optimization of the experimental parameters in the stage.Quantitative analysis of hIgG in diluted whole blood samples were made,and the results were consistent with those obtained by a traditional immunoturbidimetry method,revealing the advantages of the acquisition of RIfS signals from the back of the film and the separation of components by porous structure in the analysis of complex samples.5.The ordered porous polystyrene films were prepared by using SCCs as templates,with the optimization of preparation method and the overall thickness of the film.In addition,the interference enhancement effect of TiO₂ layer was studied and the interference signals of the ordered porous polystyrene film were successfully enhanced by the TiO₂ layer.The resulting ordered porous polystyrene film can maintain the advantages of SCC film but has larger pore size,porosity and signal response,which is more suitable for the analysis of interactions between biomolecules,and also provides more possibilities for the application of the sensing system.To sum up,a new type of interference substrate has been developed based on the interference effect of colloidal crystal films,which has highly-ordered structure,good reproducibility,stable surface properties and advantages in complex biofluid analysis,promoting the further development and application of RIfS,and providing new ideas and more possibilities for real time monitoring of biomolecular interactions.