Molecular Structure Analysis Of Polysaccharides Based On Solid-state Nanopore Technology

Saccharides is one of the four life building biomacromolecules that exist in all living things.It has important biological significance and attracted extensive research interest.Plants account for more than 80% of the total biomass of the earth,and cell wall--a special structure of plant cell--is 50%-90% made of polysaccharide.Therefore,the structure and function of plant polysaccharides play an important role in regulating plant growth and development.However,traditional methods for detecting the structure of polysaccharides have certain limitations and cannot be applied in many situations,thus it has been a great challenge to reveal the structural properties of polysaccharides,and generally effective detection methods have been elusive.This thesis introduces a solid-state nanopore based detection method for polysaccharide molecular structure determination.In this,we use a new nanopore fabrication technique,dielectric current breakdown,to prepare the nanopore for the study.Compared with several prevalent preparation methods,this method has several advantagas such as simple operation,low cost and high preparation speed.Solid-state nanopores with stable properties and low noise can be fabricated on silicon nitride thin films by using an external power supply(Keithley 2450)with adjustable current pulse and software control in a LabView environment.A controllable and automatic electro-drilling system is built,which can not only make stable preparation of solid-state nanopore by artificial intervention at any time during electro-drilling,but can also measure and record the drilling process and associated parameters.Next,acetylated-modified plant polysaccharides were detected by using the prepared solid-state nanopore sensor.The results show that the method is suitable for the identification of plant polysaccharides with different geometry and chargeproperties.The device can recognize the cell wall xylan at a concentration as low as5ng/?L,and can distinguish the xylan modification of acetylation and deacetylation.The identification of arabinoxylans and glucuronic acid xylooligosaccharides in monocots and dicotyledones was further demonstrated by experiments.A single-molecule polysaccharide analysis platform was established to process the characteristic data of polysaccharides,which opened up a new way to understand the structure of cell wall and the application of polysaccharides.Finally,in order to enhance the specific binding between solid-state nanopore and polysaccharide analyte,chemical modification experiments of nanopore were carried out.The nanopores were activated and modified by silane,then the amino acid molecules were fixed on the surface of the nanopores.The modified and bare nanopore were used to detect the samples,and the differences of the signals of the oligosaccharides and polysaccharides with different chain length were studied.The properties of polysaccharides were further studied by using solid-state nanopores,starting with viscosity and structure.It was demonstrated that the translocation signal could identify the arabinoxylan with different viscosity and the xylan with different spatial folding structure.At present,the use of solid-state nanopore to detect the sugar-related mechanism is mostly used for the sugar consumed and contained in the human body.To date,solid-state nanopore has not been applied to the structural identification of plant polysaccharides.The main innovation of this thesis is to use solid-state nanopore as sensor to identify the structure of plant polysaccharides and to study the acetylation of plant polysaccharides.The solid-state nanopore sensor,which matched the molecular size of polysaccharides,had high spatial resolution and could be used to identify the modified groups in plant polysaccharide chains.The results presented here provide a basis for further study on the effects of modified groups on plant growth and development.Compared with other existing detection methods,this method is lable-free,low cost,and highly sensitive.Solid-state nanopore can be used to detect polysaccharides with intact molecular structure,which makes up for some shortcomings of existing detection methods,andcan be combined with classical detection methods to provide a deeper understanding of the structure of polysaccharide at the molecular level.As such,this work provides a theoretical foundation and experimental technique for the development of solid-state nano-porous single-molecule sensors in the field of carbohydrate structural analysis.