Protein Immobilization On Nano Plasma Sensors And The Application In The Detection Of Receptor Like Kinases

Receptor-like protein kinases(RLKs)are the largest Arabidopsis family proteins.They bind to ligands and participate in all processes of plant growth and development.At present,only a few RLKs ligands have been discovered.The ligands,functions and mode of action of most RLKs are still unclear.Matching of receptor-like kinases and ligands is of great importance in the study of plant signaling pathways.Surface plasmon resonance(SPR)is a physical optical phenomenon.Due to its unique extraordinary optical transmission(EOT)effect,the surface plasmon resonance technology based on periodic metal nanostructures has the advantages of simple detection device and convenient operation,and has been widely used in the fields of medical diagnosis and drug screening.The immobilization efficiency of biomolecules on the surface of the sensor is one of the main factors that affect the performance of the sensor,and plays a very important role in the application of the sensor in the detection of biomolecules.This subject uses different modified substrates to functionally modify the surface of periodic nanostructured SPR sensors,and investigate the effect of different modifiers,modifiers,and functional group concentration on the efficiency of protein immobilization.The optimal protein fixation strategy was selected and applied in the detection of receptor like protein kinase.The main results are as follows:1.Study on the immobilization efficiency of protein on the sensor surface.Firstly,the surface of the sensor chip was functionalized with mercaptoundecanoic acid(MUA)and poly(Ethylene glycol)SH-PEG-COOH by SAM(Self-assembly monolayer)method.The IgG protein was used to study thenon-specific adsorption of protein on sensor surface.The carboxylated chip which activated by EDC and NHS was used as the experimental group.The chip used as adsorption control group was not activated.By detecting the SPR peak shif in transmitted spectrum,it was found that the PEG-functionalized chip has better anti-nonspecific adsorption effect than the MUA-functionalized chip.For the PEG functionalized chips,as the alkyl chain grows,the nonspecific adsorption capacity gradually increases,but the immobilization efficiency decreases.Then the effect of functional group concentrations on the immobilization efficiency was studied.Self-assembled mixed films containing long-chain PEG2000-COOH and short-chain PEG400-OH on the chip surface was constructed.The proportion of PEG2000-COOH/PEG400-OH are 1:9,1:6,1:3,1:1,3:1,6:1,9:1.The results show that when the ratio of carboxyl group to hydroxyl group is 1:6,the maximum shift of SPR peak is observed after protein immobilization.The surface of the chip was analyzed by energy dispersive spectrometer(EDS).The results show that the N element content on the chip surface is the highest when the ratio is 1:6.At the same time,GFP fluorescent protein and PS microspheres with streptavidin were used for co-identification.When the ratio of carboxyl group to hydroxyl group is 1:6,the distributions of fluorescent proteins on the chip surface were the most.The same results have been obtained for the distribution of PS microspheres.For the two-dimensional modified matrix formed by MUA and PEG on the sensor chip,the MUA functionalized chip has a high efficiency but poor ability to resist protein nonspecific adsorption.The surface anti protein nonspecific adsorption effect of the PEG functionalized chip is good,but the immobilization efficiency is relatively low.Therefore,on the basis of the PEG SAM,?-cyclodextrin is further functionalized to form a three-dimensional surface modified matrix.It was found that the three-dimensional modified matrix is superior to the two-dimensional modified matrix in both the immobilization efficiency and the anti-protein non-specific adsorption effect.2.Expression of the extracellular domain of the receptor-like protein kinase using a prokaryotic expression system.A pair of known receptor-ligands(FLS2 and flg22)was selected to study the practicability of the nanostructured SPR sensor for the detection of RLKs.It has been reported that receptor-like protein kinases bind to ligands in their extracellular domains.Therefore,this study selected FLS2 extracellular domain(24-806 AA),FER extracellular domain(28-446 AA)and Ralf 72-120 AA protein expression.A vector constructed with a His tag or a GST tag was failed to express the target proteins.Analysis of the amino acid sequences of FER and FLS2 shows that the extracellular domain of these proteins has a large number of hydrophobic amino acids.Therefore,the pSmart vectors with MBP and SUMO label were used for protein expression.3.Application of cyclodextrin-functionalized nano-SPR sensor chip in the detection of RLKs.The small peptide flg22 was immobilized on the surface of the nano SPR sensor.After reacting with FLS2,the SPR peak shifted about 3.5 nm.However,no significant shift of SPR peak was detected in the control group with small peptide RALF and FLS2.In another group of controlled experiments,FER,instead of FLS2,was reated with the surface of the sensor coupled with flg22,no shift of SPR peak was observed.These results show that the cyclodextrin-functionalized nanostructed SPR sensor can be used to detect the specific binding of receptor-like protein kinase and its ligand.