Detection Of Plant Small Peptide Ligand-Receptor Pairs Based On Gold Nanohole Arrays

Cell-to-cell signaling is essential for many processes in plant growth and environmental adaptation.Small peptides,as an important class of signaling molecules,bind to the extracellular domain of the plasma membrane receptor RLKs,and the ligand-receptor interaction activates a range of biochemical and physiological processes.Identification of these ligand-receptor pairs is crucial to advance our understanding of cell-cell communication during plant growth and development.The Arabidopsis genome encodes approximately 1,000 small peptides and more than 600 receptor-like kinases,but only a few ligand(small peptide)-receptors(receptor-like protein kinases)pairs have been identified.The identification of peptides-RLKs is technically very challenging,as the genes encoding them regularly belong to gene families with multiple members and are often low expressed in certain specific cells or during specific developmental stages.In addition,the majority of the peptides and RLKs do not have a known function and gene redundancy is widespread,limiting the application of traditional genetic methods.In recent years,some new methods have been reported for the identification of unknown receptor-ligands,but most of these methods require radioactive or fluorescent labeling,and the experimental process is cumbersome.Therefore,it is urgent to develop a sensitive and convenient technique for identification of small peptide ligand-receptor pairs.With the development of nanotechnology,nanomaterials based sensors are playing important role in biochemical detection.In particular,metallic nanohole arrays SPR sensors have attracted more attentions in recent years.It has the advantages of lable-free,high sensitivity,good biocompatibility.In particular,the detection device is simple,which overcomes the shortcomings of the traditional SPR instrument,thus,has been widely used in the biomedical applications.In this thesis,large-area ordered periodic gold nanohole arrays were prepared on glass substrate and in flexible matrix by nanosphere lithography(NSL)and nanoimprint one step method respectively.Low-cost NSL is suitable for large-scale preparation of nanohole arrays.The proposed one-step method has the advantages of high precision,and is easy to operate,thus,has the ability to take into account both structural properties and fabrication throughput.Compared to NSL,nanohole arrays prepared by one-step method exhibit higher uniformity and more stable optical properties.A SPR detection platform based on gold nanohole arrays was constructed to detect biomolecule-specific binding in three different reaction systems.The biosensor not only has an effective SPR response to Biotin-Streptavidin and Ig G-anti-Ig G binding,but also can detect the binding of ABA to its receptor PYL1 by ABA-dependent PYL1-ABI2 specific reaction.The gold nanohole arrays SPR sensor can be reused by RIE(Reaction ion etching)treatment,and the results of the three-cycle detection experiment show good repeatability.The gold nanohole arrays based SPR platform was used to detect the known peptide ligand-receptor pairs.The extracellular domain of RLKs CLV1,FER and the small peptide CLV3,RALF were expressed and purified by using E.coli prokaryotic expression system.The expressed or synthesized small peptides were immobilized on the surface of the gold nanohole arrays.The binding of the peptides to the corresponding receptors was detected by the shift of the plasma resonance transmission peak.Multiple receptor-like protein kinases were mixed and reacted with CLV3.It was found that the nanohole array SPR platform was able to detect the specific binding of CLV1 to CLV3 from the mixture containing ten kinds of RLKs.In contrast,no SPR response was obseverd in the control group.These results indicate that the nanohole arrays SPR sensor has high specificity for the detection of peptide ligand-receptor.Maltose modified gold nanoparticles were also introduced into the detection system for signal enhancement in this study.The RLK in solution to be tested was captured onto NHA surface by ligand-receptor specific binding,and the gold nanoparticles were then coupled to the NHA by the interaction of maltose on the surface of the nanoparticles and MBP in the RLK fusion protein.The SPR coupling of the gold nanoparticles and NHA enhances the SPR response of ligand-receptor binding by four times.The control experiment showed that the gold nanoparticles could not be coupled to the NHA surface for signal amplification if the solution to be tested did not contain the receptor-like kinase that specifically bound to the peptide on the NHA surface.The signal enhancement system with a detection sensitivity up to 1 p M does not require labeling of gold nanoparticles and is suitable for the detection of all RLKs in this system.Four synthetic peptides were used to screen receptors,in which SPR signal was detected when CLV3 reacted with two groups of RLKs mixture.Gold nanoparticles were further introduced,however,no signal enhancement was observed.The results of positive and negative control experiments further proved that the detected signals were not caused by the ligand-receptor specific binding.These results indicate that the gold nanoparticle based signal amplification strategy also has the capacity of distinguishing non-specific binding signals.In summary,the periodic gold NHA detection platform shows effective SPR response to biomolecular interactions,and can be applied to the detection of plant peptide ligand-RLK specific binding.Gold nanoparticles can enhance the signal and improve the specificity of detection.