Construction Of Biosensing Platform For Fungal Plasma Membrane And Its Derivatives Based On Functional Nucleic Acid Probes

Fungal plasma membrane and its derivative extracellular vesicles can reflect the physiological and functional status of fungi,and play an important role in intercellular material transport and information communication,which is one of the hot issues in current research.In recent years,membrane surface engineering technology,which can modify the surface of plasma membrane and extracellular vesicle membrane to achieve functional transformation and behavior control,has attracted the attention of many researchers.The covalent conjugation,electrostatic interaction and chemical modification methods commonly used in membrane surface engineering have serious biocompatibility problems,and also affect the biological activity.The functional nucleic acid probe composed of nucleic acid or nucleic acid like molecules has good biocompatibility and very low toxicity.It can recognize and sense the target in the measured sample and convert it into electrical signal or optical signal.In this context,this paper mainly constructed a functional nucleic acid probe which is relatively simple to synthesize,low cost,good stability and easy to modify,and used it in the anchoring and labeling of fungal plasma membrane and extracellular vesicles,so as to lay a foundation for the establishment of a general anchoring nucleic acid probe platform for fungal plasma membrane and extracellular vesicles.The main results are as follows:1.Construction of biosensing platform for Ganoderma lingzhi plasma membrane based on functional nucleic acidTAMRA perfluorocarbon nucleic acid probe(TPFN)which can anchor and label the plasma membrane of G.lingzhi was designed and synthesized to monitor the growth of G.lingzhi cell wall.Firstly,protoplasts of G.lingzhi were prepared.Introducing perfluorinated carbon chain,TPFN was synthesized by solid phase synthesis method.The preparation of TPFN was verified by NMR and MASS spectrometry.The excitation wavelength of TPFN was 560 nm and the emission wavelength was 575-700 nm.Flow cytometry detected that TPFN could be anchored to the protoplast,and strong TAMRA fluorescence was stimulated.Flow cytometry was used to detect the incubation of TPFN probe with protoplasts and cells of G.lingzhi.The results showed that TPFN probe could only anchor membrane of G.lingzhi protoplasts,which proved the specificity of TPFN.The process of protoplast growth cell wall was monitored by TPFN.The anchoring of protoplasts grown by TPFN at 0,12,24,36,48 and 60 h was measured.The results showed that,with the increase of incubation time,the cell wall of G.lingzhi gradually grew,and the exposed plasma membrane also gradually decreased,resulting in the decrease of the number of G.lingzhi protoplasts that can be bound by TPFN,corresponding to 61.5%,32.4%,30.4%,25.9%,7.25%and 0.031%respectively,The results of scanning electron microscopy also confirmed the process of cell growth.Therefore,the probe can be used as a new tool to monitor the cell wall growth of G.lingzhi.2.Construction of biosensing platform for Candida albicans plasma membrane and its derivatives based on functional nucleic acidTetrahedral DNA Nanostructure(TDN),which can be anchored and labeled on the plasma membrane of C.albicans,is synthesized to monitor the extracellular vesicles secreted by C.albicans.C.albicans protoplasts were prepared and the conditions of wall removal were optimized.The optimal conditions were determined as adding 7.5%β-mercaptoethanol(V/V),the amount of 2%snail enzyme twice the calculated value,and enzymolysis for 3 hours.The TDN probe was synthesized by combining the hydrophobic molecule cholesterol with the DNA tetrahedron nanostructure.The preparation of TDN nucleic acid probe was proved by non-denatured polyacrylamide gel electrophoresis.The excitation wavelength of TDN was 479 nm,and emission wavelength was 507-600 nm.Flow cytometry was used to detect the incubation of TDN and C.albicans protoplasts,and stimulate strong FAM fluorescence,which proved that TDN probe was feasible to anchor C.albicans protoplast;The incubation of TDN with Candida albicans protoplasts and C.andida was compared.The results showed that TDN probe combined with C.albicans protoplasts had higher fluorescence intensity,which proved the feasibility and specificity of TDN probe anchoring Calbicans protoplasts.Among them,TDN probe had the highest fluorescence intensity when binding to protoplast,which proved that TDN probe had high stability.Magnetic beads were used to enrich the extracellular vesicles of C.albicans to reach the detection limit of flow cytometry.TDN probe was used to monitor them.The results showed that TDN probe could be used as a tool to monitor the extracellular vesicles,and the combination of the two could maintain high fluorescence intensity and have stronger binding force.In this dissertation,two functional nucleic acid probes were constructed to anchor,lable and monitor the extracellular vesicles of fungal plasma membrane and its derivatives.TPFN probes can anchor protoplasts of G.lingzhi,and can be used as a new tool to monitor the growth process of G.lingzhi cell wall,which provides a new idea for molecular probes in the field of fungi.TDN probes can anchor and label C.albican protoplasts,and monitor the extracellular vesicles of C.albican,which provides a basis for the subsequent design of molecular probes to identify fungal plasma membrane and extracellular vesicles.