A Novel Fluorescent Nanoprobe For Intracellular Imaging Analysis Of D-amino Acids

D-amino acids(DAAs)tend to be present at very low levels in biological systems compared to L-amino acids.Due to the lack of sensitive and accurate chiral analysis and detection technology of amino acids in the early stage,it was difficult to detect naturally occurring DAAs,which make DAAs were considered as "non-natural amino acids" for a long time.In recent years,with the development of chiral analysis and detection methods,DAAs have been found play a significant role in food(fermented products,dairy products),various microorganisms,and the human central nervous system(cerebral cortex,hippocampus and striatum,pituitary)at high levels.In food,content level of DAAs can be used as an index to evaluate the quality of food and the production process.For example,the DAAs found in vinegar and wine can reflect the year of preparation,storage status,degree of maturity,as well as microbial contamination.In the human body,as an important co-agonist of NMDA receptors,DAAs are associated with human cognitive,learning and memory processes,which are recognized as an important biomarker and one of the potential therapeutic of some central nervous cognitive diseases such as schizophrenia and Alzheimer's disease.Therefore,more accurate,sensitive,and rapid detection and analysis of trace amounts of DAAs could be developed by further exploration of detection methods for DAAs,which will help to further reveal its subcellular localization and disease correlation in human body,and also provide new ideas and strategies for enantioselective detection and analysis of chiral amino acids.At present,DAAs analysis means of commonly used is mainly based on high performance liquid chromatography,gas chromatography and capillary electrophoresis which are rely on large equipment,while these methods have high sensitivity,but also have the disadvantages,such as time consuming operation,sample preparation need professional operation personnel and cannot be achieved in living cells(intracellular)and living(in vivo)monitoring.Enzymatic method is a kind of high enantioselective analysis method that utilizes D-amino acid oxidase(DAAO),which realizes the selection of DAAs by monitoring the products of DAAO enantiomerically catalyzed oxidation of DAAs qualitative and quantitative analysis.However,none of DAAO-based DAAs analysis methods can achieve in-situ dynamic imaging monitoring for living cells or living organisms.This is mainly due to the use of macromolecule DAAO in enzymatic method,which does not possess the ability to penetrate the cell membrane,and DAAO itself cannot produce highly sensitive detection signals such as fluorescence.Therefore,we have taken a new path through the strategy of supermolecule nano-self-assembly,designed and constructed a new type of fluorescent nanoprobe to realize the live cell imaging analysis of DAAs.Specifically,we use bio-friendly,optically transparent hollow mesoporous silica nanoparticles(HMSN)as the framework of DAAs nanoprobes,and the internal cavity of HMSN is loaded with the rate-sensitive fluorescent small molecule probe PN1 which is response to hydrogen peroxide,while the surface of HMSN is modified with DAAO through a supramolecular assembly process such as electrostatic adsorption.The overall size of the nanoprobe is about tens to hundreds of nanometers,which means it has a good ability to be taken up by cells.In the presence of DAAs,DAAO on the surface of the nanoprobe will selectively oxidize DAAs and produce the same amount of hydrogen peroxide in an equal stoichiometric ratio.And then the small hydrogen peroxide molecules will further act on the PN1 fluorescent probe molecules in the probe cavity and produce ratiometric fluorescence signals.The main work of this paper is divided into the following four parts:(1)Firstly,HMSN was prepared in three steps by the improved St(?)ber method,and then the ratio fluorescent small molecule probe PN1,which was selectively responsive to hydrogen peroxide,was loaded into the internal cavity of HMSN.Finally,the DAAO from pig kidney(pk DAAO)was coated on the surface of HMSN with the electrostatic adsorption and other supramolecular assembly modification effects between the modified amino functional group on HMSN,and finally the DAAs specific fluorescent nanoprobe DAO was successfully constructed.Subsequently,various instruments were used to characterize the properties of the constructed nanoprobe material.The experimental results show that the nanoprobe we constructed is a hollow spherical particle of uniform size,with an average hydrated diameter of about 130 nm,and an uniform mesoporous structure with a size of about 3.377 nm.The results of thermogravimetric analysis showed that the loading amount of PN1 small molecules in the internal cavity and the coating amount of DAAO on the surface of the nanoparticles were 11.91 wt% and 3.51 wt %,respectively.(2)Specific detection activity of DAO@HMSN-PN1 nanoprobe against DAAS was investigated in solution.The spectra of the nanoprobe before and after the addition of Dalanine(D-Ala)were analyzed by UV-vis absorption spectroscopy and fluorescence emission spectroscopy,which the results show that the nanoprobe has a sensitive ability to recognize DAAs.In the experiment where the nanoprobe responded to 13 different DAAs,the probe showed different affinities for different types of DAAs,except for two acidic AAs,D-aspartic acid and D-glutamic acid,as well as D-tryptophan.What's more,the nanoprobe show specific detection and analysis capability for DAAs in the presence of the signal interference of LAAs.Finally,the DAO@HMSN-PN1 nanoprobe was responded to different concentrations of DAAs in the solution,showing excellent quantitative analysis capabilities.(3)The normal human liver cell L02,liver cancer cell Hep G2 and human cervical cancer cell He La were selected as experimental cell lines to investigate the imaging and analytical ability of the intracellular DAAs of the nanoprobe.Firstly,results of cell viability experiments show that nanoprobes have low toxicity to cells.Secondly,the nanoprobe was covalently labeled with rhodamine B(Rh B)to obtain DAO@HMSN-Rh B,which was used to investigate the uptake and positioning of the nanoprobe material in the cell.Fluorescence imaging of the laser scanning confocal microscope fluorescence imaging figure showed that the nanometer can be evenly distributed in the cytoplasm after the probe enters the cell,which provides a basis for the application of intracellular imaging analysis application.Finally,the cells containing the nanoprobes were incubated with different concentrations of D-Ala.Fluorescence microscope imaging showed that the nano-probes constructed in this study had the ability of specific imaging analysis of intracellular DAAs.In this paper,mesoporous silicon nanomaterials were combined with the detection and analysis of DAAs for the first time.The D-amino acid specific fluorescent nanoprobe was constructed based on DAAO enzymatic method,which was successfully applied to the specific imaging analysis of intracellular DAAs,filling the gap in the detection and analysis of DAAs in this aspect.This strategy is the first to realize the high-chiral selective fluorescence imaging analysis of DAAs in living cells under the interference of a far excess of interfering L-amino acids,which can provide an effective new method for the research of the biological functions of DAAs in the relevant frontier.