Design And Synthesis SERS Substrate And Fluorescent Probe For Detection Of Small Biomolecules And Its Application

The sensitive and specific detection of biomarkers is necessary for disease diagnosis.Surface-enhanced Raman scattering(SERS)technology has the advantages of nondestructive testing,“fingerprint”recognition capabilities,etc.,and can detect target analytes in a short time.It has been widely used as a highly sensitive analytical method for trace analysis detection,reaction monitoring,photoelectric catalysis,and other fields.SERS can be enhanced by localized surface plasmon resonance(LSPR)to induce molecular detection level Raman signal to achieve single-molecule detection,and its enhancement factor(EF)can reach 10¹⁴-10¹⁵.When the metal nanoparticle gap is less than 10 nm,a spatial region with a very strong electromagnetic field will be generated,i.e.,a“hot spot.”Studies have shown that the key to improving the SERS substrate is to increase the number of hot spots.Therefore,the synthesis of nanomaterials with good SERS activity is essential to obtain a relatively low limit of detection(LOD).SERS has shown great potential in the field of biomarker detection(for example,protein,amino acid,and nucleic acid),and it has received widespread attention.Based on the above viewpoints,in this study,we prepared a series of SERS substrates and used them to detect small biomolecules.Conversely,based on the high sensitivity and strong recognition ability of fluorescence spectrophotometry,Fe-MIL-88NH₂material was also prepared as a fluorescent probe to rapidly and sensitively detect Cys.RhB@MOF-808material was prepared as fluorescent probe to detect dopamine(DA)and Fe³⁺.(1)We proposed proposes a new type of dual spectroscopy based on the unique spectral response of aristolochic acid I(AAI)and aristolochic acid lactam(AAT)molecules,that is,the dual mode of SERS and fluorescence measurement(FLD)Next,monitor the human protein serum and AAI in A549 cells.AAI and AAT,two harmful substances widely present in aristolochic acid(AAs),have been shown to be related to nephrotoxicity.In order to better monitor AAI,we optimized the detection conditions,including the concentration of the analyte,the p H value of the reaction,different types of reducing agents and the reaction time.And explored the reaction of Fe²⁺reduction of AAI to generate AAT(with fluorescent signal),so that AAI can be detected by SERS or FLD.This method has been successfully applied to label AAI in cells.The toxicity of AAI was tested by CCK-8 experiment,and it was found that when the concentration reached 10?M,A 549 cells began to apoptosis,and the activity of A 549 cells decreased with the increase of AAI concentration.(2)SERS spectroscopy technology,liquid-mass spectrometry technology,and animal optics experiments were used to monitor the reaction of glutathione(GSH)and AAI,and the mechanism of GSH metabolizing AAI was explored.In the organism,AAI will form a positively charged AAT-nitrogen cation,which will increase the oxidative stress of AAI to cells,leading to cell death.GSH containing the-SH group can bind to AAI,thereby reducing exposure to AAI in the A 549 cells.The results of the SERS spectroscopy showed that with the increase in GSH,the characteristic peaks of AAI gradually disappeared,but no new peaks were generated.CCK-8 test results showed that cell activity increased after adding GSH,indicating that GSH can metabolize the toxicity of AAI,and the main pathway and mechanism of GSH metabolizing AAI were confirmed by high-performance liquid chromatography-mass spectroscopy(HPLC-MS).Further,using animal optics experiments,it was found that when AAI and GSH were injected into the abdominal cavity of mice at the same time,the fluorescence of the kidney organs of the mice increased,which proved that GSH can detect AAI in-vivo.(3)We synthesized the target probe Fe-MIL-88NH₂with amino functionalization to detect Cys.The preparation method of the probe is simple,low in cost,and can quickly and sensitively detect Cys under physiological p H conditions.Using X-ray diffractometer(XRD),Fourier transform infrared spectrometer(FT-IR),scanning electron microscope(SEM)and other techniques to characterize its structure and morphology,it proved that the target material was successfully synthesized.Through the study of spectroscopic properties,we found that Fe-MIL-88NH₂can be used to detect Cys in solution with a detection limit as low as 5.33?M.This molecule can also achieve visible light excitation“off-on”detection of Cys.And because of its low cytotoxicity and good cell membrane permeability,it can be used to label Cys in A 549 cells.In addition,a portable color card has been developed for real-time detection of Cys,which can realize the naked-eye recognition of Cys.Therefore,Fe-MIL-88NH₂,as a fluorescent probe compound for detecting Cys,has potential biological application value.(4)We constructed a RhB@MOF-808 probe,which can detect dopamine(DA)and Fe³⁺based on changes in fluorescence.XRD,FT-IR,SEM and solid-state fluorescence characterization proved that RhB@MOF-808 was successfully synthesized.The preparation method of the probe is simple,low in cost,and can quickly and sensitively detect DA and Fe³⁺under physiological p H conditions.Through the study of spectroscopic properties,we found that RhB@MOF-808 can be used to detect DA and Fe³⁺in solution,with detection limits as low as 60.25 n M and 48.34 n M.By designing a“molecular logic gate”to express the fluorescence“on-off”,it shows that the molecule can also achieve visible light excitation“on-off”detection of DA and Fe³⁺.The experimental results show that RhB@MOF-808 has good cell membrane permeability and has been successfully used to label DA and Fe³⁺in Hela cells.In addition,a portable color card for real-time monitoring of DA and Fe³⁺has also been developed,which can identify DA and Fe³⁺with the naked eye.Therefore,this RhB@MOF-808 has potential biological application value as a fluorescent probe compound for detecting DA and Fe³⁺.