Plasmon Enhanced Spectroscopy Methods And Applications In Single Living Cells Analysis

The interaction of light induced electromagnetic field with metallic nanomaterials can generate collective oscillation of the conduction electrons,commonly referred to as surface plasmon resonance(SPR).This property of metal nanoparticle has drawn much attention in biosensing and single molecule imaging applications.Scattered light from the plasmon nanoparticles can be easily observed at the single particle level using dark field microscopy,so that single particle spectroscopy can be used for both qualitative and quantitative analysis of biological events in vitro and in vivo.Surface enhanced Raman scattering(SERS)is an ultra-sensitive vibrational spectroscopy technique and greatly expands the application of Raman spectroscopy.SERS offers significant advantages in biomedical applications:ultra-high sensitivity up to single molecule level,flexible excitation wavelength,hyperspectral resolution for multiplexed detection,no damage to biological samples,no background signal interference,no quenching or photobleaching,etc.In this dissertation,biomolecule recognition and enzyme kinetics were studied by plasmon resonance energy transfer based spectroscopy using plasmonic nanoprobes,and the quantitative determination of intracellular biothiols,the kinetics of drug action within cells,and the changes of intracellular pH were investigated using plasmon enhanced Raman spectroscopy.The main research contents are as follows:1.Plasmon resonance energy transfer based spectroscopy on single nanoparticles:biomolecular recognition and enzyme kineticsThe small absorption cross-sections of most molecules led to the low sensitivity of traditional optical absorption spectroscopy.This obstacle might be overcome by applying the near-field plasmon resonance energy transfer(PRET)between plasmonic nanoparticle and surrounding molecules.In this work,we utilized PRET based spectroscopy on single gold nanostars to study the specific biomolecule recognition and enzyme kinetics choosing biotin-SA pair and DNase I as models.By analyzing the changes of absorption spectra for black hole quencher 3(BHQ3),derived from spectra difference,we explored the kinetics of specific biomolecule recognition and enzyme digestion in different physiological environment,and found that the viscosities of media and the sizes of molecules play vital role in biomolecular recognition and enzyme digestion.Compared with the traditional optical absorption spectroscopy techniques,PRET based spectroscopy offers a nanoscopic resolution owing to the small size of the probe,is more sensitive achieving detection on the order of hundreds or even dozens of molecules,and can achieve high selectivity due to the specific biomolecular recognition.This method might be used in the fields of molecular diagnostics,drug discovery and clinical diagnostics.2.Simultaneously quantify multiple endogenous biothiols in single living cells by plasmon enhanced Raman spectroscopyIntracellular biothiols mediate many important physiological and pathological progresses.Due to the low content and their competing thiol-reactivity,it is still an unmet challenge to quantify them within complicated intracellular environment.Herein,we demonstrated a strategy to discriminate three biothiols,i.e.cysteine(Cys),homo-cysteine(Hcy)and glutathione(GSH),and quantify their concentrations within single living cells,by using one platform of Raman probe.By monitoring the reactive kinetics of biothiols with Raman probes,and discriminating their products with a quantitative principal component analysis(qPCA)method,these three biothiols could be simultaneously quantified in both cell lysis and single living cells.The concentrations of Cys,Hcy and GSH in single Hela cells were 158±19?M,546±67?M and 5.07±0.62 mM,respectively,which gives the precise concentrations of these three biothiols at single cell level for the first time.This method provides a general strategy of discriminating each component from a mixed system,and has potentials for quantifying any biomolecules within in vitro or in vivo biological environment.3.Revealing chemical processes and kinetics of drug action within single living cells via plasmon enhanced Raman spectroscopyBetter understanding the drug action within cells may extend our knowledge on drug action mechanisms and promote new drugs discovery.Herein,we studied the processes of drug induced chemical changes on proteins and nucleic acids in MCF-7 cells via time-resolved plasmon enhanced Raman spectroscopy in combination with principal component analysis(PCA).Using three popular chemotherapy drugs(fluorouracil,cisplatin and camptothecin)as models,chemical changes during drug action process were clearly discriminated.Reaction kinetics related to protein denaturation,conformational modification,DNA damage and their associated biomolecular events were calculated.Through rate constants and reaction delay times,the different action modes of these drugs could be distinguished.These results may provide vital insights into understanding the chemical reactions associated with drug-cell interactions.4.Monitoring the changes of pH in lysosomes during autophagy and apoptosis by plasmon enhanced Raman imagingLysosomes are acidic organelles that not only participate in intracellular degradation but also participate in various other cellular functions.The abnormal pH in the lysosomes will lead to lysosomal dysfunction,resulting in many human diseases.In this work,we prepared a simple plasmon Raman probe to statistically analyze the pH change in the lysosomes by using SERS imaging technique.Incubation and depletion method was used to localize the plasmon Raman probe to lysosomes.Autophagy and apoptosis of HeLa cells were induced by nutrient deprivation and hydrogen peroxide.Changes of pH values in the lysosomes before and after autophagy and apoptosis were analyzed in situ by SERS imaging.The pH in lysosomes during the process of autophagy decreased slightly,while the pH in lysosomes increased during apoptosis,which are consistent with the activities of the cell itself.The preparation method of the Raman probe is simple,and the statistical analysis of the pH in the lysosomes is performed by means of SERS imaging,indicating that the method has high operability and reliability.It is important approach for early diagnosis and treatment of the diseases caused by the changes of the pH in the lysosomes.