Nickel Nanowires Combined With Raman Spectroscopy:Application In Investigation Of Cytochrome C-Mediated Apoptosis

Apoptosis is one of the research hotspots in the life science,understanding the mechanism of apoptosis is helpful for the prevention and treatment of major diseases.Cytochrome c?Cyt c?is a heme protein that plays an important role in cell apoptosis.On the other hand,surface enhanced Raman spectroscopy?SERS?has shown great application potential in the field of protein detection in recent years.In this thesis,the interactions betweenredox state-dependent Cyt c and phospholipid membranes were studied by the combination of nickel nanowire?Ni NWs?and Raman spectroscopy?resonance Raman and SERS?,and a label-free SERS-based method for rapid detection of apoptotic cells was established.The main research content andresults are as follows:1.Synthesis of Ni NWs and its specific reduction activity for Cyt c?1?Ni NWs with different particle sizes were prepared by the hydrazine hydrate reduction method and its specific reduction activityforCyt c were discovered.The particle size,temperature and PH dependence of the reduction reaction were also found.?2?The selectivity of the Ni NWs for reducing heme proteins was investigated,and the specific reduction activity for Cyt c by Ni NWs was found.During the reaction,the electrons directly transferred from theNi NWs to the Fe³⁺Cyt c,meanwhileNi ions were generated.2.The interaction between Cyt c and Cardiolipin membrane was studied by Ni NWs and resonance Raman spectroscopy?1?CL and PC/PE/CL mixed liposomes were obtained by spin distillation.The interaction between Cyt c and mitochondrial membrane PC/PE/CL mixed liposomes was investigated by resonance Raman spectroscopy.It was found that the interactions between the PC/PE/CL phosphlipid membranes and Cyt c were dependent on the redox state of Cyt c.?2?The peroxidase activity induced by the binding of the phospholipid membrane toCyt c was investigated by UV-Vis spectroscopy,and it was found that the peroxidase activity of Fe²⁺Cyt c-phospholipid membrane complex was significantly higher than that of the Fe³⁺Cyt c-phospholipid membrane complex,indicating that Fe²⁺Cyt c may play a more important role than Fe³⁺Cyt c in the initiation stage of cell apoptosis.3.The combination of Ni NWsand SERS to detect the release Cytcinapoptotic mitochondria?1?HeL a cell apoptosis was induced by actinomycin D and mitochondria were isolated.Ni NWs and resonance Raman spectroscopy were combined to detect Cyt c released from mitochondria of apoptotic cells,and the relative intensity change of the characteristic peak at 750 cm^-1 was used to determine the content of Cyt c with two redox states.?2?Silver?Ag?nanoparticles were introduced into the detection system.It was found that Fe²⁺Cyt c changed into Fe³⁺Cyt c after interaction with Ag,and the total amount of releasedCyt c can be quantified by SERS.Through the Raman signal enhancement,the limit ofdetection was greatly improved,thus we established a label-free SERS-baseed method for the rapid and direct detection of the total amount of released Cyt c from mitochondria of apoptotic cells.