Electrochemical Method-SERS Coupling Detection Technique For Monitoring Exocytosis Of Single Cell

Cellular exocytosis is a ubiquitous and highly conserved process that cells communicate with each other.Neurons transmit information by the way of exocytotic process in which synaptic vesicles at the presynaptic membrane release neurotransmitters.This process precisely regulates the normal operation of the nervous system.Therefore,studying neuronal exocytosis is not only helpful for illustrating the mechanism of exocytosis,but also for researching the treatment of neurological diseases by detecting the amount and kinds of the secreted neurotransmitters.Electrochemical amperometry is the main analytical technique for detecting single cellular exocytosis.It can be used to detect membrane fusion and neurotransmitter release in the late stage of cellular exocytosis.Due to its ultra-high temporal resolution,the dynamic mechanism of vesical fusion can be deduced.Meanwhile,its high sensitivity enables quantitative analysis of the amount of the neurotransmitters.However,it is impossible to fully analyze the neurotransmitters released when only amperometry is used.On one hand,it is difficult for electrochemical method itself to detect substances without electrochemical activities;on the other hand,it is difficult to distinguish substances with similar electrochemical activities by amperometry.Neurotransmitters include catecholamine,acetylcholine,amino acids and polypeptides,etc.The differences in molecular structure result in their different molecular vibrational spectral fingerprints.Surface-enhanced Raman spectroscopy(SERS)provides fingerprint vibrational information of the analytes that can be used to identify various neurotransmitters.Moreover,it has high detection sensitivity and in situ detection capability.Coupling SERS and electrochemical methods for real-time monitoring neuronal exocytosis,a variety of neurotransmitters can be detected and the correlation between them would be explored.Considering the question and challenges above,this thesis established a novel technique in combination of electrochemical method and SERS that is capable of simultaneously obtaining electrochemical and Raman signals from a single cellular exocytotic process.The main current contents and results are list as follows:1.Based on the single cell electrochemical microscopic operating system we have,we coupled a Raman instrument with it by adding Raman optical path into the optical path of the inverted microscopy,and inducing exciting light and collecting Raman scattering light via optical fiber.Thus,we established single cell electrochemical-Raman coupling system.The electrochemical detection part of the combined system has a current noise of 4 pA that is up to exocytotic spike peak(20～50 pA)and a signal collection frequency of up to 5000 Hz,which satisfies the requirements of small current and rapid detection.The signal-to-noise ratio of the Raman system is slight lower than that of a commercial Raman instrument,but its resolution is higher.The single cell electrochemical-Raman coupling system can be used for the study of cellular exocytotic process to acquire electrochemical and Raman signals simultaneously.2.SERS active substrate was constructed on the surface of 10 μm carbon fiber electrode by electrodeposition method.The strategy we applied for deposition was rapid nucleation in high-concentration gold plating solution under high over-potential and slow-growth in low-concentration gold plating solution under low over-potential to narrow the gap between nanoparticles.Through optimizing the conditions,the electrode was deposited at-0.4 V in 5 mM HAuCl4,0.5 M H2SO4 solution for 3 s to nucleate gold nanoparticles,and at 0.75 V in 0.1 mM HAuCl4,0.5 M H2SO4 solution for 300 s to grow the nanoparticles.After deposition,the microprobe obtained maintained its electrochemical activity and had a SERS activity similar to that of the assembled one.3.Considering dopamine as the main neurotransmitter secreted during cell exocytosis,we had gotten its SERS spectrum for identification.At physiological pH,amino protonated dopamine reduces its interaction with gold,making it difficult to adsorb on the surface of solid SERS substrates.It is difficult to obtain the SERS signal of 1 mM dopamine on a solid SERS substrate even using high laser power.Iron ions can complex with dopamine,thus we modified the SERS substrate with iron ions to attract dopamine on the surface for SERS detection.The Au/Fe complex substrate can detect 10-7 M of dopamine.The detection limit of this method satisfies the detection sensitivity of dopamine release from cell exocytosis.These work establish the foundation of instruments and methodology for applying electrochemical-SERS coupling detection technology to research neuronal exocytosis and neurotransmitters secretion in the future.