Determination Of Neurotransmitters In Brain Based On Liquid/Liquid Interface Analytical Platform

The brain is the command center for all physiological functions,behaviors,and perceptions of living system,which contained many neurotransmitters that participated in the regulation of physiological activities.The content imbalance of neurotransmitters in the brain can induce brain diseases.Therefore,understanding the changes of neurotransmitters in the brain is of great research significance for analyzing the molecular mechanisms of brain function!Currently,the main method for obtaining chemical information in the brain is based on solid state interfaces.Compared to the solid/liquid interface,the liquid/liquid interface has unique properties:the liquid/liquid interface contains two solution phases,and has surface tension at the interface and strong self-healing ability with high interface repeatability.Charged ions transferred at the polarization interface to generate voltammetric signals,providing both qualitative and quantitative information.At present,sensing strategies based on liquid/liquid interfaces have been widely used for the detection of neurochemicals such as neurotransmitters.However,most of these methods cannot be applied from the sensing concept to the detection of neurotransmitters in actual brain samples,mainly due to the neurotransmitters are extremely low content(～n M)in the actual brain environment and have similar structures,making it difficult to accurately.Aiming at the key problems of numerous interference and low content during neurotransmitter analysis in the brain,this paper reasonably screened and synthesized the specific recognition probes to design and construct surface enhanced Raman spectroscopy(SERS)and electrochemical analysis method based on liquid/liquid interface to achieve the determination of a series of neurotransmitters in the brain.The main research contents are as follows:The environment of the brain is complex,which contains different kinds of chemicals,result in the severe cross-talk of the signal for detection.In order to improve the selectivity of neurotransmitter detection in vivo analysis,we established a SERS analysis platform based on liquid/liquid interface by reasonably screening specific recognition ligands,achieving high selectivity detection of serotonin(5-HT)in the rat brain.Firstly,a functionalized SERS sensing probe for 5-HT was constructed by modifying the nucleic acid adapter DNA-ROX that specifically recognized 5-HT onto the surface of silver nanoparticles(Ag NPs).The reaction of 5-HT with SERS nanoprobes caused structural expand in nucleic acid aptamers,inducing variation in SERS signal.Subsequently,the SERS nanoprobes were self-assembled at the 1,2-dichloroethane/water(DCE/W)interface to construct a multi-hot spot liquid/liquid interface SERS analysis platform.This sensor exhibited good selectivity for 5-HT,which was not affected by other neurotransmitters in vivo,and showed good linear relationship with 5-HT in the concentration range of 1-150 n M,which fulfilled the detection needs of 5-HT in the brain.Finally,this sensor was successfully realized the highly selective determination of 5-HT in brain microdialysis fluid and plasma of hypertensive model rats.The structure of neurotransmitters in the brain,such as dopamine(DA),epinephrine(EP),and norepinephrine(NE),is highly similar,making it difficult to achieve selective detection through a single probe.To solve this challenge,a ternary regulatory strategy is proposed,which constructed a liquid/liquid interface SERS platform through dual recognition of rigid molecular probes,uniform distribution of hotspots at the liquid/liquid interface,and silent SERS signal output.Firstly,rigid probe molecule 4-((4-(thiophen-3-ethynyl)-phenyl)ethyl)benzaldehyde(RP1)and 4-mercaptophenylboronic acid(MPBA)were designed and co-assembled on the surface of Au NPs to construct SERS nanoprobes with dual recognition of NE,significantly improving the selectivity of determination.Meanwhile,the rigid structure of the probe effectively fixed the gap between nanoparticles,ensuring the reproducibility of SERS analysis in sub-nanometer.Secondly,the self-assembly of functionalized nanoparticles resulted in the formation of a large area of nanoarray at the liquid/liquid interface,which was uniformly distributed and tightly arranged,significantly improving the sensitivity and reproducibility of SERS analysis.In this case,the C≡C peak located in the Raman silence region of RP1 molecule was selected as the signal output,effectively avoiding the influence of potential interfering substances in the brain and further improving the reliability of detection.This SERS sensor exhibited excellent selectivity,the DA and EP with similar structures had no effect in the detection of NE,and has a good linear relationship with NE in the range of 0.6 to 40 n M.More importantly,the constructed analysis platform exhibited high reproducibility,with a relative standard deviation less than 4.2%for 50 test results.Finally,combined with brain microdialysis technology,the liquid/liquid SERS platform successfully realized online monitoring of NE content in brain microdialysis fluid on acute stress rats.Acetylcholine(ACh)has a simple molecular structure without optical and electrical activity,which content in the brain is as low as nanomolar,and has a challenge for detection in the brain.In order to achieve the detection of ACh in the brain,based on the charged character of ACh,this work proposed a liquid/liquid interface electrochemical analysis strategy with synergistic acceleration of viral-like nanoparticles,realized a highly sensitive detection of ACh in the rat brain.First,the specific recognition ligands for ACh,25,27-bis(3-mercaptopropoxy)-26,28-dihydroxycalix[4]arene(CX4-SH),and molecular mercaptocarborane(MPC)were designed and synthesized.The MPC and CX4-SH were co-assembled on the surface of Au NPs to construct virus-like nanoparticles.On the one hand,MPC captured K~+to form nanoparticles with rich positive charges on the surface.When distributed at the DCE/W interface,it will induce the redistribution of the interface potential to drove Au/MPC+CX4-SH to transfer from the water phase of the DCE/W interface to the organic phase.On the other hand,CX4-SH specifically bound with ACh through host guest interactions,enriching trace amounts of ACh on the surface of Au/MPC+CX4-SH,significantly improving the selectivity of ACh detection and further promoting the transfer of ACh at the interface,significantly improving the sensitivity of the determination.The results indicated that the constructed electrochemical sensor has good selectivity for ACh,with a linear relationship in the range of 0.1-15 n M,the detection limit was down to 0.067 n M.Using this electrochemical sensor,we successfully monitored the variation of ACh content in rat brain microdialysis fluid during deep brain stimulation treatment of memory impairment.