Design Of Novel Surface-enhanced Raman Probes And Study On The Correlation Of Cu⁺ And Cu²⁺ In Brain Function

Copper is an essential trace element in the brain,whose uptake,transport and excretion are strictly regulated.Copper mainly exists in two forms of Cu⁺and Cu²⁺in living systems,which can be converted into each other,and the disturbance of their redox balance may cause oxidative stress and lead to neurological diseases.Although different analytical methods have been reported for the quantitative detection of Cu⁺or Cu²⁺,they still cannot satisfy the simultaneous analysis of Cu⁺and Cu²⁺in cells or in vivo,which greatly hinders us from comprehensively understanding the molecule mechanism of copper in brain science and relevant disease.Aiming at the key scientific problem of simultaneous analysis of Cu⁺and Cu²⁺in living system,highly selective ratiometric SERS probes for simultaneous analysis of Cu⁺and Cu²⁺were constructed by rationally designing and synthesizing specific recognition molecules,eventually realized the detection of copper in cells and the brain,which mainly includes the following three aspects:(1)A highly selective ratiometric SERS sensing strategy based on DNA aptamers was proposed to realize the imaging and quantitative analysis of Cu²⁺in neurons.First,the specific recognition DNA aptamer of Cu²⁺was screened and assembled on the gold nanoflowers with the size of 61±7 nm as the enhanced substrate.Then,4-MBA was selected as an internal reference molecule to be co-modified on the surface of gold nanoflowers to obtain ratiometric SERS probe.The binding of the aptamer chain to Cu²⁺caused the SERS signal of the complementary chain to decrease,while the SERS signal of the reference molecule remained unchanged,thus realizing the ratiometric determination of Cu²⁺.The sensor exhibited good selectivity for Cu²⁺,which was not affected by interferences such as other metal ions,and showed a good response to Cu²⁺in the range of 0.1-3?M,with a detection limit of 0.07±0.01?M(3?/k,n=5).In addition,the sensor also demonstrated good p H stability,temporal stability,reproducibility,and biocompatibility.Finally,based on this sensor,we achieved the highly selective determination of Cu²⁺and SERS imaging in neurons.(2)An implantable SERS probe was constructed to realize the simultaneous detection and real-time tracking of Cu⁺and Cu²⁺in the cortex of live mouse.First,two recognition molecules N,N-bis(2-(2-(ethylthio)ethyl))-2-mercaptoacetamide(ETMA)and N-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-2-mercaptoacetamide(PYMA)with the specific binding ability to Cu⁺and Cu²⁺were synthesized,and then these two molecules were co-assembled on the surface of the gold-decorated quartz tube to construct the implantable SERS probe for determining Cu⁺and Cu²⁺simultaneously.The probe presented high selectivity and good stability,and exhibited good response to both Cu⁺and Cu²⁺in the range of 0.50-10?M,with detection limits of 0.42±0.02?M and 0.34±0.02?M(3?/k,n=5),respectively,and no cross-talk was observed for simultaneous determination.Subsequently,a sensing microarray consisting of eight SERS probes was planted in the cortex of mice for the real-time detecting of the concentrations of Cu⁺and Cu²⁺.Moreover,three main pathways resulting in the increase of Cu⁺and Cu²⁺upon middle cerebral artery occlusion were found.(3)In order to study the physiological significance of intracellular Cu⁺and Cu²⁺,a SERS probe designed based on specific alkynyl functionalization was further constructed,and the simultaneous analysis of Cu⁺and Cu²⁺in mitochondria of living cells was realized.Firstly,two molecules 4-((4-((bis(2-(2-(ethylthio)ethyl))amino)methyl)phenyl)buta-1,3-diyn-1-yl)-N-(prop-2-yn-1-yl)benzamide(ETPB)and N,N-bis(pyridin-2-ylmethyl)hepta-3,6-diyn-1-amine(PHDA)for recognizing Cu⁺and Cu²⁺specifically were designed and synthesized.The efficient separation of Cu⁺and Cu²⁺Raman signals was achieved by rationally adjusting the alkynyl conjugation length and substituent.Secondly,gold nanostars with the size of 66±9 nm were prepared as the SERS substrate.Subsequently,recognition molecules(ETPB and PHDA),reference molecules(TIPP),and mitochondrial targeting molecules(PTPB)were co-modified on the gold nanostars through gold-alkyne bonds to form the highly selective SERS probes.When the probe molecules were combined with Cu⁺or Cu²⁺,the alkynyl Raman characteristic peaks at 2215 cm^-1 or 2236 cm^-1 were decreased,and at the same time,the alkynyl SERS signal of the reference molecule TIPP at 2155 cm^-1 kept unchanged,thus achieving a ratiometric determination of Cu⁺and Cu²⁺.The probe demonstrated highly selective for Cu⁺and Cu²⁺,and exhibited good response to both Cu⁺and Cu²⁺in the range of 0.50-14?M with detection limits of 0.32±0.03?M and 0.44±0.02?M(3?/k,n=5),respectively,and no cross-talk was observed for simultaneous determination.Finally,based on the SERS probe,the detection of Cu⁺and Cu²⁺and SERS imaging in mitochondrion of neuron cells were realized successfully.These research methods provide new research ideas and efficient tools for the detection of chemical substances in living system and cells,and are beneficial for in-depth and accurate analysis of the molecular mechanisms of Cu⁺and Cu²⁺in oxidative stress and other related events in brain,prevention and treatment of brain diseases.