Professor Yin Jun and Professor Liu Shenghua team of central China Normal University: glutathione specific fluorescence probe applied to dual channel biological imaging

Introduction glutathione (GSH) plays an important role in life system Using fluorescence probe and imaging technology to visually label GSH in vivo is of great significance for further understanding the function of GSH As the only output signal of fluorescence imaging, the diversity of fluorescence signal can meet different research needs Recently, Professor Yin Jun and Professor Liu Shenghua of the school of chemistry of central China Normal University and their collaborators Professor Tan Ying of Shenzhen International Graduate School of Tsinghua University and Professor Yoon juyoung of Lihua women's University of Korea, On the basis of fluorescence on and proportional fluorescence, a signal model of double fluorescence on was further proposed The first molecular probe which can detect GSH in both visible and near-infrared light channels was successfully developed, and it was used for fluorescence imaging of GSH in living cells and tissues in two channels (CHEM, 2018, 4, 1609-1628; anal Chem, 2019, 91, 11343-11348 ）。
Brief introduction of Professor Yin Jun's research group the research group has been engaged in the research of organic fluorescent dyes, photochromic materials and molecular aggregation and assembly Brief introduction of Professor Yin Jun professor and doctoral supervisor of School of chemistry, Huazhong Normal University Member of Chinese Chemical Society and American Chemical Society From 1998 to 2007, he received his Ph.D degree in the Department of chemistry, central China Normal University; from 2008 to 2010, he was engaged in post doctoral research in the Department of chemistry, National University of Singapore; from 2012 to 2013, he was engaged in scientific research as a research professor in the Department of Chemistry, Lihua women's University, South Korea Since 2010, a lot of research work has been carried out in organic fluorescent materials, fluorescent sensors, biological imaging, etc He has successively presided over and undertaken many projects, such as general program of National Natural Science Foundation, youth program, outstanding program of Hubei Natural Science Foundation, general program, Wuhan Science and technology morning light plan, etc At present, he is a young editorial board member of Chinese chemical letters and current smart materials, and a reviewer of more than 40 international journals At NAT Protoc., chem, chem SOC Rev., coord Chem Rev., J am Chem SOC., Biosens Bioelectron., anal Chem., chem Commun., org Lett., chem EUR J., J org Chem, J More than 80 papers with if > 3.0 have been published by international authoritative magazines such as mater Chem (he cited more than 4300 times, h index was 33, among which 4 papers were selected as high cited papers) It has applied for 4 Chinese invention patents and 2 authorizations Frontier research achievements: glutathione specific fluorescence probe applied to dual channel bio imaging glutathione (GSH) is a tripeptide compound containing active sulfhydryl group, which plays a key role in many physiological processes in biological system The abnormal expression of GSH in cells is related to many diseases, which can be used as an important biomarker to measure human health However, most GSH specific fluorescence probes developed so far respond to thiols by generating a fluorescence on signal in a single channel, and the change of fluorescence emission signal is usually based on the two types of single channel intensity change type and ratio type (Figure 1a) The single channel fluorescent probe with intensity change is easily limited by photobleaching, light scattering, solvent polarity, probe concentration, excitation intensity, instrument efficiency and environmental conditions, which may affect the accuracy of bioanalysis On the contrary, the ratio type fluorescence probe with the change of fluorescence signal in two emission channels can eliminate the influence of the error related to the change of measurement conditions However, although they use two-channel emission, the current ratio probes usually produce "one side up" and "one side down" fluorescence signals, which are easy to cause spectral overlap, energy scattering, self absorption and other shortcomings Therefore, the research and development of a new detection mode that can generate multi-channel fluorescence signals to avoid self absorption (energy transfer) and signal interference is of great significance for biological analysis and imaging in biological systems Recently, a team of professors Yin Jun and Liu Shenghua from central China Normal University constructed a new strategy to design a dual channel fluorescent probe (DFP) for GSH response (Figure 1b) The team envisages that in the presence of DFP, double channel fluorescence signal can be used to effectively eliminate the problems such as self absorption and signal mutual interference that are easy to encounter in the process of fluorescence signal acquisition Figure 1 Schematic diagram (b) of GSH response single channel fluorescent probe (SFP) (a) and dual channel fluorescent probe (DFP) (source: Chem, 2018, 4, 1609-1628) based on previous work (J am Chem SOC., 2014, 136, 5351-5358; NAT Protoc 2015, 10, 1742-1754; chem Commun., 2016, 52, 721-724), the team designed a two channel fluorescence probe CYP SNP (Figure 2a) with naphthalimide and indocyanine green as the visible light channel and near-infrared light channel fluorescence groups respectively, and sulfonamide as the double fluorescence group link group Using Gauss 09 program, we calculated the time-dependent density functional theory (TD-DFT) at B3LYP / 6-31G * level The results show that the probe cyp-snp only has weak fluorescence Therefore, under the action of GSH, the fluorescence activation of the probe in the visible and near-infrared channels will occur synchronously (Fig 2b) Fig 2 The operating principle and molecular orbital diagram of the probe CYP SNP (source: Chem, 2018, 4, 1609-1628) The author selected three kinds of small molecular thiols (GSH, Hcy and Cys) and a variety of amino acids as the detection objects to study the in vitro spectroscopic properties of the probe CYP SNP The results show that the probe can respond to GSH with high specificity in both visible and near-infrared channels (Fig 3a), and the detection of GSH in both channels is almost free from interference of other amino acids (Fig 3b) Fig 3 Selective experiment (a) and competitive experiment (b) (source: Chem, 2018, 4, 1609-1628) of probe CYP SNP in visible and near infrared channels to replace product 1, The 8-naphthalimide derivative NP GSH can be used as a two-photon fluorescence group Therefore, the two-photon absorption properties of the probe CYP SNP and its visible light emission product NP GSH after reaction with GSH were investigated The results show that the probe CYP SNP has excellent two-photon properties (Fig 4) Figure 4 Two photon property test of probe CYP SNP and visible light emission fluorescence product NP GSH (source: anal Chem., 2019, 91, 11343-11348) based on good in vitro test results, the author then applied probe CYP SNP to fluorescence imaging of GSH in cells The results showed that the probe CYP SNP could penetrate into the cell and react with GSH in the cell, and generate discernable green fluorescence signal (from the substitute product NP GSH) and red fluorescence signal (from the thiolysis product CYP) In addition, probe CYP SNP can be used for synchronous fluorescence imaging of endogenous GSH in green channel and red channel (Fig 5) Figure 5 Cell imaging of endogenous thiols and co localization of intracellular fluorescence signals by probe CYP SNP in green channel and red channel (source: Chem, 2018, 4, 1609-1628) The authors next explored whether probe CYP SNP still has specificity for GSH in vivo Under the same conditions, only the experimental group with GSH can produce bright fluorescent signals in both the green channel and the red channel The results showed that the probe CYP SNP could be used to specifically monitor the intracellular GSH level (Fig 6) Figure 6 Cell imaging of the response of probe CYP SNP to GSH in green and red channels (source: Chem, 2018, 4, In 1609-1628) cationic cyanine ir-780 dye structure, the positive charge of the cation has a charge attraction to the negative potential of the cell mitochondrial membrane, so the author has verified the mitochondrial imaging ability of the probe CYP SNP containing the cationic structural unit The results show that the probe CYP SNP has the ability to target endogenous GSH in mitochondria in both the green fluorescence signal in the visible channel and the red fluorescence signal in the near infrared channel (Fig 7) Figure 7 (a) fluorescence signal of probe CYP SNP in red channel and co localization fluorescence imaging of commercial mitochondrial green localization probe mitotracker green; (b) fluorescence signal of probe CYP SNP in green channel and co localization fluorescence imaging of commercial mitochondrial red localization probe mitotracker red (source: Chem, 2018, 4, 1609-1628) then, the author continued to use mitotrackerred red, a commercial mitochondrial red localization probe, as a reference, to simultaneously co locate the probe CYP SNP in two channels The results show that the fluorescence signals of probe CYP SNP in two different channels can specifically label the subcellular organelle mitochondria in living cells at the same time, and successfully realize synchronous fluorescence imaging of the same subcellular organelle in living cells in two completely different fluorescence channels (Fig 8) Figure 8 Simultaneous co localization of probe CYP SNP and commercial mitochondrial red localization probe mitotracker red in green and red channels (source: Chem, 2018, 4, 1609-1628) using the two-photon emission characteristics of the probe CYP SNP in the visible region and the single photon near-infrared emission characteristics of the near-infrared region, the author co incubated HepG2 cells or HeLa cells with the probe CYP SNP, and observed the bright green fluorescence signal in the visible channel excited by two-photon at 810 nm, 633 nm Bright red fluorescence is also shown in the near-infrared channel excited by a single photon (Fig 9) The above results show that the probe CYP SNP has two-photon fluorescence imaging ability in visible channel and one photon fluorescence imaging ability in near-infrared channel Figure 9 Two photon imaging of probe CYP SNP in visible channel and single photon imaging in near infrared channel (A) HepG2 cells; (b) HeLa cells (source: anal Chem., 2019, 91, 11343-11348) Compared with traditional single photon visible fluorescence imaging, two-photon fluorescence imaging and near-infrared fluorescence imaging have the advantages of deeper tissue penetration Therefore, the author further studied the tissue imaging ability of the probe by incubating the freshly prepared mouse liver sections with the probe CYP SNP The results show that the probe CYP SNP can be applied to two-photon tissue imaging in the visible light channel and tissue imaging in the near-infrared channel to achieve two-channel tissue imaging of endogenous GSH in two completely different channels (FIG 10) Figure 10 Two photon fluorescence imaging (a) in visible light channel and single photon fluorescence imaging (b) in near infrared channel of GSH in mouse liver tissue by probe CYP SNP (source: anal Chem., 2019, 91, 11343-11348) using the advantages of the probe CYP SNP in near-infrared emission, the author injected the probe CYP SNP into mice through tail vein, and then applied it to the visualization of GSH in living animals through the small animal living imaging system Real time imaging results in vivo and organ formation in vitro