The biogenic monoamines dopamine （DA）, norepinephrine （NE） and serotonin （5-hydroxytryptamine, 5-HT） are major neuromodulators in the mammalian central nervous system （CNS）. DA containing neurons are found in ...The biogenic monoamines dopamine （DA）, norepinephrine （NE） and serotonin （5-hydroxytryptamine, 5-HT） are major neuromodulators in the mammalian central nervous system （CNS）. DA containing neurons are found in i） the mesolimbic system in which cell bodies in the ventral tegmental area （VTA） project axons into the amygdala, cortex, hippocampus and the nucleus accumbens; and ii） the nigrostriatal system in which cell bodies located in the substantia nigra pars compacta send their axons into the dorsolateral parts of the striatum （Bjorklund and Dunnett, 2007）. The central noradrenergic neurons are concentrated in distinct brainstem nuclei with the locus coeruleus （LC） being the most prominent nucleus which projects a diffusely arborizing axonal network to most areas of the CNS （Szabadi, 2013）.展开更多
The use of tetra-substituted ammo aluminum phthalocyanine (TAAIPc) as a new red-region fluorescent substrate for horseradish peroxidase (HRP)-based enzyme-linked immunosorbent assay was investigated. TAAIPc displayed ...The use of tetra-substituted ammo aluminum phthalocyanine (TAAIPc) as a new red-region fluorescent substrate for horseradish peroxidase (HRP)-based enzyme-linked immunosorbent assay was investigated. TAAIPc displayed an excitation maximum at 610 nm and emission maximum at 678 nm in a strong acidic medium. In the presence of HRP, trace amounts of H2O2 could rapidly and significantly react with TAAIPc, thus quenching the fluorescence of TAAIPc. The Michaelis-Menten parameters Km and Vmax were measured to be 2.82 × 10?6 mol/L?1 and 6.0 × 10?9 mol·L?1, respectively. In this paper, TAAIPc was used in an HRP-based enzyme-linked immunosorbent assay (ELISA) of α-fetoprotein (AFP) in human serum with satisfactory results. AFP could be determined in the concentration range of 0.5–200 ng/mL with a detection limit of 0.2 ng/mL, which was close to that of radioimmunoassay. The advantage of proposed method was strongly minimizing the interference resulting from background fluorescence or scattering light and had a high analytical sensitivity.展开更多
The combination of horseradish peroxidase(HRP)and a fluorescence substrate has been attracting great interests in developing sensitive biochemical analysis and immunoassays.10-Acetyl-3,7-dihydroxyphenoxazine(ADHP or A...The combination of horseradish peroxidase(HRP)and a fluorescence substrate has been attracting great interests in developing sensitive biochemical analysis and immunoassays.10-Acetyl-3,7-dihydroxyphenoxazine(ADHP or Amplex red)is the most sensitive fluorogenic substrate known for HRP in current market,however,it suffers from some drawbacks,such as non-specific reactivity to carboxylesterase and limited fluorescence stability.In the present study,a novel HRP substrate10-cyclopropylcarbonyl-dichloro-dihydroxyphenoxazine(AR-2),has been prepared,which exhibited improved sensitivity than ADHP in sensing HRP.Moreover,the fluorescence of AR-2/HRP demonstrated improved tolerance to physiological relevant p H fluctuation as compared to ADHP/HRP.Successful detection of uric acid/urate oxidase reaction indicated excellent application prospect of AR-2/HRP for monitoring H_(2)O_(2)-generating biochemical reactions.More interestingly,an enzyme-linked immunosorbent assay(ELISA)using AR-2 as the fluorescence reporter has been successfully used in detecting IgG against severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)from human serum samples.Overall,AR-2 exhibits improved performances over the commercial ADHP,which will be an ideal alternative to ADHP in HRP-based fluorescence biochemical analysis and immunoassays.展开更多
Estrogen sulfotransferase(SULT1E1),an essential conjugative enzyme in mammals,plays a crucial role in both estrogen homeostasis and xenobiotic metabolism.Deciphering the dynamic changes in SULT1E1function under specif...Estrogen sulfotransferase(SULT1E1),an essential conjugative enzyme in mammals,plays a crucial role in both estrogen homeostasis and xenobiotic metabolism.Deciphering the dynamic changes in SULT1E1function under specific physiological or pathological conditions and discovering SULT1E1 modulators require practical and highly efficient tools for sensing SULT1E1 in biological context.Herein,we showcase a scaffold-seeking and structural optimization strategy for the rational engineering of isoform-specific fluorescent substrates for SULT1E1.First,docking-based virtual screening coupled with biochemical assays suggested that N–butyl–4-hydroxyphenyl-1,8-naphthalimide(HPN) was a suitable scaffold for constructing the fluorescent substrates for SULT1E1,but this fluorophore could be metabolized by multiple SULT isoforms.To develop isoform-specific substrates for SULT1E1,various substituents were introduced on the north part of HPN to explore the structure-enzyme specificity relationships of HPN derivatives as SULT1E1substrates.After molecular docking and experimental validation,an isoform-specific fluorescent substrate(HPN10) for SULT1E1 was successfully engineered.HPN10 demonstrated exceptional isoform-specificity,ultra-high sensitivity,and favorable signal-to-noise ratio(212).HPN10 excelled in the precise sensing of SULT1E1 activities in complex biological matrices,including cellular specimens and liver preparations.HPN10 immensely facilitated the discovery and characterization of SULT1E1 inhibitors,while tetrabromobisphenol A(TBBPA,half inhibitory concentration(IC_(50)) = 31.5 ± 3.4 nmol/L) was identified as a potent SULT1E1 inhibitor that could strongly block SULT1E1 activities in living cells.Collectively,this work presents a practical and efficient strategy for the rational engineering of isoform-specific fluorescent substrates for target conjugative enzyme(s),while HPN10 emerges as a reliable SULT1E1-activatable tool for functional sensing and drug discovery.展开更多
基金supported by the Deutsche Forschungsgemeinschaft(SFB636)to PS and FM
文摘The biogenic monoamines dopamine （DA）, norepinephrine （NE） and serotonin （5-hydroxytryptamine, 5-HT） are major neuromodulators in the mammalian central nervous system （CNS）. DA containing neurons are found in i） the mesolimbic system in which cell bodies in the ventral tegmental area （VTA） project axons into the amygdala, cortex, hippocampus and the nucleus accumbens; and ii） the nigrostriatal system in which cell bodies located in the substantia nigra pars compacta send their axons into the dorsolateral parts of the striatum （Bjorklund and Dunnett, 2007）. The central noradrenergic neurons are concentrated in distinct brainstem nuclei with the locus coeruleus （LC） being the most prominent nucleus which projects a diffusely arborizing axonal network to most areas of the CNS （Szabadi, 2013）.
文摘The use of tetra-substituted ammo aluminum phthalocyanine (TAAIPc) as a new red-region fluorescent substrate for horseradish peroxidase (HRP)-based enzyme-linked immunosorbent assay was investigated. TAAIPc displayed an excitation maximum at 610 nm and emission maximum at 678 nm in a strong acidic medium. In the presence of HRP, trace amounts of H2O2 could rapidly and significantly react with TAAIPc, thus quenching the fluorescence of TAAIPc. The Michaelis-Menten parameters Km and Vmax were measured to be 2.82 × 10?6 mol/L?1 and 6.0 × 10?9 mol·L?1, respectively. In this paper, TAAIPc was used in an HRP-based enzyme-linked immunosorbent assay (ELISA) of α-fetoprotein (AFP) in human serum with satisfactory results. AFP could be determined in the concentration range of 0.5–200 ng/mL with a detection limit of 0.2 ng/mL, which was close to that of radioimmunoassay. The advantage of proposed method was strongly minimizing the interference resulting from background fluorescence or scattering light and had a high analytical sensitivity.
基金the funding support from Key-Area Research and Development Program of Guangdong Province(No.2022B1111020003)Guangzhou Talents Program for Innovation and Entrepreneurship(No.2021-L010)the Foshan“Blue Ocean Talent Program”for Innovation and Entrepreneurship(No.2230032002063)。
文摘The combination of horseradish peroxidase(HRP)and a fluorescence substrate has been attracting great interests in developing sensitive biochemical analysis and immunoassays.10-Acetyl-3,7-dihydroxyphenoxazine(ADHP or Amplex red)is the most sensitive fluorogenic substrate known for HRP in current market,however,it suffers from some drawbacks,such as non-specific reactivity to carboxylesterase and limited fluorescence stability.In the present study,a novel HRP substrate10-cyclopropylcarbonyl-dichloro-dihydroxyphenoxazine(AR-2),has been prepared,which exhibited improved sensitivity than ADHP in sensing HRP.Moreover,the fluorescence of AR-2/HRP demonstrated improved tolerance to physiological relevant p H fluctuation as compared to ADHP/HRP.Successful detection of uric acid/urate oxidase reaction indicated excellent application prospect of AR-2/HRP for monitoring H_(2)O_(2)-generating biochemical reactions.More interestingly,an enzyme-linked immunosorbent assay(ELISA)using AR-2 as the fluorescence reporter has been successfully used in detecting IgG against severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)from human serum samples.Overall,AR-2 exhibits improved performances over the commercial ADHP,which will be an ideal alternative to ADHP in HRP-based fluorescence biochemical analysis and immunoassays.
基金supported by Natural Science Foundation of China (Nos.U23A20516,82273897,81922070)Organizational Key Research and Development Program of Shanghai University of Traditional Chinese Medicine (No.2023YZZ02)+3 种基金the State Key Laboratory of Fine Chemicals,Dalian University of Technology (Nos.KF2202,KF2414)Shanghai Municipal Health Commission’s TCM research project (No.2022CX005)Future Plan for Traditional Chinese Medicine development of Science and Technology of Shanghai Municipal Hospital of TCM (Nos.WL-XJRY-2021010 K,WLGNDBZPY-2022001 K)Shanghai Jing’an District Health Commission (No.2024QT03)。
文摘Estrogen sulfotransferase(SULT1E1),an essential conjugative enzyme in mammals,plays a crucial role in both estrogen homeostasis and xenobiotic metabolism.Deciphering the dynamic changes in SULT1E1function under specific physiological or pathological conditions and discovering SULT1E1 modulators require practical and highly efficient tools for sensing SULT1E1 in biological context.Herein,we showcase a scaffold-seeking and structural optimization strategy for the rational engineering of isoform-specific fluorescent substrates for SULT1E1.First,docking-based virtual screening coupled with biochemical assays suggested that N–butyl–4-hydroxyphenyl-1,8-naphthalimide(HPN) was a suitable scaffold for constructing the fluorescent substrates for SULT1E1,but this fluorophore could be metabolized by multiple SULT isoforms.To develop isoform-specific substrates for SULT1E1,various substituents were introduced on the north part of HPN to explore the structure-enzyme specificity relationships of HPN derivatives as SULT1E1substrates.After molecular docking and experimental validation,an isoform-specific fluorescent substrate(HPN10) for SULT1E1 was successfully engineered.HPN10 demonstrated exceptional isoform-specificity,ultra-high sensitivity,and favorable signal-to-noise ratio(212).HPN10 excelled in the precise sensing of SULT1E1 activities in complex biological matrices,including cellular specimens and liver preparations.HPN10 immensely facilitated the discovery and characterization of SULT1E1 inhibitors,while tetrabromobisphenol A(TBBPA,half inhibitory concentration(IC_(50)) = 31.5 ± 3.4 nmol/L) was identified as a potent SULT1E1 inhibitor that could strongly block SULT1E1 activities in living cells.Collectively,this work presents a practical and efficient strategy for the rational engineering of isoform-specific fluorescent substrates for target conjugative enzyme(s),while HPN10 emerges as a reliable SULT1E1-activatable tool for functional sensing and drug discovery.
