Enzymes are macromolecular biological catalysts which can accelerate chemical reactions in living organisms. Almost all the physiological metabolism activities in the cell need enzymes to sustain life via rapid cataly...Enzymes are macromolecular biological catalysts which can accelerate chemical reactions in living organisms. Almost all the physiological metabolism activities in the cell need enzymes to sustain life via rapid catalysis. Currently, medical research has proved that abnormal enzyme activity is associated with numerous diseases, such as Parkinson’s disease(PD), Alzheimer’s disease(AD) and cancers. On the other hand, early diagnosis of those diseases is of great significance to improve the survival rate and cure rate.In the current diagnostic tools, two-photon fluorescent probes(TPFPs) are developing rapidly due to their unique advantages, such as higher spatial resolution, deeper imaging depth, and lower biotoxicity.Therefore, the design and synthesis of two-photon(TP) small molecule enzymatic probes have broad prospects for early diagnosis and treatment of diseases. As of now, scientists have developed many TP small molecule enzymatic probes. This review aims to summarize the TP small molecule enzymatic probes and expound the reaction mechanism.展开更多
Protein labeling by using a protein tag and tag-specific fluorescent probes is increasingly becoming a useful technique for the real-time imaging of proteins in living cells. SNAP-tag as one of the most prominent fusi...Protein labeling by using a protein tag and tag-specific fluorescent probes is increasingly becoming a useful technique for the real-time imaging of proteins in living cells. SNAP-tag as one of the most prominent fusion tags has been widely used and already commercially available. Recently, various fluorogenic probes for SNAP-tag based protein labeling were reported. Owing to turn-on fluorescence response, fluorogenic probes for SNAP-tag minimize the fluorescence background caused by unreacted or nonspecifically bound probes and allow for direct imaging in living cells without wash-out steps. Thus,real-time analysis of protein localization, dynamics and interactions has been made possible by SNAP-tag fluorogenic probes. In this review,we describe the design strategies of fluorogenic probes for SNAP-tag and their applications in cellular protein labeling.展开更多
Monoamine oxidase is flavoenzymes, widely distributed in mammals. It is well recognized that MAOs serve an important role in metabolism that they have close relationship with health .Along with the discoveries between...Monoamine oxidase is flavoenzymes, widely distributed in mammals. It is well recognized that MAOs serve an important role in metabolism that they have close relationship with health .Along with the discoveries between MAOs and neurotic disease, more and more studies have been jumped in .In this paper, we design a new probe for assaying the activities of MAOs. The results showed that the probe [7-（3-aminopropoxy）coumarin] is simple, effective and sensitive for MAOB.展开更多
The application of a new fluorogenic probe-based PCR assay (PCR duplex scorpion primer assay) to the detection of Hepatitis B virus (HBV) DNA in human sera was described. Duplex scorpion primer is a modified variant o...The application of a new fluorogenic probe-based PCR assay (PCR duplex scorpion primer assay) to the detection of Hepatitis B virus (HBV) DNA in human sera was described. Duplex scorpion primer is a modified variant of duplex Amplifluor, and the incorporation of a PCR stopper between probe and primer sequences improve the detection specificity and sensitivity. Combined with PCR amplification, this probe can give unambiguous positive results for the reactions initiated with more than 20 HBV molecules. In addition, the particular unimolecular probing mechanism of this probe makes the use of short target-specific probe sequence possible, which will render this probe applicable in some specific systems.展开更多
Monitoring mitochondrial derived copper（Ⅱ） in live cells is highly demanded, but accurately detecting is unmet due to the interference with cytoplasmic copper（Ⅱ）. Herein, we have reported the design,synthesis an...Monitoring mitochondrial derived copper（Ⅱ） in live cells is highly demanded, but accurately detecting is unmet due to the interference with cytoplasmic copper（Ⅱ）. Herein, we have reported the design,synthesis and characterization of photocontrollable fluorogenic probe, MCu-3, which is equipped with a photo-labile group（nitrobenzyl group） and mitochondria targeting unit（triphenylphosphonium salt）.This novel probe showed an intense fluorescence enhancement in response to copper（Ⅱ） without interference from other metal cations in the biological condition（p H 6–9）. The detection limit is 1.7 ×10^（-7） mol/L in HEPES buffer. The confocal fluorescence imaging results demonstrated MCu-3 can visualize mitochondrial copper（Ⅱ） in live mammalian cells. The clear advantage of our photocontrollable method is successful to avoid the influence of cytoplasmic copper（Ⅱ） during mitochondria specific detection.展开更多
Porcine lipoprotein lipase （LPL） cDNA was cloned as the standard for real-time quantifying LPL mRNA and the TaqMan-fluorescence quantitative PCR assay for detection was established. The total RNA extracted from Long...Porcine lipoprotein lipase （LPL） cDNA was cloned as the standard for real-time quantifying LPL mRNA and the TaqMan-fluorescence quantitative PCR assay for detection was established. The total RNA extracted from Longissimus dorsi of porcine was reverse-transcribed to cDNA. LPL cDNA was ligated with pGM-T vector and transformed into Escherichia coli TOP10. Plasmid DNA extracted from positive clones was verified by PCR amplification and sequenced. LPL was amplified by real-time fluorescence quantitative PCR from the plasmid DNA. The concentration of DNA template purified was detected by analyzing absorbance in 260 nm and then the combined plasmid was diluted to series as standard for fluorescence quantitative PCR （FQ-PCR）. The method of LPL mRNA real-time PCR was well established, which detected as low as 103 with the linear range 10^3 to 10^10 copies. The standard curves showed high correlations （R2 = 0.9871）. A series of standards for real-time PCR analysis have been constructed successfially, and real-time TaqMan-fluorescence quantitative RT-PCR is reliable to quantitatively evaluate FQ-PCR mRNA in L. dorsi of porcine.展开更多
基金financially supported by the National Natural Science Foundation of China (No. 81672508)Jiangsu Provincial Foundation for Distinguished Young Scholars (No. BK20170041)+2 种基金Natural Science Foundation of Shaanxi Province (No. 2019JM-016)China-Sweden Joint Mobility Project (No. 51811530018)Fundamental Research Funds for the Central Universities
文摘Enzymes are macromolecular biological catalysts which can accelerate chemical reactions in living organisms. Almost all the physiological metabolism activities in the cell need enzymes to sustain life via rapid catalysis. Currently, medical research has proved that abnormal enzyme activity is associated with numerous diseases, such as Parkinson’s disease(PD), Alzheimer’s disease(AD) and cancers. On the other hand, early diagnosis of those diseases is of great significance to improve the survival rate and cure rate.In the current diagnostic tools, two-photon fluorescent probes(TPFPs) are developing rapidly due to their unique advantages, such as higher spatial resolution, deeper imaging depth, and lower biotoxicity.Therefore, the design and synthesis of two-photon(TP) small molecule enzymatic probes have broad prospects for early diagnosis and treatment of diseases. As of now, scientists have developed many TP small molecule enzymatic probes. This review aims to summarize the TP small molecule enzymatic probes and expound the reaction mechanism.
基金supports from the National Natural Science Foundation of China (Nos. 21422606 and 21502189)Dalian Cultivation Fund for Distinguished Young Scholars (Nos. 2014J11JH130 and 2015J12JH205)
文摘Protein labeling by using a protein tag and tag-specific fluorescent probes is increasingly becoming a useful technique for the real-time imaging of proteins in living cells. SNAP-tag as one of the most prominent fusion tags has been widely used and already commercially available. Recently, various fluorogenic probes for SNAP-tag based protein labeling were reported. Owing to turn-on fluorescence response, fluorogenic probes for SNAP-tag minimize the fluorescence background caused by unreacted or nonspecifically bound probes and allow for direct imaging in living cells without wash-out steps. Thus,real-time analysis of protein localization, dynamics and interactions has been made possible by SNAP-tag fluorogenic probes. In this review,we describe the design strategies of fluorogenic probes for SNAP-tag and their applications in cellular protein labeling.
基金the project sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars Fund,Zhejiang Province(No.Z01105002)Returned Overseas Chinese Scholars Fund.
文摘Monoamine oxidase is flavoenzymes, widely distributed in mammals. It is well recognized that MAOs serve an important role in metabolism that they have close relationship with health .Along with the discoveries between MAOs and neurotic disease, more and more studies have been jumped in .In this paper, we design a new probe for assaying the activities of MAOs. The results showed that the probe [7-（3-aminopropoxy）coumarin] is simple, effective and sensitive for MAOB.
基金the National Natural Science Foundation of China (No. 20075012) and the Outstanding Scholar Program of Nankai University.
文摘The application of a new fluorogenic probe-based PCR assay (PCR duplex scorpion primer assay) to the detection of Hepatitis B virus (HBV) DNA in human sera was described. Duplex scorpion primer is a modified variant of duplex Amplifluor, and the incorporation of a PCR stopper between probe and primer sequences improve the detection specificity and sensitivity. Combined with PCR amplification, this probe can give unambiguous positive results for the reactions initiated with more than 20 HBV molecules. In addition, the particular unimolecular probing mechanism of this probe makes the use of short target-specific probe sequence possible, which will render this probe applicable in some specific systems.
基金supported by the National Natural Science Foundation of China (Nos. 81672508, 61505076)Natural Science Foundation of Jiangsu Province (No. BK20140951)+1 种基金Key University Science Research Project of Jiangsu Province (No. 16KJA180004)SICAM Fellowship & Scholarship by Jiangsu National Synergetic Innovation Center for Advanced Materials
文摘Monitoring mitochondrial derived copper（Ⅱ） in live cells is highly demanded, but accurately detecting is unmet due to the interference with cytoplasmic copper（Ⅱ）. Herein, we have reported the design,synthesis and characterization of photocontrollable fluorogenic probe, MCu-3, which is equipped with a photo-labile group（nitrobenzyl group） and mitochondria targeting unit（triphenylphosphonium salt）.This novel probe showed an intense fluorescence enhancement in response to copper（Ⅱ） without interference from other metal cations in the biological condition（p H 6–9）. The detection limit is 1.7 ×10^（-7） mol/L in HEPES buffer. The confocal fluorescence imaging results demonstrated MCu-3 can visualize mitochondrial copper（Ⅱ） in live mammalian cells. The clear advantage of our photocontrollable method is successful to avoid the influence of cytoplasmic copper（Ⅱ） during mitochondria specific detection.
基金support provided by the 973 Program of China (2004CB117500)
文摘Porcine lipoprotein lipase （LPL） cDNA was cloned as the standard for real-time quantifying LPL mRNA and the TaqMan-fluorescence quantitative PCR assay for detection was established. The total RNA extracted from Longissimus dorsi of porcine was reverse-transcribed to cDNA. LPL cDNA was ligated with pGM-T vector and transformed into Escherichia coli TOP10. Plasmid DNA extracted from positive clones was verified by PCR amplification and sequenced. LPL was amplified by real-time fluorescence quantitative PCR from the plasmid DNA. The concentration of DNA template purified was detected by analyzing absorbance in 260 nm and then the combined plasmid was diluted to series as standard for fluorescence quantitative PCR （FQ-PCR）. The method of LPL mRNA real-time PCR was well established, which detected as low as 103 with the linear range 10^3 to 10^10 copies. The standard curves showed high correlations （R2 = 0.9871）. A series of standards for real-time PCR analysis have been constructed successfially, and real-time TaqMan-fluorescence quantitative RT-PCR is reliable to quantitatively evaluate FQ-PCR mRNA in L. dorsi of porcine.
