Hepatic ischemia-reperfusion injury(HIRI)is the cause of postoperative hepatic dysfunction and failure,and even death.As an important biological effector molecule,hydrogen sulfide(H_(2)S)of mitochondria as a gasotrans...Hepatic ischemia-reperfusion injury(HIRI)is the cause of postoperative hepatic dysfunction and failure,and even death.As an important biological effector molecule,hydrogen sulfide(H_(2)S)of mitochondria as a gasotransmitter that is usually used to protect against acute HIRI injury.However,the exact relationship between HIRI and mitochondrial H_(2)S remains tangled due to the lack of an effective analytical method.Herein,we have fabricated a mitochondria-targeted H_(2)S-activatable fluorogenic probe(Mito-GW)to explore the stability of mitochondrial H_(2)S and track the changes of mitochondrial H_(2)S during the HIRI.By virtue of pyridinium electropositivity and its amphiphilicity,Mito-GW could accumulate in mitochondria.It goes through an analyte-prompted immolation when reacts with H_(2)S,resulting in the releasing of the fluorophore(GW).Therefore,the extent of Mito-GW conversion to GW can be used to evaluate the changes of mitochondrial H_(2)S level in living cells and tissues.As proof-of-principle,we have used MitoGW to demonstrate the mitochondria H_(2)S-levels increase and then decrease during HIRI in vitro and in vivo.Our research highlights the tremendous potential of Mito-GW as a mitochondrial H_(2)S fluorogenic probe in elucidating the pathogenesis of HIRI,providing a powerful tool for promoting future research on hepatology.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
Protein self-labeling tags achieve selective fusion and labeling of target proteins through genetic coding technology,but require exogenous fluorescent probes with fluorogenicity for protein tag binding to have the pe...Protein self-labeling tags achieve selective fusion and labeling of target proteins through genetic coding technology,but require exogenous fluorescent probes with fluorogenicity for protein tag binding to have the performance of wash-free fluorescence imaging in live cells.In this paper,we reported a fluorogenic probe 1 capable of ratiometric fluorescence recognition of SNAP-tag proteins.In this probe,the O6-benzylguanine derivative of 3-hydroxy-1,8-naphthalimide underwent a selective covalent linkage reaction with SNAP-tag protein.The hydroxyl group on the naphthalimide fluorophore formed a hydrogen bond with the functional group near the protein cavity.The excited state proton transfer occurred after illumination,to obtain the ratio fluorescence signal from blue emission to red emission,realizing the wash-free fluorescence imaging of the target proteins.展开更多
Peroxynitrite(ONOO^(-))contributes to oxidative stress and neurodegeneration in Parkinson's disease(PD).Developing a peroxynitrite probe would enable in situ visualization of the overwhelming ONOO^(-)flux and unde...Peroxynitrite(ONOO^(-))contributes to oxidative stress and neurodegeneration in Parkinson's disease(PD).Developing a peroxynitrite probe would enable in situ visualization of the overwhelming ONOO^(-)flux and understanding of the ONOO^(-)stress-induced neuropathology of PD.Herein,a novelα-ketoamide-based fluorogenic probe(DFlu)was designed for ONOO^(-)monitoring in multiple PD models.The results demonstrated that DFlu exhibits a fluorescence turn-on response to ONOO^(-)with high specificity and sensitivity.The efficacy of DFlu for intracellular ONOO^(-)imaging was demonstrated systematically.The results showed that DFlu can successfully visualize endogenous and exogenous ONOO^(-)in cells derived from chemical and biochemical routes.More importantly,the two-photon excitation ability of DFlu has been well demonstrated by monitoring exogenous/endogenous ONOO^(-)production and scavenging in live zebraflsh PD models.This work provides a reliable and promisingα-ketoamide-based optical tool for identifying variations of ONOO^(-)in PD models.展开更多
H2S is an essential gas signal molecule in cells,and viscosity is a key internal environmental parameter.Recent studies have shown that H_(2)S acts as a cytoarchitecture agent and gas transmitter in many tissues,e.g.,...H2S is an essential gas signal molecule in cells,and viscosity is a key internal environmental parameter.Recent studies have shown that H_(2)S acts as a cytoarchitecture agent and gas transmitter in many tissues,e.g.,as a regulator of neuroendocrine in the brain for mediating vascular tone in blood vessels.Mitochondrial viscosity is an important parameter for judging whether mitochondrial function is normal.It has been reported that oxidative stress and mitochondrial dysfunction are connected with Parkinson’s disease(PD),and the protective role of H_(2)S in PD models has been extensively demonstrated.Herein,Mito-HS,a new two-photon fluorescent probe was demonstrated to detect cross-talk between the two channels of mitochondrial viscosity and H_(2)S content.Moreover,this probe could detect the relative amount of and changes in mitochondrial H2S in situ due to the reduced mitochondrial targeting ability after reaction with H_(2)S.The results show that H2S in mitochondria is inversely related to viscosity.The PD model has a lower H2S in mitochondria and a higher mitochondrial viscosity than did the normal.This result is important for our deep understanding of PD and its causes.展开更多
Imaging dynamics of membrane proteins of live cells in a wash-free and real-time manner has been a challenging task. Herein, we report unprecedented applications of malachite green(MG), an organic dye widely used in p...Imaging dynamics of membrane proteins of live cells in a wash-free and real-time manner has been a challenging task. Herein, we report unprecedented applications of malachite green(MG), an organic dye widely used in pigment industry, as a switchable fluorophore to monitor membrane enzymes or noncatalytic proteins in live cells. Conformationally flexible MG is non-fluorescent in aqueous solution, yet covalent binding with endogenous proteins of cells significantly enhances its fluorescence at 670 nm by restricting flexibility of dye. Integrating a phosphate-caged quinone methide precursor with MG yielded a covalent labeling fluorogenic probe, allowing real-time imaging of membrane alkaline phosphatase(ALP,a model catalytic protein) activity in live cells with over 100-fold enhancement of fluorescence intensity.Moreover, MG is also applicable to image non-catalytic protein by conjugation with protein-specific ligand. A fluorogenic probe consisted of c-RGDf K peptide and MG proved to be compatible with wash-free and real-time visualization of non-catalytic integrin α_(v)β_(3) in live cells with high contrast.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
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
Hepatitis and arthritis are prevalent inflammatory diseases,and the utilization of fluorogenic probes incorporating hydrogen sulfide(H_(2)S)as a crucial mediator of inflammation presents significant opportunities for ...Hepatitis and arthritis are prevalent inflammatory diseases,and the utilization of fluorogenic probes incorporating hydrogen sulfide(H_(2)S)as a crucial mediator of inflammation presents significant opportunities for early detection.However,the poor in vivo biodistribution and limited targeted efficacy of molecule probes for inflammation imaging severely impede their ability to differentiate the extent of inflammation and provide real-time monitoring of inflammatory levels.Therefore,we developed a highly efficient H_(2)S-activated near-infrared(NIR)fluorogenic probe(hCy-DNP)for real-time tracking and capturing fluctuations in H_(2)S levels within inflammatory lesions.hCy-DNP demonstrates an exceptionally sensitive fluorescence response to H_(2)S expression,enabling specific differentiation between various levels of lipopolysaccharide(LPS)-stimulated early hepatitis models in situ,while also facilitating visual monitoring for diagnosis and efficacy evaluation of arthritis.Therefore,hCy-DNP offers an innovative tool for exploring early diagnosis and evaluating treatment effectiveness across diverse inflammatory diseases.展开更多
Hepatic ischemia-reperfusion injury(HIRI)is characterized by mitochondrial dysfunction and oxidative stress.Monitoring mitochondrial hydrogen peroxide(mtH_(2)O_(2))levels in real-time through super-resolution imaging ...Hepatic ischemia-reperfusion injury(HIRI)is characterized by mitochondrial dysfunction and oxidative stress.Monitoring mitochondrial hydrogen peroxide(mtH_(2)O_(2))levels in real-time through super-resolution imaging is crucial for elucidating its distribution in live cells and its mechanism of action during HIRI.However,low-background fluorogenic probes have been overlooked in the context of super-resolution imaging.In this study,we developed a low-background fluorogenic probe(MitoWG)with the potential for super-resolution morphology-correlated mitochondrial identification to track the fluctuates of mtH_(2)O_(2)in HIRI.Activation of the desirable fluorescence properties of the probe by mtH_(2)O_(2)was confirmed using structural illumination microscopy(SIM),enabling high-quality mitochondrial imaging with exceptional specificity and sensitivity.Fluctuations in mtH_(2)O_(2)levels were successfully observed in both cellular and rat models of HIRI.Furthermore,we associated the decline in mitochondrial redox homeostasis with accelerated mtH_(2)O_(2)production during HIRI,which triggered mitophagy deficiency and led to cell death.In conclusion,Mito-WG possesses excellent photophysical and low-background properties for SIM imaging,making it a promising tool for mtH_(2)O_(2)tracking in HIRI research and clinical diagnosis.展开更多
Fluorogenic probes with"off-on"fluorescence signals have emerged as powerful tools for biosensing and bioimaging of biomolecules in living systems.Conventional single-target probes,however,often suffer from ...Fluorogenic probes with"off-on"fluorescence signals have emerged as powerful tools for biosensing and bioimaging of biomolecules in living systems.Conventional single-target probes,however,often suffer from false-positive signals due to non-specific activation in non-target tissues or the diffusion of activated fluorescent products.To address these limitations,dual-targeted fluorogenic probes(DTFPs)have been developed,which simultaneously target two biomarkers to enhance detection specificity and minimize false-positive outcomes.DTFPs are designed to activate or retain fluorescence only when both biomarkers are present within a targeted region,enabling precise in vivo imaging of pathological conditions such as tumors and inflammation.This review highlights recent advances in DTFP development,focusing on their design principles,activation mechanisms,and applications in biosensing and bioimaging.We also discuss current challenges and future directions for DTFP research,aiming to inspire the design of next-generation probes with improved accuracy and specificity.By providing a comprehensive overview of DTFPs,this review seeks to advance their potential for transformative applications in biomedical imaging and diagnostics.展开更多
Fluorogenic probes have shown great potential in imaging biological species as well as in diagnosing diseases,especially cancers.However,the fluorogenic mechanisms are largely limited to a few photophysical processes ...Fluorogenic probes have shown great potential in imaging biological species as well as in diagnosing diseases,especially cancers.However,the fluorogenic mechanisms are largely limited to a few photophysical processes to date,typically including photoinduced electron transfer(PeT),fluorescence resonant energy transfer(FRET),and intramolecular charge transfer(ICT).Herein,by calculations and experiments,we set forth that the inhibition of the excited-stateπ-conjugation in meso-ester Si-rhodamine SiR-COOM or the de-π-conjugation in meso-ester cyanine 5 Cy5-COOM via the“ester-to-carboxylate”conversion can operate as a general fluorogenic mechanism to fabricate fluorogenic probes.Based on the mechanism and considering the higher chemical stability of Cy5-COOM than that of SiR-COOM,we developed,as a proof-of-concept,three fluorogenic probes Cy5-APN,Cy5-GGT,and Cy5-NTR on the basis of the Cy5-COOM platform for sensing cancer biomarkers aminopeptidase N(APN),γ-glutamyltranspeptidase(GGT),and nitroreductase(NTR),respectively,and demonstrated their outstanding performances in distinguishing between cancerous and normal tissues with the high tumor-tonormal tissue ratios in the range of 9−14.展开更多
Polarity,as a crucial environmental characteristic,plays a significant role in numerous cellular physiological processes.Abnormal changes in polarity are closely associated with various diseases.However,existing tools...Polarity,as a crucial environmental characteristic,plays a significant role in numerous cellular physiological processes.Abnormal changes in polarity are closely associated with various diseases.However,existing tools still have certain limitations that hinder accurate detection of polarity.Therefore,there is a pressing need to develop powerful tools for precisely monitoring changes in polarity.In this study,we developed two dual-emissive fluorogenic dyes by innovatively introducing 1,3-dithio-2-heteroarsenic cyclopentane and 1,2-diselenocyclopentane respectively into the near-infrared(NIR)coumarin-benzopyranium skeleton to enhance their cellular uptake capability.Additionally,we synthesized the polarity-sensitive dual-emissive fluorogenic probe CSFNS,which exhibits high cellular uptake rate,by modifying the spironolactone(Aldactone)structure of CBA into spirolactam.CSFNS not only demonstrates excellent polarity sensitivity in vitro but is also successfully applied to visually monitor the polarity changes in various types of living cells,including healthy cells,cancer cells and drug-induced senescent cells.展开更多
Single-molecule fluorescence microscopy enables the direct observation of individual reaction events at the surface of a catalyst.It has become a powerful tool to image in real time both intraand interparticle heterog...Single-molecule fluorescence microscopy enables the direct observation of individual reaction events at the surface of a catalyst.It has become a powerful tool to image in real time both intraand interparticle heterogeneity among different nanoscale catalyst particles.Single-molecule fluorescence microscopy of heterogeneous catalysts relies on the detection of chemically activated fluorogenic probes that are converted from a nonfluorescent state into a highly fluorescent state through a reaction mediated at the catalyst surface.This review article describes challenges and opportunities in using such fluorogenic probes as proxies to develop structure−activity relationships in nanoscale electrocatalysts and photocatalysts.We compare single-molecule fluorescence microscopy to other microscopies for imaging catalysis in situ to highlight the distinct advantages and limitations of this technique.We describe correlative imaging between super-resolution activity maps obtained from multiple fluorogenic probes to understand the chemical origins behind spatial variations in activity that are frequently observed for nanoscale catalysts.Fluorogenic probes,originally developed for biological imaging,are introduced that can detect products such as carbon monoxide,nitrite,and ammonia,which are generated by electro-and photocatalysts for fuel production and environmental remediation.We conclude by describing how single-molecule imaging can provide mechanistic insights for a broader scope of catalytic systems,such as single-atom catalysts.展开更多
A variety of bioorthogonal chemical tools have been developed and widely used in the study of biological phenomena in situ.Tetrazine bioorthogonal chemistry exhibits ultrafast reaction kinetics,excellent biocompatibil...A variety of bioorthogonal chemical tools have been developed and widely used in the study of biological phenomena in situ.Tetrazine bioorthogonal chemistry exhibits ultrafast reaction kinetics,excellent biocompatibility,and precise optical regulatory capabilities.Fluorogenic tetrazine bioorthogonal probes have achieved particularly diverse applications in bioimaging and disease diagnosis and treatment.This Viewpoint briefly introduces the characteristics and advantages of tetrazine bioorthogonal chemistry,some design strategies of fluorogenic tetrazine probes,and the status of applications of these tools to in vivo imaging,as well as disease diagnosis and treatment.Finally,we discuss challenges and propose future trends in the field of fluorogenic tetrazine probes.This Viewpoint offers insights into the development of new bioorthogonal tools for chemical biology research and for the design of new drugs.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
Herein we presented a general strategy for in situ assembly of intramolecular charge-transfer(ICT)-based light-up fluorophores via bioorthogonal Suzuki-Miyaura cross-coupling reaction.By introducing iodo group at the ...Herein we presented a general strategy for in situ assembly of intramolecular charge-transfer(ICT)-based light-up fluorophores via bioorthogonal Suzuki-Miyaura cross-coupling reaction.By introducing iodo group at the appropriate position,five fluorophores with different scaffolds including naphthalimide,coumarin,naphthalene sulfonate,nitrobenzoxadiazole,and acetonaphthone,were designed as bioorthogonal multicolor fluorogenic probes,which could produce significant fluorescence enhancement and high fluorescence quantum yield after Suzuki-Miyaura reaction with aryl boronic acid or boronate.Manipulating the substituents andπscaffold in the fluorophores allows fine-tuning of their photophysical properties.With this strategy,we succeeded in peptide conjugation,no-wash fluorogenic protein labeling,and mitochondria-selective bioorthogonal imaging in live cells.展开更多
As one of the major causes of antimicrobial resistance,β‐lactamase develops rapidly among bacteria.Detection of β‐lactamase in an efficient and low‐cost point‐of‐care testing(POCT)way is urgently needed.However...As one of the major causes of antimicrobial resistance,β‐lactamase develops rapidly among bacteria.Detection of β‐lactamase in an efficient and low‐cost point‐of‐care testing(POCT)way is urgently needed.However,due to the volatile environmental factors,the quantitative measurement of current POCT is often inaccurate.Herein,we demonstrate an artificial intelligence(AI)‐assisted mobile health system that consists of a paper‐basedβ‐lactamase fluorogenic probe analytical device and a smartphone‐based AI cloud.An ultrafast broad‐spectrum fluorogenic probe(B1)that could respond toβ‐lactamase within 20 s was first synthesized,and the detection limit was determined to be 0.13 nmol/L.Meanwhile,a three‐dimensional microfluidic paper‐based analytical device was fabricated for integration of B1.Also,a smartphone‐based AI cloud was developed to correct errors automatically and output results intelligently.This smart system could calibrate the temperature and pH in theβ‐lactamase level detection in complex samples and mice infected with various bacteria,which shows the problem‐solving ability in interdisciplinary research,and demonstrates potential clinical benefits.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
基金financially supported by the National Natural Science Foundation of China(Nos.22077101,22004099)the Joint Research Funds of Department of Science&Technology of Shaanxi Province and Northwestern Polytechnical University(Nos.2020GXLH-Z-008,2020GXLH-Z-021,2020GXLH-Z-023)+4 种基金Natural Science Foundation of Shaanxi Province(No.2022JM-130)The Natural Science Foundation of Ningbo(Nos.202003N4049,202003N4065)the Open Project Program of Wuhan National Laboratory for Optoelectronics(Nos.2020WNLOKF023,2022WNLOKF009)Innovation Foundation for Doctor Dissertation of Northwestern Polytechnical University(No.CX2022034)Innovation Capability Support Program of Shaanxi(No.2023-CX-PT-23)。
文摘Hepatic ischemia-reperfusion injury(HIRI)is the cause of postoperative hepatic dysfunction and failure,and even death.As an important biological effector molecule,hydrogen sulfide(H_(2)S)of mitochondria as a gasotransmitter that is usually used to protect against acute HIRI injury.However,the exact relationship between HIRI and mitochondrial H_(2)S remains tangled due to the lack of an effective analytical method.Herein,we have fabricated a mitochondria-targeted H_(2)S-activatable fluorogenic probe(Mito-GW)to explore the stability of mitochondrial H_(2)S and track the changes of mitochondrial H_(2)S during the HIRI.By virtue of pyridinium electropositivity and its amphiphilicity,Mito-GW could accumulate in mitochondria.It goes through an analyte-prompted immolation when reacts with H_(2)S,resulting in the releasing of the fluorophore(GW).Therefore,the extent of Mito-GW conversion to GW can be used to evaluate the changes of mitochondrial H_(2)S level in living cells and tissues.As proof-of-principle,we have used MitoGW to demonstrate the mitochondria H_(2)S-levels increase and then decrease during HIRI in vitro and in vivo.Our research highlights the tremendous potential of Mito-GW as a mitochondrial H_(2)S fluorogenic probe in elucidating the pathogenesis of HIRI,providing a powerful tool for promoting future research on hepatology.
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supported by the National Natural Science Foundation of China(Nos.22225806,22078314 and 22278394)Dalian Institute of Chemical Physics(Nos.DICPI202227 and DICPI202142)。
文摘Protein self-labeling tags achieve selective fusion and labeling of target proteins through genetic coding technology,but require exogenous fluorescent probes with fluorogenicity for protein tag binding to have the performance of wash-free fluorescence imaging in live cells.In this paper,we reported a fluorogenic probe 1 capable of ratiometric fluorescence recognition of SNAP-tag proteins.In this probe,the O6-benzylguanine derivative of 3-hydroxy-1,8-naphthalimide underwent a selective covalent linkage reaction with SNAP-tag protein.The hydroxyl group on the naphthalimide fluorophore formed a hydrogen bond with the functional group near the protein cavity.The excited state proton transfer occurred after illumination,to obtain the ratio fluorescence signal from blue emission to red emission,realizing the wash-free fluorescence imaging of the target proteins.
基金supported by the National Natural Science Foundation of China(22077101)the Open Project Program of Wuhan National Laboratory for Optoelectronics(No.2020WNLOKF023)+4 种基金Natural Science Foundation of Shaanxi Province(2022JM-130)the Joint Research Funds of Department of Science&Technology of Shaanxi Province and Northwestern Polytechnical University(2020GXLH-Z-008,2020GXLH-Z-021,2020GXLH-Z-023)the China Postdoctoral Science Foundation(2022M711595,2022M722595)Postdoctoral Research Funding Schemes of Jiangsu Province(2021K036A)The Natural Science Foundation of Ningbo(202003N4049,202003N4065).
文摘Peroxynitrite(ONOO^(-))contributes to oxidative stress and neurodegeneration in Parkinson's disease(PD).Developing a peroxynitrite probe would enable in situ visualization of the overwhelming ONOO^(-)flux and understanding of the ONOO^(-)stress-induced neuropathology of PD.Herein,a novelα-ketoamide-based fluorogenic probe(DFlu)was designed for ONOO^(-)monitoring in multiple PD models.The results demonstrated that DFlu exhibits a fluorescence turn-on response to ONOO^(-)with high specificity and sensitivity.The efficacy of DFlu for intracellular ONOO^(-)imaging was demonstrated systematically.The results showed that DFlu can successfully visualize endogenous and exogenous ONOO^(-)in cells derived from chemical and biochemical routes.More importantly,the two-photon excitation ability of DFlu has been well demonstrated by monitoring exogenous/endogenous ONOO^(-)production and scavenging in live zebraflsh PD models.This work provides a reliable and promisingα-ketoamide-based optical tool for identifying variations of ONOO^(-)in PD models.
基金the National Natural Science Foundation of China(Nos.81672508,21675085)Jiangsu Provincial Foundation for Distinguished Young Scholars(Nos.BK20170041,BK20170042)+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H2S is an essential gas signal molecule in cells,and viscosity is a key internal environmental parameter.Recent studies have shown that H_(2)S acts as a cytoarchitecture agent and gas transmitter in many tissues,e.g.,as a regulator of neuroendocrine in the brain for mediating vascular tone in blood vessels.Mitochondrial viscosity is an important parameter for judging whether mitochondrial function is normal.It has been reported that oxidative stress and mitochondrial dysfunction are connected with Parkinson’s disease(PD),and the protective role of H_(2)S in PD models has been extensively demonstrated.Herein,Mito-HS,a new two-photon fluorescent probe was demonstrated to detect cross-talk between the two channels of mitochondrial viscosity and H_(2)S content.Moreover,this probe could detect the relative amount of and changes in mitochondrial H2S in situ due to the reduced mitochondrial targeting ability after reaction with H_(2)S.The results show that H2S in mitochondria is inversely related to viscosity.The PD model has a lower H2S in mitochondria and a higher mitochondrial viscosity than did the normal.This result is important for our deep understanding of PD and its causes.
基金financially supported by the National Natural Science Foundation of China (No. 22077031)the Research Program of State Key Laboratory of Bioreactor Engineeringthe Fundamental Research Funds for the Central Universities。
文摘Imaging dynamics of membrane proteins of live cells in a wash-free and real-time manner has been a challenging task. Herein, we report unprecedented applications of malachite green(MG), an organic dye widely used in pigment industry, as a switchable fluorophore to monitor membrane enzymes or noncatalytic proteins in live cells. Conformationally flexible MG is non-fluorescent in aqueous solution, yet covalent binding with endogenous proteins of cells significantly enhances its fluorescence at 670 nm by restricting flexibility of dye. Integrating a phosphate-caged quinone methide precursor with MG yielded a covalent labeling fluorogenic probe, allowing real-time imaging of membrane alkaline phosphatase(ALP,a model catalytic protein) activity in live cells with over 100-fold enhancement of fluorescence intensity.Moreover, MG is also applicable to image non-catalytic protein by conjugation with protein-specific ligand. A fluorogenic probe consisted of c-RGDf K peptide and MG proved to be compatible with wash-free and real-time visualization of non-catalytic integrin α_(v)β_(3) in live cells with high contrast.
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National Key Research&Development Program of China,Grant/Award Numbers:2021YFA1201000,2022YFA1207300National Natural Science Foundation of China,Grant/Award Numbers:22274005,U2067214,22334001,U2167222The Beijing Outstanding Young Scientist Program,Grant/Award Number:BJJWZYJH01201910005017。
文摘Hepatitis and arthritis are prevalent inflammatory diseases,and the utilization of fluorogenic probes incorporating hydrogen sulfide(H_(2)S)as a crucial mediator of inflammation presents significant opportunities for early detection.However,the poor in vivo biodistribution and limited targeted efficacy of molecule probes for inflammation imaging severely impede their ability to differentiate the extent of inflammation and provide real-time monitoring of inflammatory levels.Therefore,we developed a highly efficient H_(2)S-activated near-infrared(NIR)fluorogenic probe(hCy-DNP)for real-time tracking and capturing fluctuations in H_(2)S levels within inflammatory lesions.hCy-DNP demonstrates an exceptionally sensitive fluorescence response to H_(2)S expression,enabling specific differentiation between various levels of lipopolysaccharide(LPS)-stimulated early hepatitis models in situ,while also facilitating visual monitoring for diagnosis and efficacy evaluation of arthritis.Therefore,hCy-DNP offers an innovative tool for exploring early diagnosis and evaluating treatment effectiveness across diverse inflammatory diseases.
基金supported by the National Natural Science Foundation of China(62288102,22077101,62475216)the Natural Science Foundation of Shaanxi Province(2022JM-130)+7 种基金the Key Research and Development Program of Shaanxi(2024GH-ZDXM-37)the Innovation Capability Support Program of Shaanxi(2023-CX-PT-23)the Natural Science Basic Research Program of Shaanxi(2024JC-YBQN-0919)the Open Project Program of the Analytical&Testing Center of Northwestern Polytechnical University(2020T018)the Postdoctoral Fellowship Program of China Postdoctoral Science Foundation(CPSF)(GZC20240889)the Innovation Foundation for Doctor Dissertation of Northwestern Polytechnical University(CX2022034,CX2023098)the Startup Program of XMU(L.L.)Fundamental Research Funds for the Central Universities。
文摘Hepatic ischemia-reperfusion injury(HIRI)is characterized by mitochondrial dysfunction and oxidative stress.Monitoring mitochondrial hydrogen peroxide(mtH_(2)O_(2))levels in real-time through super-resolution imaging is crucial for elucidating its distribution in live cells and its mechanism of action during HIRI.However,low-background fluorogenic probes have been overlooked in the context of super-resolution imaging.In this study,we developed a low-background fluorogenic probe(MitoWG)with the potential for super-resolution morphology-correlated mitochondrial identification to track the fluctuates of mtH_(2)O_(2)in HIRI.Activation of the desirable fluorescence properties of the probe by mtH_(2)O_(2)was confirmed using structural illumination microscopy(SIM),enabling high-quality mitochondrial imaging with exceptional specificity and sensitivity.Fluctuations in mtH_(2)O_(2)levels were successfully observed in both cellular and rat models of HIRI.Furthermore,we associated the decline in mitochondrial redox homeostasis with accelerated mtH_(2)O_(2)production during HIRI,which triggered mitophagy deficiency and led to cell death.In conclusion,Mito-WG possesses excellent photophysical and low-background properties for SIM imaging,making it a promising tool for mtH_(2)O_(2)tracking in HIRI research and clinical diagnosis.
基金the National Natural Science Foundation of China(22137003 and 21922406)Natural Science Foundation of Jiangsu Province(BK20200301 and BK20190055)the Fundamental Research Funds for the Central Universities(020514380251)are acknowledged.
文摘Fluorogenic probes with"off-on"fluorescence signals have emerged as powerful tools for biosensing and bioimaging of biomolecules in living systems.Conventional single-target probes,however,often suffer from false-positive signals due to non-specific activation in non-target tissues or the diffusion of activated fluorescent products.To address these limitations,dual-targeted fluorogenic probes(DTFPs)have been developed,which simultaneously target two biomarkers to enhance detection specificity and minimize false-positive outcomes.DTFPs are designed to activate or retain fluorescence only when both biomarkers are present within a targeted region,enabling precise in vivo imaging of pathological conditions such as tumors and inflammation.This review highlights recent advances in DTFP development,focusing on their design principles,activation mechanisms,and applications in biosensing and bioimaging.We also discuss current challenges and future directions for DTFP research,aiming to inspire the design of next-generation probes with improved accuracy and specificity.By providing a comprehensive overview of DTFPs,this review seeks to advance their potential for transformative applications in biomedical imaging and diagnostics.
基金supported by the National Natural Science Foundation of China(Nos.22277070,22274091,22007061)the Youth Talent Support Program of Shanxi Province,the Program for the Top Young Academic Leaders of Higher Learning Institutions of Shanxi,and the fundamental research program of Shanxi province(20210302123445).
文摘Fluorogenic probes have shown great potential in imaging biological species as well as in diagnosing diseases,especially cancers.However,the fluorogenic mechanisms are largely limited to a few photophysical processes to date,typically including photoinduced electron transfer(PeT),fluorescence resonant energy transfer(FRET),and intramolecular charge transfer(ICT).Herein,by calculations and experiments,we set forth that the inhibition of the excited-stateπ-conjugation in meso-ester Si-rhodamine SiR-COOM or the de-π-conjugation in meso-ester cyanine 5 Cy5-COOM via the“ester-to-carboxylate”conversion can operate as a general fluorogenic mechanism to fabricate fluorogenic probes.Based on the mechanism and considering the higher chemical stability of Cy5-COOM than that of SiR-COOM,we developed,as a proof-of-concept,three fluorogenic probes Cy5-APN,Cy5-GGT,and Cy5-NTR on the basis of the Cy5-COOM platform for sensing cancer biomarkers aminopeptidase N(APN),γ-glutamyltranspeptidase(GGT),and nitroreductase(NTR),respectively,and demonstrated their outstanding performances in distinguishing between cancerous and normal tissues with the high tumor-tonormal tissue ratios in the range of 9−14.
基金the financial support from the National Natural Science Foundation of China(Nos.22477101,22277098 and 22037002)the Natural Science Basic Research Plan for Distinguished Young Scholars in Shaanxi Province of China(No.2020JC-38)the Shaanxi Fundamental Science Research Project for Chemistry and Biology(No.22JHQ070)。
文摘Polarity,as a crucial environmental characteristic,plays a significant role in numerous cellular physiological processes.Abnormal changes in polarity are closely associated with various diseases.However,existing tools still have certain limitations that hinder accurate detection of polarity.Therefore,there is a pressing need to develop powerful tools for precisely monitoring changes in polarity.In this study,we developed two dual-emissive fluorogenic dyes by innovatively introducing 1,3-dithio-2-heteroarsenic cyclopentane and 1,2-diselenocyclopentane respectively into the near-infrared(NIR)coumarin-benzopyranium skeleton to enhance their cellular uptake capability.Additionally,we synthesized the polarity-sensitive dual-emissive fluorogenic probe CSFNS,which exhibits high cellular uptake rate,by modifying the spironolactone(Aldactone)structure of CBA into spirolactam.CSFNS not only demonstrates excellent polarity sensitivity in vitro but is also successfully applied to visually monitor the polarity changes in various types of living cells,including healthy cells,cancer cells and drug-induced senescent cells.
基金supported by the National Science Foundation(NSF)under grant no.CHE-1753344 to B.S.The Authors thank J.S.Bates for useful discussions regarding SACs and EXAFS.
文摘Single-molecule fluorescence microscopy enables the direct observation of individual reaction events at the surface of a catalyst.It has become a powerful tool to image in real time both intraand interparticle heterogeneity among different nanoscale catalyst particles.Single-molecule fluorescence microscopy of heterogeneous catalysts relies on the detection of chemically activated fluorogenic probes that are converted from a nonfluorescent state into a highly fluorescent state through a reaction mediated at the catalyst surface.This review article describes challenges and opportunities in using such fluorogenic probes as proxies to develop structure−activity relationships in nanoscale electrocatalysts and photocatalysts.We compare single-molecule fluorescence microscopy to other microscopies for imaging catalysis in situ to highlight the distinct advantages and limitations of this technique.We describe correlative imaging between super-resolution activity maps obtained from multiple fluorogenic probes to understand the chemical origins behind spatial variations in activity that are frequently observed for nanoscale catalysts.Fluorogenic probes,originally developed for biological imaging,are introduced that can detect products such as carbon monoxide,nitrite,and ammonia,which are generated by electro-and photocatalysts for fuel production and environmental remediation.We conclude by describing how single-molecule imaging can provide mechanistic insights for a broader scope of catalytic systems,such as single-atom catalysts.
基金support from the National Natural Science Foundation of China(No.22277083)Natural Science Foundation of Sichuan Province(Grant Nos.2023NSFSC53332024NSFTD0023).
文摘A variety of bioorthogonal chemical tools have been developed and widely used in the study of biological phenomena in situ.Tetrazine bioorthogonal chemistry exhibits ultrafast reaction kinetics,excellent biocompatibility,and precise optical regulatory capabilities.Fluorogenic tetrazine bioorthogonal probes have achieved particularly diverse applications in bioimaging and disease diagnosis and treatment.This Viewpoint briefly introduces the characteristics and advantages of tetrazine bioorthogonal chemistry,some design strategies of fluorogenic tetrazine probes,and the status of applications of these tools to in vivo imaging,as well as disease diagnosis and treatment.Finally,we discuss challenges and propose future trends in the field of fluorogenic tetrazine probes.This Viewpoint offers insights into the development of new bioorthogonal tools for chemical biology research and for the design of new drugs.
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.
基金supported by the Beijing Nova Program(No.Z201100006820049)the National Natural Science Foundation of China(No.21907109)the CAMS Innovation Fund for Graduate Students(No.2019–1007–03)
文摘Herein we presented a general strategy for in situ assembly of intramolecular charge-transfer(ICT)-based light-up fluorophores via bioorthogonal Suzuki-Miyaura cross-coupling reaction.By introducing iodo group at the appropriate position,five fluorophores with different scaffolds including naphthalimide,coumarin,naphthalene sulfonate,nitrobenzoxadiazole,and acetonaphthone,were designed as bioorthogonal multicolor fluorogenic probes,which could produce significant fluorescence enhancement and high fluorescence quantum yield after Suzuki-Miyaura reaction with aryl boronic acid or boronate.Manipulating the substituents andπscaffold in the fluorophores allows fine-tuning of their photophysical properties.With this strategy,we succeeded in peptide conjugation,no-wash fluorogenic protein labeling,and mitochondria-selective bioorthogonal imaging in live cells.
基金supported by the National Key R&D Program of China(No.2020YFA0709900)the National Natural Science Foundation of China(Nos.62288102,22077101,and 52073230)+3 种基金the Joint Research Funds of Department of Science&Technology of Shaanxi Province and Northwestern Polytechnical University(Nos.2020GXLH‐Z‐008 and 2020GXLH‐Z‐013)the Key Research and Development Program of Shaanxi(No.2022ZDLGY13‐04)Shanxi Provincial Science Fund for Distinguished Young Scholars(No.2023‐JC‐JQ‐32)Fundamental Research Funds for the Central Universities,and the Innovation Foundation for Doctorate Dissertation of Northwestern Polytechnical University(No.CX2021121).
文摘As one of the major causes of antimicrobial resistance,β‐lactamase develops rapidly among bacteria.Detection of β‐lactamase in an efficient and low‐cost point‐of‐care testing(POCT)way is urgently needed.However,due to the volatile environmental factors,the quantitative measurement of current POCT is often inaccurate.Herein,we demonstrate an artificial intelligence(AI)‐assisted mobile health system that consists of a paper‐basedβ‐lactamase fluorogenic probe analytical device and a smartphone‐based AI cloud.An ultrafast broad‐spectrum fluorogenic probe(B1)that could respond toβ‐lactamase within 20 s was first synthesized,and the detection limit was determined to be 0.13 nmol/L.Meanwhile,a three‐dimensional microfluidic paper‐based analytical device was fabricated for integration of B1.Also,a smartphone‐based AI cloud was developed to correct errors automatically and output results intelligently.This smart system could calibrate the temperature and pH in theβ‐lactamase level detection in complex samples and mice infected with various bacteria,which shows the problem‐solving ability in interdisciplinary research,and demonstrates potential clinical benefits.
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.
