With increasing industrial activities,mercury has been largely discharged into environment and caused serious environmental problems.The growing level of mercury pollution has become a huge threat to human health due ...With increasing industrial activities,mercury has been largely discharged into environment and caused serious environmental problems.The growing level of mercury pollution has become a huge threat to human health due to its significant biotoxicity.Therefore,the simple and fast means for on-site monitoring discharged mercury pollution are highly necessary to protect human beings from its pernicious effects in time.Herein,a"turn off"fluorescent biosensor(mCherry L199 C)for sensing Hg2+was successfully designed based on direct modification of the chromophore environment of fluorescent protein mCherry.For rapid screening and characterization,the designed variant of mCherry(mCherry L199 C)was directly expressed on outer-membrane of Escherichia coli cells by cell surface display technique.The fluorescent biosensor was characterized to have favorable response to Hg2+at micromole level among other metal ions and over a broad pH range.Further,the cells of the fluorescent biosensor were encapsulated in alginate hydrogel to develop the cells-alginate hydrogel-based paper.The cells-alginate hydrogel-based paper could detect mercury pollution in 5 min with simple operation process and inexpensive equipment,and it could keep fluorescence and activity stable at 4℃ for 24 hr,which would be a high-throughput screening tool in preliminarily reporting the presence of mercury pollution in natural setting.展开更多
Detection of N-acyl homoserine lactones （AHLs） is useful for understanding quorum sensing （QS） behaviors, including biofilm formation, virulence and metabolism. For detecting AHLs and indicating the host cells in ...Detection of N-acyl homoserine lactones （AHLs） is useful for understanding quorum sensing （QS） behaviors, including biofilm formation, virulence and metabolism. For detecting AHLs and indicating the host cells in situ, we constructed the plasmid pUCGMA2T1-4 to make a dual fluorescent whole- cell biosensor based on the AhlI/R AHL system of Pseudomonas syringae pv. syringae B728a. The plasmid contains three components： constitutively expressed enptll：：gfP for indicating host cells, Pahll：：mcherry that produces red fluorescence in response to AHL, and the ahIR gene that encodes an AHL regulatory protein. Meanwhile, two copies of T1-4 （four tandem copies of a transcriptional terminator） were added into the plasmid to reduce background. The results showed that when the plasmid was placed into Escherichia coli, the dual fluorescence whole-cell biosensor was able to respond with red fluorescence within 6 hr to 5 × 10^-8-1 × 10^-5 mol/L of 3OC6-HSL. Bright green fluorescence indicated the host cells. Furthermore, when the plasmid was transferred to wild- type Pseudomonas PhTA125 （an AHL-producing bacterium）, it also showed both green and red fluorescence. This result demonstrates that this plasmid can be used to construct whole-cell indicators that can indicate the AHL response and spatial behaviors of microbes in a mi tal niche展开更多
Here, we report an efficient fluorescence biosensor for chondroitin sulfate（CHS） based on polyelectrolyte microspheres of carboxymethyl cellulose（CMC） and chitosan（CS） composites inducing the aggregation of grap...Here, we report an efficient fluorescence biosensor for chondroitin sulfate（CHS） based on polyelectrolyte microspheres of carboxymethyl cellulose（CMC） and chitosan（CS） composites inducing the aggregation of graphene quantum dots（GQDs）, calling CMC/CS-GQDs. The polyelectrolyte microspheres（CMC/CS microspheres） were fabricated by using anioniccationic electrostatic attraction between CMC and CS by high voltage electrostatic spray technology. The aggregating process of GQDs was based on the anionic-cationic electrostatic attraction as well. After combing with the polyelectrolyte microspheres, the fluorescence of GQDs disappeared. CHS, which widely consists in the cell surface of human beings and animals, carries a large number of negative charges on the surface. The addition of CHS enabled CHS and GQDs to compete with each other to composite with the CMC/CS microshpheres. As a result of the higher surface charge density of CHS, CMC/CS-CHS formed accompanied by the release of GQDs, and the fluorescence of the system recovered. The CHS content was detected by analyzing the system＇s fluorescence recovery, which suggested that the obtained fluorescence biosensor can accurately detect the concentration of CHS. The test results showed that the linear range of the fluorescence recovery for this biosensor with respect to CHS was 0~12.00 mg/mL, and the detection limit was 10-8 M. Besides, to test the stability of the biosensor, the CMC/CS-GQDs micropsheres persisted for one month, with a low fluorescence quenching of 9.48%. These results suggested that CMC/CS-GQDs can be utilized as efficient fluorescence biosensor for the detection of CHS. Moreover, the detection method was simple and efficient, and could be widely popularized.展开更多
Ratiometric DNA biosensors,which utilize DNA as the recognition element and integrate dual signals from diverse sensing platforms including fluorescence,electrochemistry,and surface-enhanced Raman scattering(SERS),hav...Ratiometric DNA biosensors,which utilize DNA as the recognition element and integrate dual signals from diverse sensing platforms including fluorescence,electrochemistry,and surface-enhanced Raman scattering(SERS),have demonstrated remarkable proficiency in detecting a wide range of targets.In this review,we showcase the significant progress achieved by ratiometric biosensors.Firstly,ratiometric biosensors have made notable advancements in analyzing real samples.These include the analysis of pH values near cancer cells,quantification of miRNA in human cell lysates,detection of human telomerase RNA in cell extracts,and performing DNA logicgated in situ bioimaging on cell membranes.Secondly,excellent sensitivity has been attained through the utilization of effective amplification methods such as RCA,HCR,and CHA,among others.Thirdly,the construction of stable reference signals has resulted in significantly improved precision for ratiometric biosensors.This breakthrough has overcome matrix effects,enabling reliable detection in real samples with high selectivity.This review provides a comprehensive overview of recent advancements in strategies employed by ratiometric DNA biosensors.We present three types of biosensors based on distinct sensing platforms:fluorescent,electrochemical,and SERS biosensors.Additionally,we discuss future directions and primary challenges in the development of ratiometric DNA biosensors.展开更多
A reliable and sensitive strategy which can assess nucleic acid levels in living cells would be essential for fundamental research of biomedical applications. Some nanomaterial-based fluorescence biosensors recently d...A reliable and sensitive strategy which can assess nucleic acid levels in living cells would be essential for fundamental research of biomedical applications. Some nanomaterial-based fluorescence biosensors recently developed for detecting nucleic acids, however, are often with expensive, complicated and timeconsuming preparation process. Here, by using a facile bottom-up synthesis method, a two-dimensional(2 D) coordination polymer(CP) nanosheet, [Cu(tz)](Htz = 1,2,4-triazole), was successfully prepared after optimizing reaction conditions. These ultrathin CP nanosheets with thickness of 4.7 ± 1.1 nm could readily form nanosensors by assembly with DNA probes, which exhibited a low limit of detection(LOD)for p53 DNA fragment as 144 pmol/L. Furthermore, by integrating [Cu(tz)] nanosheets with hybridization chain reaction(HCR) probes, mi R-21, one kind of micro RNA upregulated in many cancer cells, can be sensitively detected with a LOD of 100 pmol/L and monitored in living cells, giving consistent results with those obtained by quantitative reverse-transcription polymerase chain reaction(q RT-PCR) analysis.Thus [Cu(tz)] nanosheets, which not only possess much better nucleic acids sensing performance than bulk cystals, but also exhibit nucleic acid delivery functions, could be used as a novel nanoplatform in biomedical imaging and sensing applications.展开更多
Sensitive detection and accurate diagnosis/prognosis of glioma remain urgent challenges.Herein,dispersed magnetic covalent organic framework nanospheres(MCOF)with uniformed Fe3O4 nano-assembly as cores and high-crysta...Sensitive detection and accurate diagnosis/prognosis of glioma remain urgent challenges.Herein,dispersed magnetic covalent organic framework nanospheres(MCOF)with uniformed Fe3O4 nano-assembly as cores and high-crystalline COF as shells were prepared by monomer-mediated in-situ interface growth strategy.Based on the unique interaction between MCOF and hairpin DNA,a fluorescent signal amplified miRNA biosensor was constructed.It could realize the sensitive detection of miRNA-182 in different matrixes,where the detection limit,linearity range and determination coefficient(R^(2))in real blood samples reached 20 fM,0.1 pM-10 pM and 0.991,respectively.Also,it possessed good stability and precision as observed from the low intra-day/inter-day RSD and high extraction recovery.As a result,it could quantify miRNA-182 in serum of glioma patients,the concentration of which was significantly higher than that of healthy people and obviously decreased after surgery.Finally,a proof-of-concept capillary chip system using this biosensor was proposed to realize the visualized detection of miRNA-182 in microsample.These findings suggest a robust way for sensitive detection and accurate diagnosis/prognosis of glioma.展开更多
G protein-coupled receptors(GPCRs),crucial for diverse physiological responses,have traditionally been investigated in their monomeric form.However,some GPCRs can form heteromers,revealing complexity in their function...G protein-coupled receptors(GPCRs),crucial for diverse physiological responses,have traditionally been investigated in their monomeric form.However,some GPCRs can form heteromers,revealing complexity in their functional characteristics such as ligand binding properties,downstream signaling pathways,and trafficking.Understanding GPCR heteromers is crucial in both physiological contexts and drug development.Here,we review the methodologies for investigating physical interactions in GPCR heteromers,including co-immunoprecipitation,proximity ligation assays,interfering peptide approaches,and live cell imaging techniques based on resonance energy transfer and bimolecular fluorescence complementation.In addition,we discuss recent advances in live cell imaging techniques for exploring functional features of GPCR heteromers,for example,circularly permuted fluorescent protein-based GPCR biosensors,TRUPATH,and nanobody-based GPCR biosensors.These advanced biosensors and live cell imaging technologies promise a deeper understanding of GPCR heteromers,urging a reassessment of their physiological importance and pharmacological relevance.展开更多
Background:Commitment to a new cell cycle is controlled by a number of cellular signals.Mitogen-activated protein kinase(MAPK)pathways,which transduce multiple extracellular cues,have been shown to be interconnected w...Background:Commitment to a new cell cycle is controlled by a number of cellular signals.Mitogen-activated protein kinase(MAPK)pathways,which transduce multiple extracellular cues,have been shown to be interconnected with the cell cycle and can modulate its progression.Methods:In budding yeast,we have introduced fluorescent biosensors that monitor in real time the signaling activity of the MAPKs Fus3 and Kssl and the cyclin-dependent kinase(CDK)in individual cells.We have quantified in hundreds of live single cells the interplay between the MAPKs regulating the mating response and the CDK controlling cell cycle progression.Results:Different patterns of MAPK activity dynamics could be identified by clustering cells based on their CDK activity,denoting the tight relationship between these two cellular signals.Our data suggest that beyond the already well-established mechanisms of regulation between the MAPK and the CDK,additional mechanisms remain to be identified.Conclusion:A tight interplay between MAPK pathways and the cell cycle is essential to control cellular proliferation and cell fate decisions.展开更多
基金supported by the Natural Science Foundation of Guangxi Province(No.2018JJB120049)National Natural Science Foundation of China(No.31800631)+1 种基金the BAGUI Scholar Program of Guangxi Province of China。
文摘With increasing industrial activities,mercury has been largely discharged into environment and caused serious environmental problems.The growing level of mercury pollution has become a huge threat to human health due to its significant biotoxicity.Therefore,the simple and fast means for on-site monitoring discharged mercury pollution are highly necessary to protect human beings from its pernicious effects in time.Herein,a"turn off"fluorescent biosensor(mCherry L199 C)for sensing Hg2+was successfully designed based on direct modification of the chromophore environment of fluorescent protein mCherry.For rapid screening and characterization,the designed variant of mCherry(mCherry L199 C)was directly expressed on outer-membrane of Escherichia coli cells by cell surface display technique.The fluorescent biosensor was characterized to have favorable response to Hg2+at micromole level among other metal ions and over a broad pH range.Further,the cells of the fluorescent biosensor were encapsulated in alginate hydrogel to develop the cells-alginate hydrogel-based paper.The cells-alginate hydrogel-based paper could detect mercury pollution in 5 min with simple operation process and inexpensive equipment,and it could keep fluorescence and activity stable at 4℃ for 24 hr,which would be a high-throughput screening tool in preliminarily reporting the presence of mercury pollution in natural setting.
基金supported by the National Natural Science Foundation of China (No. 2117145)
文摘Detection of N-acyl homoserine lactones （AHLs） is useful for understanding quorum sensing （QS） behaviors, including biofilm formation, virulence and metabolism. For detecting AHLs and indicating the host cells in situ, we constructed the plasmid pUCGMA2T1-4 to make a dual fluorescent whole- cell biosensor based on the AhlI/R AHL system of Pseudomonas syringae pv. syringae B728a. The plasmid contains three components： constitutively expressed enptll：：gfP for indicating host cells, Pahll：：mcherry that produces red fluorescence in response to AHL, and the ahIR gene that encodes an AHL regulatory protein. Meanwhile, two copies of T1-4 （four tandem copies of a transcriptional terminator） were added into the plasmid to reduce background. The results showed that when the plasmid was placed into Escherichia coli, the dual fluorescence whole-cell biosensor was able to respond with red fluorescence within 6 hr to 5 × 10^-8-1 × 10^-5 mol/L of 3OC6-HSL. Bright green fluorescence indicated the host cells. Furthermore, when the plasmid was transferred to wild- type Pseudomonas PhTA125 （an AHL-producing bacterium）, it also showed both green and red fluorescence. This result demonstrates that this plasmid can be used to construct whole-cell indicators that can indicate the AHL response and spatial behaviors of microbes in a mi tal niche
文摘Here, we report an efficient fluorescence biosensor for chondroitin sulfate（CHS） based on polyelectrolyte microspheres of carboxymethyl cellulose（CMC） and chitosan（CS） composites inducing the aggregation of graphene quantum dots（GQDs）, calling CMC/CS-GQDs. The polyelectrolyte microspheres（CMC/CS microspheres） were fabricated by using anioniccationic electrostatic attraction between CMC and CS by high voltage electrostatic spray technology. The aggregating process of GQDs was based on the anionic-cationic electrostatic attraction as well. After combing with the polyelectrolyte microspheres, the fluorescence of GQDs disappeared. CHS, which widely consists in the cell surface of human beings and animals, carries a large number of negative charges on the surface. The addition of CHS enabled CHS and GQDs to compete with each other to composite with the CMC/CS microshpheres. As a result of the higher surface charge density of CHS, CMC/CS-CHS formed accompanied by the release of GQDs, and the fluorescence of the system recovered. The CHS content was detected by analyzing the system＇s fluorescence recovery, which suggested that the obtained fluorescence biosensor can accurately detect the concentration of CHS. The test results showed that the linear range of the fluorescence recovery for this biosensor with respect to CHS was 0~12.00 mg/mL, and the detection limit was 10-8 M. Besides, to test the stability of the biosensor, the CMC/CS-GQDs micropsheres persisted for one month, with a low fluorescence quenching of 9.48%. These results suggested that CMC/CS-GQDs can be utilized as efficient fluorescence biosensor for the detection of CHS. Moreover, the detection method was simple and efficient, and could be widely popularized.
基金financially supported by:The National Quality Infrastructure Program of China(2021YFF0600705 NQI)the Natural Science Foundation of China(No.22074093).
文摘Ratiometric DNA biosensors,which utilize DNA as the recognition element and integrate dual signals from diverse sensing platforms including fluorescence,electrochemistry,and surface-enhanced Raman scattering(SERS),have demonstrated remarkable proficiency in detecting a wide range of targets.In this review,we showcase the significant progress achieved by ratiometric biosensors.Firstly,ratiometric biosensors have made notable advancements in analyzing real samples.These include the analysis of pH values near cancer cells,quantification of miRNA in human cell lysates,detection of human telomerase RNA in cell extracts,and performing DNA logicgated in situ bioimaging on cell membranes.Secondly,excellent sensitivity has been attained through the utilization of effective amplification methods such as RCA,HCR,and CHA,among others.Thirdly,the construction of stable reference signals has resulted in significantly improved precision for ratiometric biosensors.This breakthrough has overcome matrix effects,enabling reliable detection in real samples with high selectivity.This review provides a comprehensive overview of recent advancements in strategies employed by ratiometric DNA biosensors.We present three types of biosensors based on distinct sensing platforms:fluorescent,electrochemical,and SERS biosensors.Additionally,we discuss future directions and primary challenges in the development of ratiometric DNA biosensors.
基金supported by the National Key Research and Development Program of China (No.2018YFA0902801)the National Natural Science Foundations of China (Nos.21775169,21801259 and 21974153)+4 种基金the Scientific Technology Project of Shenzhen City (No.JCYJ20200109142410170)the Scientific Technology Project of Guangzhou City (No.202103000003)the Guangdong Natural Science Foundation (Nos.2018A030313290,2019A1515010587)the Guangdong Science and Technology Plan Project (No.2020B1212060077)the Fundamental Research Funds for the Central Universities,SYSU (No.19lgpy142)。
文摘A reliable and sensitive strategy which can assess nucleic acid levels in living cells would be essential for fundamental research of biomedical applications. Some nanomaterial-based fluorescence biosensors recently developed for detecting nucleic acids, however, are often with expensive, complicated and timeconsuming preparation process. Here, by using a facile bottom-up synthesis method, a two-dimensional(2 D) coordination polymer(CP) nanosheet, [Cu(tz)](Htz = 1,2,4-triazole), was successfully prepared after optimizing reaction conditions. These ultrathin CP nanosheets with thickness of 4.7 ± 1.1 nm could readily form nanosensors by assembly with DNA probes, which exhibited a low limit of detection(LOD)for p53 DNA fragment as 144 pmol/L. Furthermore, by integrating [Cu(tz)] nanosheets with hybridization chain reaction(HCR) probes, mi R-21, one kind of micro RNA upregulated in many cancer cells, can be sensitively detected with a LOD of 100 pmol/L and monitored in living cells, giving consistent results with those obtained by quantitative reverse-transcription polymerase chain reaction(q RT-PCR) analysis.Thus [Cu(tz)] nanosheets, which not only possess much better nucleic acids sensing performance than bulk cystals, but also exhibit nucleic acid delivery functions, could be used as a novel nanoplatform in biomedical imaging and sensing applications.
基金supported by National Natural Science Foundation of China(31922044,81861138040,81773280,82172746 and 81974453)Program of Shanghai Academic Research Leader(20XD1420500)+1 种基金Natural Science Foundation of Shanghai(19ZR1471600)Jiangsu Provincial Science and Technology Department Social Development-Clinical Frontier Technology(BE2020769).
文摘Sensitive detection and accurate diagnosis/prognosis of glioma remain urgent challenges.Herein,dispersed magnetic covalent organic framework nanospheres(MCOF)with uniformed Fe3O4 nano-assembly as cores and high-crystalline COF as shells were prepared by monomer-mediated in-situ interface growth strategy.Based on the unique interaction between MCOF and hairpin DNA,a fluorescent signal amplified miRNA biosensor was constructed.It could realize the sensitive detection of miRNA-182 in different matrixes,where the detection limit,linearity range and determination coefficient(R^(2))in real blood samples reached 20 fM,0.1 pM-10 pM and 0.991,respectively.Also,it possessed good stability and precision as observed from the low intra-day/inter-day RSD and high extraction recovery.As a result,it could quantify miRNA-182 in serum of glioma patients,the concentration of which was significantly higher than that of healthy people and obviously decreased after surgery.Finally,a proof-of-concept capillary chip system using this biosensor was proposed to realize the visualized detection of miRNA-182 in microsample.These findings suggest a robust way for sensitive detection and accurate diagnosis/prognosis of glioma.
基金supported by the National Research Foundation of Korea(NRF)grant RS-2023-00227950,RS-2024-00338426,RS-2024-00403094,New Faculty Startup Fund from Seoul National University,and Seoul National University Bundang Hospital(J.S.).
文摘G protein-coupled receptors(GPCRs),crucial for diverse physiological responses,have traditionally been investigated in their monomeric form.However,some GPCRs can form heteromers,revealing complexity in their functional characteristics such as ligand binding properties,downstream signaling pathways,and trafficking.Understanding GPCR heteromers is crucial in both physiological contexts and drug development.Here,we review the methodologies for investigating physical interactions in GPCR heteromers,including co-immunoprecipitation,proximity ligation assays,interfering peptide approaches,and live cell imaging techniques based on resonance energy transfer and bimolecular fluorescence complementation.In addition,we discuss recent advances in live cell imaging techniques for exploring functional features of GPCR heteromers,for example,circularly permuted fluorescent protein-based GPCR biosensors,TRUPATH,and nanobody-based GPCR biosensors.These advanced biosensors and live cell imaging technologies promise a deeper understanding of GPCR heteromers,urging a reassessment of their physiological importance and pharmacological relevance.
基金We thank all members of the Pelet and Martin labs for helpful discussions and comments on the manuscripts,Marta Schmitt and Clemence Viaridel for technical assistance.This study was supported by Swiss National Science Foundation grants(PP00P3139121)and the University of Lausanne.
文摘Background:Commitment to a new cell cycle is controlled by a number of cellular signals.Mitogen-activated protein kinase(MAPK)pathways,which transduce multiple extracellular cues,have been shown to be interconnected with the cell cycle and can modulate its progression.Methods:In budding yeast,we have introduced fluorescent biosensors that monitor in real time the signaling activity of the MAPKs Fus3 and Kssl and the cyclin-dependent kinase(CDK)in individual cells.We have quantified in hundreds of live single cells the interplay between the MAPKs regulating the mating response and the CDK controlling cell cycle progression.Results:Different patterns of MAPK activity dynamics could be identified by clustering cells based on their CDK activity,denoting the tight relationship between these two cellular signals.Our data suggest that beyond the already well-established mechanisms of regulation between the MAPK and the CDK,additional mechanisms remain to be identified.Conclusion:A tight interplay between MAPK pathways and the cell cycle is essential to control cellular proliferation and cell fate decisions.
