We propose a concept for ligase detection by conversion of aggregation-based homogeneous analysis into surface-tethered electrochemical assay through streptavidin(SA)-biotin interaction.Sortase A(Srt A)served as the m...We propose a concept for ligase detection by conversion of aggregation-based homogeneous analysis into surface-tethered electrochemical assay through streptavidin(SA)-biotin interaction.Sortase A(Srt A)served as the model analyte and two biotinylated peptides(bio-LPETGG and GGGK-bio)were used as the substrates.Srt A-catalyzed ligation of the peptide substrates led to the generation of bio-LPETGGGKbio.The ligation product(bio-LPETGGGK-bio)induced the aggregation and color change of SA-modified gold nanoparticles(Au NPs)through the SA-biotin interactions,which could be assayed by the colorimetric method.Furthermore,we found that the bio-LPETGGGK-bio could trigger the assembly of tetrameric SA proteins with the formation of the(SA-bio-LPETGGGK-bio)nassemblies through the same interactions.The above results were further confirmed by atomic force microscopy and fluorescent imaging.The insulated assemblies were in-situ fabricated at the SA-modified gold electrode,thus hindering the electron transfer of[Fe(CN)_(6)]^(3-/4-) and leading to an increase in the electron-transfer resistance.The capability of the method for the detection of Srt A both in vitro and Staphylococcus aureus(S.aureus)has been demonstrated.Srt A with a concentration down to 1 pmol/L has been determined by the electrochemical analysis,which is lower than that achieved by the colorimetric assay(50 pmol/L).By integrating the advantages of homogeneous reaction and heterogeneous detection,the strategy serves as an ideal means for the fabrication of various sensing platforms by adopting biotin-labeled and sequence-specific peptide or nucleic acid substrates.展开更多
The specific binding of receptor to ligand covalently attached to surface with different surface densities was studied using streptavidin-biotin model pair. Biotinylated substrates with different spacer thicknesses as...The specific binding of receptor to ligand covalently attached to surface with different surface densities was studied using streptavidin-biotin model pair. Biotinylated substrates with different spacer thicknesses as formed through a simple reaction between amine immobilized surfaces and N-hydroxysucciimide groups at the end of biotin modifi ed PEG in anhydrous organic solutions（＂grafting to＂ technique）. The amount of the specifi cally adsorbed protein was measured as a function of spacer thickness between hard surface and biotin moieties. It has been shown that the amount of specifically adsorbed streptavidin decreases with the increase spacer thickness and the protein adsorbs onto the functionalized surfaces in a single molecular manner. It provides an interesting model system for studying single molecular interactions.展开更多
The rupture force of the streptavidin-biotin complex was investigated using atomic force microscopy (AFM). The most frequently observed rupture force (MFOF), which is essential for the evaluation of the potential land...The rupture force of the streptavidin-biotin complex was investigated using atomic force microscopy (AFM). The most frequently observed rupture force (MFOF), which is essential for the evaluation of the potential landscape, was evaluated by processing 22,500 force curves using two methods. One method is a conventional method, which is usually built in commercial AFM systems, i.e., difference between the baseline value and the minimum force value in the force curve. The other is a detection of rupture events based on a fuzzy logic algorithm to detect the rupture event from analyzing the shape of the force curves. Our statistical analysis revealed that the conventional method exhibited a significant artifact, which is the increase in the population of small forces comparable to thermal noise of cantilevers, resulting in a smaller MFOF. Based on this finding, we discuss the choice of a method and its effecton the illustrated potential landscapes of ligand-receptor complexes.展开更多
Two kinds of streptavidin magnetic particles,namely streptavidin GoldMag particles and streptavidin amino terminal particles were prepared by the methods of physical adsorption and covalent interaction respectively.Th...Two kinds of streptavidin magnetic particles,namely streptavidin GoldMag particles and streptavidin amino terminal particles were prepared by the methods of physical adsorption and covalent interaction respectively.The streptavidin coated on magnetic particle surface,crucial to many applications,was greatly influenced by the choice of the different buffer.Compared with DynalbeadsM-270 streptavidin, the binding capacity for biotin of different streptavidin magnetic particles was determined by enzyme inhibition method,and the coupling capacity and activity of biotinylated oligonucleotide on their sur- face were also analyzed.The results indicated that the streptavidin GoldMag particle prepared by physical adsorption was stable in STE(NaCl-Tris-EDTA)buffer that was frequently used in nucleic acid hybridization and detection.The streptavidin amino terminal particles prepared by covalent interaction could be used both in STE buffer and PBS(phosphate buffered saline)buffer.The biotin binding ca- pacity for 1 mg of streptavidin GoldMag particles and streptavidin amino terminal particles was 4950 and 5115 pmol respectively.The capacity of biotinylated oligonucleotide(24 bp)coupled on 1 mg of GoldMag and amino terminal magnetic particles was 2839 and 2978 pmol separately.These data were about 6-7 times higher than those of DynabeadsM-270 streptavidin.The hybridization results with FITC-labeled complementary probe on magnetic particle surface demonstrated that the oligonucleotide coupled on streptavidin magnetic particles had high biological activity.展开更多
Thiol terminated oligonucleotide was immobilized to gold surface by self assembly method. A novel amplification strategy was introduced for improving the sensitivity of DNA hybridization using biotin labeled protein...Thiol terminated oligonucleotide was immobilized to gold surface by self assembly method. A novel amplification strategy was introduced for improving the sensitivity of DNA hybridization using biotin labeled protein streptavidin network complex. This complex can be formed in a cross linking network of molecules so that the amplification of the response signal will be realized due to the big molecular size of the complex. It could be proved from the impedance technique that this amplification strategy caused dramatic improvement of the detection sensitivity. These results give significant advances in the generality and sensitivity as it is applied to biosensing.展开更多
DNA 5-formylcytosine(5fC)is a prominent epigenetic modification within biological systems.Recent investigations have shed light on its pivotal role in governing cell fate,gene expression,and disease pathways.However,o...DNA 5-formylcytosine(5fC)is a prominent epigenetic modification within biological systems.Recent investigations have shed light on its pivotal role in governing cell fate,gene expression,and disease pathways.However,our comprehension of the precise control of the 5f site structure to influence its functionality remains limited.In this study,we have successfully achieved precise control over 5fc activity by harnessing the interaction between streptavidin and biotin.This research underscores the potential application of interactions between biomacromolecules and small molecules in advancing the field of DNA epigenetic functional regulation.展开更多
The extracellular vesicles show great potential as a noninvasive biomarker for the early detection of cancer.Hence,there is an urgent requirement to create biosensors that are time-saving,simple,and easily scalable in...The extracellular vesicles show great potential as a noninvasive biomarker for the early detection of cancer.Hence,there is an urgent requirement to create biosensors that are time-saving,simple,and easily scalable in order to accomplish rapid,sensitive,and quantitative detection of extracellular vesicles.In this study,we present a self-propelled DNA walker powered by endonuclease Nt.Bbv CI,which enables the development of a“signal on”sensing platform for the rapid and highly sensitive detection of extracellular vesicles.The DNA motor employed tracks made of streptavidin magnetic beads,which consisted of substrate strands labeled with fluorescein and motor strands locked by aptamers.The aptamer recognition of the target protein on extracellular vesicles unlocked the motor strand,initiating the DNA motor process.After replacing the optimal buffer solution containing the endonuclease Nt.BbvC I,the motor strands autonomously moved along the streptavidin magnetic beads track,continuously releasing fluorescent molecules and producing detectable fluorescence signals.Under optimal conditions,the detection range was from 2×10~4particles/mL to 2×10~9particles/mL,with a detection limit of 2.9×10~3particles/mL,demonstrating excellent selectivity.This method has demonstrated good selectivity in different tumorderived extracellular vesicles and performs well in complex biological samples.The ability to effectively analyze surface proteins of extracellular vesicles in a short period of time gives our DNA walker a tremendous potential for developing simple and cost-effective clinical diagnostic devices.展开更多
基金the National Natural Science Foundation of China(Nos.22076221,21876208)the Program for Innovative Research Team of Science and Technology in the University of Henan Province(No.21IRTSTHN005)the Hunan Provincial Science and Technology Plan Project,China(No.2019TP1001)。
文摘We propose a concept for ligase detection by conversion of aggregation-based homogeneous analysis into surface-tethered electrochemical assay through streptavidin(SA)-biotin interaction.Sortase A(Srt A)served as the model analyte and two biotinylated peptides(bio-LPETGG and GGGK-bio)were used as the substrates.Srt A-catalyzed ligation of the peptide substrates led to the generation of bio-LPETGGGKbio.The ligation product(bio-LPETGGGK-bio)induced the aggregation and color change of SA-modified gold nanoparticles(Au NPs)through the SA-biotin interactions,which could be assayed by the colorimetric method.Furthermore,we found that the bio-LPETGGGK-bio could trigger the assembly of tetrameric SA proteins with the formation of the(SA-bio-LPETGGGK-bio)nassemblies through the same interactions.The above results were further confirmed by atomic force microscopy and fluorescent imaging.The insulated assemblies were in-situ fabricated at the SA-modified gold electrode,thus hindering the electron transfer of[Fe(CN)_(6)]^(3-/4-) and leading to an increase in the electron-transfer resistance.The capability of the method for the detection of Srt A both in vitro and Staphylococcus aureus(S.aureus)has been demonstrated.Srt A with a concentration down to 1 pmol/L has been determined by the electrochemical analysis,which is lower than that achieved by the colorimetric assay(50 pmol/L).By integrating the advantages of homogeneous reaction and heterogeneous detection,the strategy serves as an ideal means for the fabrication of various sensing platforms by adopting biotin-labeled and sequence-specific peptide or nucleic acid substrates.
文摘The specific binding of receptor to ligand covalently attached to surface with different surface densities was studied using streptavidin-biotin model pair. Biotinylated substrates with different spacer thicknesses as formed through a simple reaction between amine immobilized surfaces and N-hydroxysucciimide groups at the end of biotin modifi ed PEG in anhydrous organic solutions（＂grafting to＂ technique）. The amount of the specifi cally adsorbed protein was measured as a function of spacer thickness between hard surface and biotin moieties. It has been shown that the amount of specifically adsorbed streptavidin decreases with the increase spacer thickness and the protein adsorbs onto the functionalized surfaces in a single molecular manner. It provides an interesting model system for studying single molecular interactions.
文摘The rupture force of the streptavidin-biotin complex was investigated using atomic force microscopy (AFM). The most frequently observed rupture force (MFOF), which is essential for the evaluation of the potential landscape, was evaluated by processing 22,500 force curves using two methods. One method is a conventional method, which is usually built in commercial AFM systems, i.e., difference between the baseline value and the minimum force value in the force curve. The other is a detection of rupture events based on a fuzzy logic algorithm to detect the rupture event from analyzing the shape of the force curves. Our statistical analysis revealed that the conventional method exhibited a significant artifact, which is the increase in the population of small forces comparable to thermal noise of cantilevers, resulting in a smaller MFOF. Based on this finding, we discuss the choice of a method and its effecton the illustrated potential landscapes of ligand-receptor complexes.
基金the National Natural Science Foundation of China(Grant No.20273050)the National High Technology Research and Development Programof China(Grant No.2005AA205220)
文摘Two kinds of streptavidin magnetic particles,namely streptavidin GoldMag particles and streptavidin amino terminal particles were prepared by the methods of physical adsorption and covalent interaction respectively.The streptavidin coated on magnetic particle surface,crucial to many applications,was greatly influenced by the choice of the different buffer.Compared with DynalbeadsM-270 streptavidin, the binding capacity for biotin of different streptavidin magnetic particles was determined by enzyme inhibition method,and the coupling capacity and activity of biotinylated oligonucleotide on their sur- face were also analyzed.The results indicated that the streptavidin GoldMag particle prepared by physical adsorption was stable in STE(NaCl-Tris-EDTA)buffer that was frequently used in nucleic acid hybridization and detection.The streptavidin amino terminal particles prepared by covalent interaction could be used both in STE buffer and PBS(phosphate buffered saline)buffer.The biotin binding ca- pacity for 1 mg of streptavidin GoldMag particles and streptavidin amino terminal particles was 4950 and 5115 pmol respectively.The capacity of biotinylated oligonucleotide(24 bp)coupled on 1 mg of GoldMag and amino terminal magnetic particles was 2839 and 2978 pmol separately.These data were about 6-7 times higher than those of DynabeadsM-270 streptavidin.The hybridization results with FITC-labeled complementary probe on magnetic particle surface demonstrated that the oligonucleotide coupled on streptavidin magnetic particles had high biological activity.
基金ProjectsupportedbytheNationalNaturalScienceFoundationofChina (No .2 0 0 75 0 2 7)
文摘Thiol terminated oligonucleotide was immobilized to gold surface by self assembly method. A novel amplification strategy was introduced for improving the sensitivity of DNA hybridization using biotin labeled protein streptavidin network complex. This complex can be formed in a cross linking network of molecules so that the amplification of the response signal will be realized due to the big molecular size of the complex. It could be proved from the impedance technique that this amplification strategy caused dramatic improvement of the detection sensitivity. These results give significant advances in the generality and sensitivity as it is applied to biosensing.
基金the National Natural Science Foundation of China(Nos.22177089,21721005,92153303,22037004,22177088)the Fundamental Research Funds for the Central Universities(2042021kf0211)Translational Medicine and Interdisciplinary Research Joint Fund of Zhongnan Hospital of Wuhan University(Grant No.ZNJC202309).
文摘DNA 5-formylcytosine(5fC)is a prominent epigenetic modification within biological systems.Recent investigations have shed light on its pivotal role in governing cell fate,gene expression,and disease pathways.However,our comprehension of the precise control of the 5f site structure to influence its functionality remains limited.In this study,we have successfully achieved precise control over 5fc activity by harnessing the interaction between streptavidin and biotin.This research underscores the potential application of interactions between biomacromolecules and small molecules in advancing the field of DNA epigenetic functional regulation.
基金supported financially by the National Natural Science Foundation of China(NSFC,Nos.62071119 and 62075098)the National Key Research and Development Program of China(Nos.2017YFA0205301 and 2018YFC1602905)。
文摘The extracellular vesicles show great potential as a noninvasive biomarker for the early detection of cancer.Hence,there is an urgent requirement to create biosensors that are time-saving,simple,and easily scalable in order to accomplish rapid,sensitive,and quantitative detection of extracellular vesicles.In this study,we present a self-propelled DNA walker powered by endonuclease Nt.Bbv CI,which enables the development of a“signal on”sensing platform for the rapid and highly sensitive detection of extracellular vesicles.The DNA motor employed tracks made of streptavidin magnetic beads,which consisted of substrate strands labeled with fluorescein and motor strands locked by aptamers.The aptamer recognition of the target protein on extracellular vesicles unlocked the motor strand,initiating the DNA motor process.After replacing the optimal buffer solution containing the endonuclease Nt.BbvC I,the motor strands autonomously moved along the streptavidin magnetic beads track,continuously releasing fluorescent molecules and producing detectable fluorescence signals.Under optimal conditions,the detection range was from 2×10~4particles/mL to 2×10~9particles/mL,with a detection limit of 2.9×10~3particles/mL,demonstrating excellent selectivity.This method has demonstrated good selectivity in different tumorderived extracellular vesicles and performs well in complex biological samples.The ability to effectively analyze surface proteins of extracellular vesicles in a short period of time gives our DNA walker a tremendous potential for developing simple and cost-effective clinical diagnostic devices.
