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
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
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
Kanamycin is an aminoglycoside antibiotic used increasingly in human and veterinary medicine. However, kanamycin residues in food can cause serious side effects, Here we reported the preparation of polyclonal antibody...Kanamycin is an aminoglycoside antibiotic used increasingly in human and veterinary medicine. However, kanamycin residues in food can cause serious side effects, Here we reported the preparation of polyclonal antibody and the development of an indirect competitive biotin-streptavidin-amplified-based enzyme-linked immunosorbent assay（BA-ELISA） that can sensitively and specifically detect kanamycin residues in milk and honey. The immtmogen and coating antigen were synthesized by covalently linking kanamycin to carrier proteins using the carbodiimide method. The anti-kanamycin polyclonal antibodies were obtained from immunized rabbits. The key assay parameters were investigated and optimized. The results show that under optimum conditions, the limit of detection for kanamycin is 0.07 ng/mL and the ICs0 is 6.48 ng/mL. Cross-reactivity values of the antibody with four kanamycin analogues are all 〈1%. Trace amounts of kanamycin in milk and honey samples can be detected by this novel BA-ELISA method successfully with satisfactory recoveries of 91.0%-103.3%. The developed protocol was also validated against liquid chromatography-mass spectrometry, returning a significant correlation. These results indicate that BA-ELISA is a viable option for monitoring kanamycin residues in milk and honey.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
Natural and syntlietic nanopores are increasingly popular tools in biosensors. In this work, the DNA supersandwich structure, which was made from two specially designed probes has been used to be fabricated in solid n...Natural and syntlietic nanopores are increasingly popular tools in biosensors. In this work, the DNA supersandwich structure, which was made from two specially designed probes has been used to be fabricated in solid nanopores. Integrating the idea of affinity between streptavidin and biotin, the DNA supersandwich structure with biotins was successfully constructed for streptavidin detection, and the limitation of detection was found to be 10 fmol/L. This nanodevice allows specific, sensitive and versatile detection of diverse analytes with easy operations, thus we believe that it could be developed to detect some disease-related molecular targets and play a considerable role in biotechnology.展开更多
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
核酸适配体是人工合成的短链核酸,作为分子识别元件,能够与各类靶标物质高特异性、高亲和力的结合,分为单链DNA和RNA两种类型。其中单链DNA适配体由于其稳定性比RNA适配体更好而更受欢迎,因此得到广泛应用。核酸适配体筛选通常是通过配...核酸适配体是人工合成的短链核酸,作为分子识别元件,能够与各类靶标物质高特异性、高亲和力的结合,分为单链DNA和RNA两种类型。其中单链DNA适配体由于其稳定性比RNA适配体更好而更受欢迎,因此得到广泛应用。核酸适配体筛选通常是通过配体指数富集系统进化技术(systematic evolution of ligands by exponential enrichment,SELEX)实现的,筛选能否成功在很大程度上取决于其最关键的单链制备步骤,即将双链DNA转化为相应的单链DNA。目前,存在许多方法可以制备单链DNA,包括热变性法、生物素-链霉亲和素亲和分离法、变性胶电泳分离法、核酸外切酶消化法、不对称聚合酶链式反应(polymerase chain reaction,PCR)法等。本文在总结文献报道的基础上,具体阐述了各种单链DNA制备方法的原理、优缺点及近5年的应用情况,并对这些单链DNA制备方法进行了比较和展望,以期能为成功筛选各类靶标的核酸适配体提供参考。展开更多
New reagents for immunofluorescence analysis of carbazole series containing fluorinated β-dicarbonyl fragments and carboxylic substituent groups separated by spacers of different lengths from the light-gathering carb...New reagents for immunofluorescence analysis of carbazole series containing fluorinated β-dicarbonyl fragments and carboxylic substituent groups separated by spacers of different lengths from the light-gathering carbazole scaffold have been developed. The markers in complex with Eu<sup>3+</sup> ions possess stability in the aqueous phase, intense and prolonged luminescence (τ 550 - 570 μs) with characteristic emission maxima in the region of 615 nm and excitation wavelengths in the region of 380 - 390 nm, which distinguishes them from most of the analogs used. In the study of marker conjugation with streptavidin, a reagent containing 4 - 5 europium labeling complexes based on spacer-containing carbazole tetraketone was obtained. The marker-doped silicate nanoparticles exhibit intense and long-lived luminescence in the characteristic region.展开更多
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(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Supported by the National Natural Science Foundation of China（No.21675008）, the Natural Science Foundation of Beijing, China（No.2132048） and the Fundamental Research Funds for the Central Universities, China（No.JDl 516）.
文摘Kanamycin is an aminoglycoside antibiotic used increasingly in human and veterinary medicine. However, kanamycin residues in food can cause serious side effects, Here we reported the preparation of polyclonal antibody and the development of an indirect competitive biotin-streptavidin-amplified-based enzyme-linked immunosorbent assay（BA-ELISA） that can sensitively and specifically detect kanamycin residues in milk and honey. The immtmogen and coating antigen were synthesized by covalently linking kanamycin to carrier proteins using the carbodiimide method. The anti-kanamycin polyclonal antibodies were obtained from immunized rabbits. The key assay parameters were investigated and optimized. The results show that under optimum conditions, the limit of detection for kanamycin is 0.07 ng/mL and the ICs0 is 6.48 ng/mL. Cross-reactivity values of the antibody with four kanamycin analogues are all 〈1%. Trace amounts of kanamycin in milk and honey samples can be detected by this novel BA-ELISA method successfully with satisfactory recoveries of 91.0%-103.3%. The developed protocol was also validated against liquid chromatography-mass spectrometry, returning a significant correlation. These results indicate that BA-ELISA is a viable option for monitoring kanamycin residues in milk and honey.
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Supported by the National Natural Science Foundation of China(No.21505101)the Natural Science Foundation of Zhejiang Province,China(No.LQ16B050003).
文摘Natural and syntlietic nanopores are increasingly popular tools in biosensors. In this work, the DNA supersandwich structure, which was made from two specially designed probes has been used to be fabricated in solid nanopores. Integrating the idea of affinity between streptavidin and biotin, the DNA supersandwich structure with biotins was successfully constructed for streptavidin detection, and the limitation of detection was found to be 10 fmol/L. This nanodevice allows specific, sensitive and versatile detection of diverse analytes with easy operations, thus we believe that it could be developed to detect some disease-related molecular targets and play a considerable role in biotechnology.
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.
文摘核酸适配体是人工合成的短链核酸,作为分子识别元件,能够与各类靶标物质高特异性、高亲和力的结合,分为单链DNA和RNA两种类型。其中单链DNA适配体由于其稳定性比RNA适配体更好而更受欢迎,因此得到广泛应用。核酸适配体筛选通常是通过配体指数富集系统进化技术(systematic evolution of ligands by exponential enrichment,SELEX)实现的,筛选能否成功在很大程度上取决于其最关键的单链制备步骤,即将双链DNA转化为相应的单链DNA。目前,存在许多方法可以制备单链DNA,包括热变性法、生物素-链霉亲和素亲和分离法、变性胶电泳分离法、核酸外切酶消化法、不对称聚合酶链式反应(polymerase chain reaction,PCR)法等。本文在总结文献报道的基础上,具体阐述了各种单链DNA制备方法的原理、优缺点及近5年的应用情况,并对这些单链DNA制备方法进行了比较和展望,以期能为成功筛选各类靶标的核酸适配体提供参考。
文摘New reagents for immunofluorescence analysis of carbazole series containing fluorinated β-dicarbonyl fragments and carboxylic substituent groups separated by spacers of different lengths from the light-gathering carbazole scaffold have been developed. The markers in complex with Eu<sup>3+</sup> ions possess stability in the aqueous phase, intense and prolonged luminescence (τ 550 - 570 μs) with characteristic emission maxima in the region of 615 nm and excitation wavelengths in the region of 380 - 390 nm, which distinguishes them from most of the analogs used. In the study of marker conjugation with streptavidin, a reagent containing 4 - 5 europium labeling complexes based on spacer-containing carbazole tetraketone was obtained. The marker-doped silicate nanoparticles exhibit intense and long-lived luminescence in the characteristic region.
