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Real-time profiling Anti-EpCAM Based Immune capture, from Molecules to Cells using MP-SPR
摘要： Antibodies of epithelial cell-adhesion-molecule (anti-EpCAM)-based interfaces have proven to be highly efficient at capturing circulating tumor cells (CTCs). To achieve the bonding of anti-EpCAM to the interface, biotin and streptavidin are used to modify the surface. These processes are critical to subsequent cell-capture efficiencies. However, quantitative research on the interactions between biotin, streptavidin and biotinylated anti-EpCAM on the interface is lacking. In this work, the thermodynamics and kinetics of biomolecular interactions were determined by using surface plasmon resonance. The equilibrium binding affinities for biotinylated anti-EpCAM to streptavidin and streptavidin to biotin (illustrated by biotin-PEG400-thiol) were found to be 2.75×10^6 M^-1 and 8.82×10^6 M^-1, respectively. Each streptavidin can bind up to 2.30 biotinylated anti-EpCAM under thermodynamic equilibrium. The findings provide useful information to optimize the modification of anti-EpCAM and improve the capture efficiency of CTCs.
关键词： surface plasmon resonance,antibodies of epithelial cell-adhesion-molecule,cell capture,biotin–streptavidin interaction,thermodynamics,kinetics
更新于2025-09-23 15:23:52
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Identification of Protein Targets of Bioactive Small Molecules Using Randomly Photomodified Probes
摘要： Identifying protein targets of bioactive small molecules often requires complex, lengthy development of affinity probes. We present a method for stochastic modification of small molecules of interest with a photoactivatable phenyldiazirine linker. The resulting isomeric mixture is conjugated to a hydrophilic copolymer decorated with biotin and a fluorophore. We validated this approach using known inhibitors of several medicinally relevant enzymes. At least a portion of the stochastic derivatives retained their binding to the target, enabling target visualization, isolation, and identification. Moreover, the mix of stochastic probes could be separated into fractions and tested for binding affinity. The structure of the active probe could be determined and the probe re-synthesized to improve binding efficiency. Our approach can thus enable rapid target isolation, identification, and visualization, while providing information required for subsequent synthesis of an optimized probe.
关键词： bioactive small molecules,target isolation,protein targets,target identification,stochastic modification,target visualization,photoactivatable phenyldiazirine linker,biotin,fluorophore,hydrophilic copolymer
更新于2025-09-23 15:21:21
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Silver nanosol SERS quantitative analysis of ultratrace biotin coupled N-doped carbon dots catalytic amplification with affinity reaction
摘要： Highly catalytic and stable N-doped carbon dots (N-CDs) were prepared rapidly by microwave procedure using glucose as precursor and ammonium sulfite as N-dopant. The reduction of AgNO3 by trisodium citrate (TCA) was slow to form nanosilver (AgNP), and the N-CDs exhibited strong catalysis of the AgNP reaction. The formed AgNPs were used as indicator in the presence of Vitoria blue B (VBB) molecule probe with a SERS peak at 1615 cm?1. With the increase of nanocatalyst N-CDs concentration, the AgNP reaction speed up, and the SERS peak of VBB enhanced linearly due to formation of more AgNPs as substrate. In the presence of avidin (Ad), the SERS peak weakened. Upon addition of biotin, the SERS peak enhanced due to turn on the indicator nanoreaction. The enhanced SERS signal had a good linear relationship with the biotin concentration in range of 0.0006–0.021 ng/mL, with a detection limit of 0.3 pg/mL.
关键词： Nanocatalysis,N-doped carbon dots,SERS,Affinity reaction,Biotin
更新于2025-09-23 15:19:57
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Large-Scale Purification of Photon-Upconversion Nanoparticles by Gel Electrophoresis for Analog and Digital Bioassays
摘要： The performance of photon-upconversion nanoparticles (UCNPs) as background-free luminescent labels in bioanalytical applications strongly depends on the preparation of well-defined and water-dispersible nanoconjugates. We have exploited the separation power of agarose gel electrophoresis to purify milligram amounts of homogeneous UCNPs covered with carboxylated silica, biotin or streptavidin with recovery rates of 30 to 50%. Clusters containing discrete numbers of UCNPs were isolated from the gel and reanalyzed by agarose gel electrophoresis, single nanoparticle upconversion microscopy and additional complementary methods. The purified nanoconjugates improved conventional (analog) bioaffinity assays and provided highly monodisperse conjugates for assays that rely on counting individual UCNPs (digital assays).
关键词： streptavidin,bioconjugate,Agarose gel electrophoresis,purification,digital bioassay,biotin
更新于2025-09-23 15:19:57
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A Plasmonic Approach to Study Protein Interaction Kinetics through the Dimerization of Functionalized Ag Nanoparticles
摘要： Understanding the kinetics of protein interactions plays a key role in biology with significant implications for the design of analytical methods for disease monitoring and diagnosis in medical care, research and industrial applications. Herein, we introduce a novel plasmonic approach to study the binding kinetics of protein-ligand interactions following the formation of silver nanoparticles (Ag nps) dimers by UV-Vis spectroscopy that can be used as probes for antigen detection and quantification. to illustrate and test the method, the kinetics of the prototype biotin-streptavidin (Biot-StV) pair interaction was studied. controlled aggregates (dimers) of StV functionalized Ag nps were produced by adding stoichiometric quantities of gliadin-specific biotinylated antibodies (IgG-Biot). The dimerization kinetics was studied in a systematic way as a function of Ag NPs size and at different concentrations of IgG-Biot. The kinetics data have shown to be consistent with a complex reaction mechanism in which only the Ag NPs attached to the IgG-Biot located in a specific STV site are able to form dimers. These results help in elucidating a complex reaction mechanism involved in the dimerization kinetics of functionalized Ag nps, which can serve as probes in surface plasmon resonance-based bioassays for the detection and quantification of different biomarkers or analytes of interest.
关键词： biotin-streptavidin interaction,silver nanoparticles,dimerization,protein interaction kinetics,plasmonic approach,surface plasmon resonance,UV-Vis spectroscopy
更新于2025-09-11 14:15:04
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Proteomic Analysis of S-Palmitoylated Proteins in Ocular Lens Reveals Palmitoylation of AQP5 and MP20
摘要： The purpose of this study was to characterize the palmitoyl-proteome in lens fiber cells. S-palmitoylation is the most common form of protein S-acylation and the reversible nature of this modification functions as a molecular switch to regulate many biological processes. This modification could play important roles in regulating protein functions and protein–protein interactions in the lens. The palmitoyl-proteome of bovine lens fiber cells was investigated by combining acyl-biotin exchange (ABE) chemistry and mass-spectrometry analysis. Due to the possibility of false-positive results from ABE experiment, a method was also developed for direct detection of palmitoylated peptides by mass spectrometry for validating palmitoylation of lens proteins MP20 and AQP5. Palmitoylation levels on AQP5 in different regions of the lens were quantified after iodoacetamide (IAA)-palmitate exchange. The ABE experiment identified 174 potential palmitoylated proteins. These proteins include 39 well-characterized palmitoylated proteins, 92 previously reported palmitoylated proteins in other tissues, and 43 newly identified potential palmitoylated proteins including two important transmembrane proteins in the lens, AQP5 and MP20. Further analysis by direct detection of palmitoylated peptides confirmed palmitoylation of AQP5 on C6 and palmitoylation of MP20 on C159. Palmitoylation of AQP5 was found to only occur in a narrow region of the inner lens cortex and does not occur in the lens epithelium, in the lens outer cortex, or in the lens nucleus. AQP5 and MP20 are among 174 palmitoylated proteins found in bovine lens fiber cells. This modification to AQP5 and MP20 may play a role in their translocation from the cytoplasm to cell membranes during fiber cell differentiation.
关键词： palmitoyl proteome,AQP5 and MP20,S-palmitoylation,acyl-biotin exchange
更新于2025-09-11 14:15:04
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[IEEE 2018 IEEE 15th International Conference on Group IV Photonics (GFP) - Cancun (2018.8.29-2018.8.31)] 2018 IEEE 15th International Conference on Group IV Photonics (GFP) - System-Level Integrated Active Silicon Photonic Biosensor for Detecting Small Molecule Interactions
摘要： We present a system-level integration of active silicon photonic biosensors. With on-chip photodetectors, sensors are characterized in the photovoltaic mode. A biotin-avidin affinity assay is employed to exemplify the detection of small molecule interactions, showing a detection limit in the order of 10?5 M.
关键词： photovoltaic mode,small molecule interactions,detection limit,silicon photonic biosensors,biotin-avidin affinity assay
更新于2025-09-04 15:30:14
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A reversible fluorescent probe for directly monitoring protein-small molecules interaction utilizing vibration-induced emission
摘要： The interactions between proteins and small molecules play an important role in the regulation of various cellular processes and modern drug discovery. Herein, we have developed a novel strategy for real-time monitoring the protein-ligand interaction based on vibration-induced emission (VIE) mechanism. These results demonstrated that the probe DPAC-DB could directly visualize the binding process in biotin-avidin system, which was undisturbed to other environmental stimuli, such as pH, interference species and temperature. Furthermore, the unique VIE effect caused by dramatic and reversible intramolecular vibrations enabled our strategy possible to build up the bio-logic gate. This method to efficiently control the emission of VIE-based probe through the reversible noncovalent protein-small molecules interactions opens new avenues for the development of potent protein pharmaceuticals and small molecule drugs.
关键词： Reversible,Vibration-induced emission (VIE),Biotin-avidin system,Bio-logic gate
更新于2025-09-04 15:30:14