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Preparation and evaluation of fluorescent poly(p-phenyleneethylene) covalently coated microspheres with reactive sites for bioconjugation
摘要： Fluorescent microspheres with reactive sites for interacting with biomolecules are greatly demanded in flow cytometry based suspension array. Aiming to develop new method for preparing fluorescent microspheres, two poly(p-phenyleneethylene) (PPE) conjugated polymers (CPs) with pedant carboxylic groups were synthesized via Sonogarshira coupling and followed with hydrolysis of ester groups; then the conjugated polymers were immobilized onto monodispersed amino-modified porous poly(glycidylmethacrylate) (APGMA) microspheres via coupling reaction between carboxylic and amino groups to give APGMA-CP fluorescent microspheres. The fluorescent microspheres were found to have good photo- and thermal stability as well as negligible influence from rigorous washing. The emission was uniform all across the inner and surface of the spheres. To evaluate the effectiveness of bioconjugation on the fluorescent microspheres, fluorescein isothiocyanate isomer I (FITC) labeled bovine serum albumin (BSA) (BSA-FITC) was chosen as the representative biomolecule to react with the fluorescent microspheres to give APGMA-CP-BSA-FITC. In the flow cytometry study, fluorescence compensation between the V500 and FITC detectors (receiving signals from fluorophores excited by 405 nm and 488 nm, respectively), to remove the interference between the emission of FITC and CPs, was realized using singly-stained microspheres. Finally, APGMA-CP-BSA-FITC microspheres were found to be double positive for CP and FITC with very high percentage (>95%), suggesting the bioconjugation is very effective. This study provides a facile method for simultaneous introduction of fluorescence and reactive sites onto the microspheres, which is very promising to be used as general strategy for fabricating fluorescence microspheres for application in high-throughput technology.
关键词： Fluorescence,Bioconjugation,Flow Cytometry,Microsphere,Conjugated Polymer
更新于2025-09-23 15:22:29
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Dextran-Functionalized Semiconductor Quantum Dot Bioconjugates for Bioanalysis and Imaging
摘要： The prerequisites for maximizing the advantageous optical properties of colloidal semiconductor quantum dots (QDs) in biological applications are effective surface functionalization and bioconjugation strategies. Functionalization with dextran has been highly successful with some nanoparticle materials, but has had very limited application with QDs. Here, we report the preparation, characterization, and proof-of-concept applications of dextran-functionalized QDs. Multiple approaches to dextran ligands were evaluated, including performance with respect to colloidal stability across a range of pH, nonspecific binding with proteins and cells, and microinjection into cells and viability assays. Multiple bioconjugation strategies were demonstrated and applied, including covalent coupling to develop a simple pH sensor, binding of polyhistidine-tagged peptides to the QD for energy transfer-based proteolytic activity assays, and binding with tetrameric antibody complexes (TACs) to enable a sandwich immunoassay and cell immunolabeling and imaging. Our results show that dextran ligands are highly promising for the functionalization of QDs, and that the design of the ligands is tailorable to help optimally meet the requirements of applications.
关键词： dextran,quantum dots,colloidal stability,imaging,bioconjugation,bioanalysis
更新于2025-09-23 15:19:57
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Synthesis and Characterization of Thiolate-Protected Gold Nanoparticles of Controlled Diameter
摘要： Gold nanoparticles (AuNPs) have been the focus of many studies owing to their unique optical and electronic properties and versatile applications. However, synthesis of stable and homogeneous AuNPs with a particular choice of size is still a challenge. In this study we describe a direct synthesis approach to produce stable and monodisperse water-soluble AuNPs with a tightly controlled diameter in the 1.7?2.4 nm range. We controlled the size by changing only the sodium hydroxide (NaOH) concentration in the synthesis. Gel electrophoresis, transmission electron microscopy (TEM), and solution X-ray scattering showed that the AuNPs had narrow size-distributions. We further showed that AuNPs of the di?erent sizes were clearly distinguishable in TEM micrographs, paving the way to dual-target labeling. The reactivity of the AuNPs toward DNA and proteins was also demonstrated. We utilized this reactivity to label tail-anchored proteins embedded in the membrane of the anticancer nanodrug Doxil as a means to target it to speci?c cell types. The gold-labeling enabled the precise localization of the tail-anchored proteins in cryo-TEM images of the therapeutic liposomes.
关键词： thiolate-protected,bioconjugation,cryo-TEM,tail-anchored proteins,synthesis,size control,Gold nanoparticles,Doxil
更新于2025-09-16 10:30:52
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Synthesis and evaluation of zirconium-89 labelled and long-lived GLP-1 receptor agonists for PET imaging
摘要： Introduction: Lately, zirconium-89 has shown great promise as a radionuclide for PET applications of long circulating biomolecules. Here, the design and synthesis of protracted and long-lived GLP-1 receptor agonists conjugated to desferrioxamine and labelled with zirconium-89 is presented with the purpose of studying their in vivo distribution by PET imaging. The labelled conjugates were evaluated and compared to a non-labelled GLP-1 receptor agonist in both in vitro and in vivo assays to certify that the modification did not significantly alter the peptides’ structure or function. Finally, the zirconium-89 labelled peptides were employed in PET imaging, providing visual verification of their in vivo biodistribution. Methods: The evaluation of the radiolabelled peptides and comparison to their non-labelled parent peptide was performed by in vitro assays measuring binding and agonistic potency to the GLP-1 receptor, physicochemical studies aiming at elucidating change in peptide structure upon bioconjugation and labelling as well as an in vivo food in-take study illustrating the compounds’ pharmacodynamic properties. The biodistribution of the labelled GLP-1 analogues was determined by ex vivo biodistribution and in vivo PET imaging. Results: The results indicate that it is surprisingly feasible to design and synthesize a protracted, zirconium-89 labelled GLP-1 receptor agonist without losing in vitro potency or affinity as compared to a non-labelled parent peptide. Physicochemical properties as well as pharmacodynamic properties are also maintained. The biodistribution in rats show high accumulation of radiolabelled peptide in well-perfused organs such as the liver, kidney, heart and lungs. The PET imaging study confirmed the findings from the biodistribution study with a significant high uptake in kidneys and presence of activity in liver, heart and larger blood vessels. Conclusions and Advances in Knowledge: This initial study indicates the potential to monitor the in vivo distribution of long-circulating incretin hormones using zirconium-89 based PET.
关键词： GLP-1,Bioconjugation,PET,Molecular imaging,Zirconium-89,Radiolabelling
更新于2025-09-12 10:27:22
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Electrostatic Repulsion Controls Efficiency of Cu‐free Click‐Reaction with Azide‐Modified Semiconductor Quantum Dots
摘要： Determination of factors influencing the efficiency of conjugation of various molecules with colloidal semiconductor quantum dots (QDs) is an important step toward their biomedical application. We have utilized controlled strain promoted [3+2] azid-alkyne cycloaddition (SPAAC) as an instrument for studying the influence of charge interaction between QDs and molecules on the efficiency of their conjugation. Azide-modified polymer-encapsulated core-shell CdSe/ZnS QDs, bicyclononyne(BCN)-modified JOE dye and BCN-BHQ1(Black Hole Quencher 1)-modified oligonucleotide duplex were used as model objects for conjugation. Strong surface negative charge of carboxylic QDs was shown to suppress efficient conjugation with negatively charged dsDNA or JOE dye (2',7'-dimethoxy-4',5'-dichloro-fluorescein) molecules. Utilization of zwitter-ionic QDs with reduced surface charge allows significantly enhance the efficiency of QDs conjugation with dsDNA.
关键词： bioconjugation,QDs,click-chemistry,zeta potential,SPAAC
更新于2025-09-12 10:27:22
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A Robust and General Approach to Quantitatively Conjugate Enzymes to Plasmonic Nanoparticles
摘要： Bioconjugates of plasmonic nanoparticles have received considerable attention due to their potential biomedical applications. Succesfull bioconjugation requires control over the number and activity of the conjugated proteins, and the colloidal stability of the particles. In practice, this requires re-optimization of the conjugation protocol for each combination of protein and nanoparticle. Here we report a robust and general protocol that allows for the conjugation of a range of proteins to di?erent types of nanoparticles using very short polyethylene-glycol(PEG) linkers, while simultaneously preserving protein activity and colloidal stability. The use of short linkers ensures that the protein is located close to the particle surface, where their refractive index sensitivity and near-?eld enhancement is maximal. We demonstrate that the use a Tween20 containing stabilizing bu?er is critical in maintaining colloidal stability and protein function throughout the protocol. We obtain quantitative control over the average number of enzymes per particle by either varying the number of functional groups on the particle, or the enzyme concentration during incubation. This new route of preparing quantitative protein-nanoparticle bioconjugates paves the way to develop rational and quantitative strategies to functionalize nanoparticles for applications in sensing, medical diagnostics and drug delivery.
关键词： medical diagnostics,drug delivery,quantitative control,protein activity,plasmonic nanoparticles,colloidal stability,sensing,bioconjugation,PEG linkers,Tween20
更新于2025-09-12 10:27:22
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Evaluating glucose and mannose profiles in Candida species using quantum dots conjugated with Cramoll lectin as fluorescent nanoprobes
摘要： Glycoconjugates found on cell walls of Candida species are fundamental for their pathogenicity. Laborious techniques have been employed to investigate the sugar composition of these microorganisms. Herein, we prepared a nanotool, based on the fluorescence of quantum dots (QDs) combined with the specificity of Cramoll lectin, to evaluate glucose/mannose profiles on three Candida species. The QDs-Cramoll conjugates presented specificity and bright fluorescence emission. The lectin preserved its biological activity after the conjugation process mediated by adsorption interactions. The labeling of Candida species was analyzed by fluorescence microscopy and quantified by flow cytometry. Morphological analyses of yeasts labeled with QDs-Cramoll conjugates indicated that C. glabrata (2.7 μm) was smaller when compared to C. albicans (4.0 μm) and C. parapsilosis sensu stricto (3.8 μm). Also, C. parapsilosis population was heterogeneous, presenting rod-shaped blastoconidia. More than 90% of cells of the three species were labeled by conjugates. Inhibition and saturation assays indicated that C. parapsilosis had a higher content of exposed glucose/mannose than the other two species. Therefore, QDs-Cramoll conjugates demonstrated to be effective fluorescent nanoprobes for evaluation of glucose/mannose constitution on the cell walls of fungal species frequently involved in candidiasis.
关键词： Candida,Cratylia mollis,Bioconjugation,Semiconductor nanocrystals,Lectin
更新于2025-09-11 14:15:04
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Quantifying the Initial Unfolding of Bacteriorhodopsin Reveals Retinal Stabilization
摘要： The forces that stabilize membrane proteins remain elusive to precise quantification. Particularly important but poorly resolved are the forces present during a membrane protein’s initial unfolding, where the most native set of interactions are present. We developed a high-precision, atomic force microscopy assay to study the initial unfolding of bacteriorhodopsin. We discovered rapid near-equilibrium folding between the first three unfolding states that corresponded to the unfolding of 5 and 8 amino-acids respectively when using a cantilever optimized for 2-μs resolution. Interestingly, the third of these states was retinal stabilized and previously undetected despite being the most mechanically stable state in the whole unfolding pathway, supporting 150 pN for >1 min. We expect that this ability to measure the rapid and reversible dynamics in the initial unfolding of bacteriorhodopsin provides a platform for quantifying the energetics of membrane proteins under native-like conditions.
关键词： protein folding,single molecule force spectroscopy,site-specific bioconjugation,membrane proteins,atomic force microscopy
更新于2025-09-04 15:30:14