Bifunctional Linker Research Articles

Bifunctional Rhodamine Linker Simplifies Colocalization Studies in Single-Molecule Imaging

Xanthene dyes (e.g., fluoresceins, rhodamines and rhodols) containing linkers for bio-conjugation are used extensively as fluorescent labels and probes to study biological systems. We have synthesized unsymmetrical, single isomer, bright, photostable rhodamine dyes. The asymmetric nature of the dye allowed us to introduce two independent functional groups into the rhodamine dye system. This, in turn, enabled us to generate a biotinylated and fluorescently-labeled protein, ready to be immobilized onto a streptavidin-coated surface for imaging.We demonstrated the utility of this bifunctional dye in a simple colocalization experiment. We showed that that by using anti-NGAL antibody attached to the biotin-linked rhodamine dye (bt-Rh-mAb), we can easily determine the surface density of this capture antibody on a glass slide. We reacted several different concentrations of bt-Rh-mAb onto a streptavidin surface, washed, and took single-molecule images. Simply counting the fluorophores generated a good estimate of the surface concentration of capture antibody. Without the fluorescent label, the surface concentration of the capture antibody must be either assumed or determined through a more complex and less direct process. We also conducted binding experiments using Cy5-labeled NGAL under equilibrium conditions. This colocalization measurement provided the fraction bound and thus a quick estimate of the dissociation constant.Lastly, we performed a sandwich immunoassay utilizing the bt-Rh-mAb capture antibody and a Cy5-labeled detection antibody. Colocalization analysis provided the analyte concentration and simultaneously reduced the contribution of nonspecific surface binding since “Cy5-only signals” were excluded. The bifunctional rhodamine linker is therefore a useful reagent to simplify a wide range of single-molecule experiments.

Antimicrobial peptide melimine coating for titanium and its in vivo antibacterial activity in rodent subcutaneous infection models

Implant-associated infections represent a significant health problem and financial burden on healthcare systems. Current strategies for the treatment or prevention of such infections are still inadequate and new strategies are needed in this era of antibiotic resistance. Melimine, a synthetic antimicrobial peptide with broad spectrum activity against bacteria, fungi and protozoa, has been shown to be a promising candidate for development as antimicrobial coating for biomedical devices and implants. In this study, the in vitro and in vivo antimicrobial activity of melimine-coated titanium was tested. The titanium surface was amine-functionalised with 3-aminopropyltriethoxysilane (APTES) followed by reaction with a bifunctional linker 4-(N-maleimidomethyl)cyclohexane-1-carboxylic 3-sulfo-n-hydroxysuccinimide ester (Sulfo-SMCC) to yield a maleimide functionalised surface. Melimine was then tethered to the surface via a thioether linkage through a Michael addition reaction of the cysteine at its N-terminus with the maleimide moiety. Melimine coating significantly reduced in vitro adhesion and biofilm formation of Pseudomonas aeruginosa by up to 62% and Staphylococcus aureus by up to 84% on the titanium substrates compared to the blank (p < 0.05). The activity was maintained after ethylene oxide gas sterilisation. The coating was also challenged in both mouse and rat subcutaneous infection models and was able to reduce the bacterial load by up to 2 log10 compared to the uncoated surface (p < 0.05). Melimine coating is a promising candidate for development as a surface antimicrobial that can withstand industrial sterilisation while showing good biocompatibility.

Photoresponsive Formation of an Intermolecular Minimal G-Quadruplex Motif.

The ability of three different bifunctional azobenzene linkers to enable the photoreversible formation of a defined intermolecular two-tetrad G-quadruplex upon UV/Vis irradiation was investigated. Circular dichroism and NMR spectroscopic data showed the formation of G-quadruplexes with K(+) ions at room temperature in all three cases with the corresponding azobenzene linker in an E conformation. However, only the para-para-substituted azobenzene derivative enables photoswitching between a nonpolymorphic, stacked, tetramolecular G-quadruplex and an unstructured state after E-Z isomerization.

Photoresponsive Formation of an Intermolecular Minimal G‐Quadruplex Motif

AbstractThe ability of three different bifunctional azobenzene linkers to enable the photoreversible formation of a defined intermolecular two‐tetrad G‐quadruplex upon UV/Vis irradiation was investigated. Circular dichroism and NMR spectroscopic data showed the formation of G‐quadruplexes with K+ ions at room temperature in all three cases with the corresponding azobenzene linker in an E conformation. However, only the para–para‐substituted azobenzene derivative enables photoswitching between a nonpolymorphic, stacked, tetramolecular G‐quadruplex and an unstructured state after E–Z isomerization.

Self-organization of Au–CdSe hybrid nanoflowers at different length scales via bi-functional diamine linkers

This work introduces a series of molecular bridging bi-functional linkers to produce laterally self-assembled nanostructures of the Au–CdSe nanoflowers on different length scales ranging from 10 nm to 100 microns. Assembly of Au nanocrystals within amorphous CdSe rods is found in the early stages of the growth of the Au–CdSe nanoflowers. The Au–CdSe nanoflowers are formed through a one-pot low temperature (150 °C) process where CdSe clusters are adsorbed on the surface of the Au cores, and they then start to form multiple arms and branches resulting in flower-shaped hybrid nanostructures. More complex assembly at a micron length scale can be achieved by means of bi-functional capping agents with appropriate alkyl chain lengths, such as 1,12-diaminododecane.

A Facile Approach to Functionalize Cell Membrane-Coated Nanoparticles.

Convenient strategies to provide cell membrane-coated nanoparticles (CM-NPs) with multi-functionalities beyond the natural function of cell membranes would dramatically expand the application of this emerging class of nanomaterials. We have developed a facile approach to functionalize CM-NPs by chemically modifying live cell membranes prior to CM-NP fabrication using a bifunctional linker, succinimidyl-[(N-maleimidopropionamido)-polyethyleneglycol] ester (NHS-PEG-Maleimide). This method is particularly suitable to conjugate large bioactive molecules such as proteins on cell membranes as it establishes a strong anchorage and enable the control of linker length, a critical parameter for maximizing the function of anchored proteins. As a proof of concept, we show the conjugation of human recombinant hyaluronidase, PH20 (rHuPH20) on red blood cell (RBC) membranes and demonstrate that long linker (MW: 3400) is superior to short linker (MW: 425) for maintaining enzyme activity, while minimizing the changes to cell membranes. When the modified membranes were fabricated into RBC membrane-coated nanoparticles (RBCM-NPs), the conjugated rHuPH20 can assist NP diffusion more efficiently than free rHuPH20 in matrix-mimicking gels and the pericellular hyaluronic acid matrix of PC3 prostate cancer cells. After quenching the unreacted chemical groups with polyethylene glycol, we demonstrated that the rHuPH20 modification does not reduce the ultra-long blood circulation time of RBCM-NPs. Therefore, this surface engineering approach provides a platform to functionlize CM-NPs without sacrificing the natural function of cell membranes.

Modular, polymer-directed nanoparticle assembly for fabricating metamaterials.

We achieve the fabrication of plasmonic meta-atoms by utilizing a novel, modular approach to nanoparticle self-assembly that utilizes polymer templating to control meta-atom size and geometry. Ag nanocubes are deposited and embedded into a polymer thin-film, where the polymer embedding depth is used to dictate which nanocube faces are available for further nanocrystal binding. Horizontal and vertical nanocube dimers were successfully fabricated with remarkably high yield using a bifunctional molecular linker to bind a second nanocube. Surface plasmon coupling can be readily tuned by varying the size, shape, and orientation of the second nanoparticle. We show that meta-atoms can be fabricated to exhibit angle- and polarization-dependent optical properties. This scalable technique for meta-atom assembly can be used to fabricate large-area metasurfaces for polarization- and phase-sensitive applications, such as optical sensing.

PH-Reversible Cationic RNase A Conjugates for Enhanced Cellular Delivery and Tumor Cell Killing.

Intracellularly-acting therapeutic proteins are considered promising alternatives for the treatment of various diseases. Major limitations of their application are low efficiency of intracellular delivery and possible reduction of protein activity during derivatization. Herein, we report pH-sensitive covalent modification of proteins with a histidine-rich cationic oligomer (689) for efficient intracellular transduction and traceless release of functional proteins. Enhanced Green fluorescent protein (EGFP), as model for the visualization of protein transduction, and RNase A, as therapeutic protein with antitumoral effect, were modified with the pH-sensitive bifunctional AzMMMan linker and varying amounts of cationic oligomer. The modification degree showed impact on the internalization and cellular distribution of EGFP as well as the biological effect of RNase A conjugates, which mediated considerable toxicity against cancer cells at optimal ratio. The presented conjugates demonstrate their qualification to achieve efficient intracellular delivery and controlled release without protein inactivation and potential prospective applications in protein-based therapies.

Self-assembled thin film of imidazolium ionic liquid on a silicon surface: Low friction and remarkable wear-resistivity

Imidazolium-hexafluorophosphate (ImPF6) ionic liquid thin film is prepared on a silicon surface using 3-chloropropyltrimethoxysilane as a bifunctional chemical linker. XPS result revealed the covalent grafting of ImPF6 thin film on a silicon surface. The atomic force microscopic images demonstrated that the ImPF6 thin film is composed of nanoscopic pads/clusters with height of 3–7nm. Microtribological properties in terms of coefficient of friction and wear-resistivity are probed at the mean Hertzian contact pressure of 0.35–0.6GPa under the rotational sliding contact. The ImPF6 thin film exhibited low and steady coefficient of friction (μ=0.11) along with remarkable wear-resistivity to protect the underlying silicon substrate. The low shear strength of ImPF6 thin film, the covalent interaction between ImPF6 ionic liquid thin film and underlying silicon substrate, and its regular grafting collectively reduced the friction and improved the anti-wear property. The covalently grafted ionic liquid thin film further shows immense potential to expand the durability and lifetime of M/NEMS based devices with significant reduction of the friction.

Self‐Assembly of Graphene Oxide on Silicon Substrate via Covalent Interaction: Low Friction and Remarkable Wear‐Resistivity

Few‐layer graphene oxide (GO) is assembled on the silicon surface by a self‐assembly approach via covalent interaction using 3‐aminopropyltrimethoxysilane (APTMS) as a bifunctional chemical linker. X‐ray photoelectron spectroscopy results suggest chemical interactions between oxygen functionalities of GO and amino group of APTMS thin film. The oxygen functionalities of GO thin film are eliminated by vacuum ultraviolet (VUV) photon exposure. Topographic images reveal efficient grafting of GO on the silicon and suggest the presence of few layers in the GO thin film along with wrinkles and folds. Microtribological properties of VUV‐reduced GO (rGO) thin film are probed under the mean contact pressure of 0.3–0.6 GPa. The rGO thin film exhibits low and steady friction (0.12–0.15) compared to that of bare silicon (0.6). The rGO thin film could survive for ≈37 000 laps at 100 mN load, revealing its remarkable wear‐resistivity. Microscopic images and carbon mapping reveal the deposition of delaminated graphene lamellae on the counter steel ball surface. The low friction and excellent wear‐resistivity of rGO thin film are collectively attributed to low‐resistance to shear between the neighboring lamellae of rGO, full coverage and strong interaction of rGO thin film with silicon, and deposition of delaminated graphene lamellae on the counter steel surface.

Two Comparable Isostructural Microporous Metal–Organic Frameworks: Better Luminescent Sensor and Higher Adsorption Selectivity for the Fluorine‐Decorated Framework

AbstractTwo isostructural 3D metal–organic frameworks (MOFs), {[Zn3(tzba)3(dabco)]·5DMF·3H2O} (1) and {[Zn3(F‐tzba)3(dabco)]·5DMA·3H2O} (2) [tzba = 4‐(1H‐tetrazol‐5‐yl) benzolate, F‐tzba = 2‐fluoro‐4‐(1H‐tetrazol‐5‐yl) benzolate, dabco = triethylenediamine], were solvothermally synthesized from a tetrazolate–carboxylate bifunctional linker and a diamine ligand. The framework contains an uncommon metal–tetrazolate–carboxylate linear trinuclear Zn3(ttaz)3(COO)3 (ttaz = tetrazolyl) cluster, which serves as an eight‐connected node to afford a 3D porous network. Both 1 and 2 show strong photoluminescence and selective luminescent quenching for Cu2+ ions and nitrobenzene relative to that shown by other metal ions (e.g., Na+, K+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Cd2+, and Pb2+) and organic solvents (e.g., methanol, ethanol, acetonitrile, n‐propanol, 2‐propanol, ethyl acetate, toluene, dichloromethane, N,N′‐dimethylformamide, and N‐methylpyrrolidone). It is interesting to find that relative to 1, 2 decorated with F atoms exhibits more efficient luminescent sensing for these two guests. Furthermore, 2 also adsorbs more CO2 and has higher CO2/CH4 adsorption selectivities than 1 under the same conditions, which can be ascribed to the CO2‐philic F sites in the pores of 2. These advantages make 2 a promising material in both luminescent sensors and CO2 separation from a CO2/CH4 mixture.

Enhanced Photoluminescence Property for Quantum Dot-Gold Nanoparticle Hybrid.

In this paper, we have synthesized ZnCdSeS quantum dots (QDs)-gold nanoparticle (Au NPs) hybrids in aqueous solution via bi-functional linker mercaptoacetic acid (MPA). The absorption peaks of ZnCdSeS QDs and Au are both located at 520 nm. It is investigated that PL intensity of QD-Au hybrid can be affected by the amounts of Au and pH value of hybrid solution. The located surface plasmon resonance (LSPR) effect of QD-Au NPs has been demonstrated by increased fluorescence intensity. The phenomenon of fluorescence enhancement can be maximized under the optimized pH value of 8.5. LSPR-enhanced photoluminescence property of QD-Au hybrid will be beneficial for the potential applications in the area of biological imaging and detection.

Synthetic and Immunological Studies of Mycobacterial Lipoarabinomannan Oligosaccharides and Their Protein Conjugates.

Lipoarabinomannan (LAM) is one of the major constituents of the Mycobacterium tuberculosis cell wall and an attractive molecular scaffold for antituberculosis drug and vaccine development. In this paper, a convergent strategy was developed for the synthesis of LAM oligosaccharides with an α-1,2-linked dimannopyranose cap at the nonreducing end. The strategy was highlighted by efficient coupling of separately prepared nonreducing end and reducing end oligosaccharides. Glycosylations were mainly achieved with thioglycoside donors, which gave excellent yields and stereoselectivity even for reactions between complex oligosaccharides. The strategy was utilized to successfully synthesize tetra-, hepta-, and undecasaccharides of LAM from d-arabinose in 10, 15, and 14 longest linear steps and 7.84, 7.50, and 2.59% overall yields, respectively. The resultant oligosaccharides with a free amino group at their reducing end were effectively conjugated with carrier proteins, including bovine serum albumin and keyhole limpet hemocyanin (KLH), via a bifunctional linker. Preliminary immunological studies on the KLH conjugates revealed that they could elicit robust antibody responses in mice and that the antigen structure had some influence on their immunological property, thus verifying the potential of the oligosaccharides for vaccine development and other immunological studies.

Plasmonic Effects of Infiltrated Silver Nanoparticles Inside TiO2 Film: Enhanced Photovoltaic Performance in DSSCs

The plasmonic effects of infiltrated silver (Ag) nanoparticles, with different contents, inside a nanostructured TiO2 film on the photovoltaic performance of dye‐sensitized solar cells (DSSCs) are explored. The synthesized Ag nanoparticles are immobilized onto deposited TiO2 nanoparticles by a new strategy using 3‐mercaptopropionic acid (MPA), a bifunctional linker molecule. Transmission electron microscope (TEM) images show that monodispersed Ag and polydispersed TiO2 nanoparticles have an average diameter of 12 ± 3 nm and 5 ± 1 nm, respectively. Moreover, Fourier transform infrared spectroscopy (FTIR) analysis reveals that Ag nanoparticles were successfully functionalized and capped with MPA. Optical studies on the MPA‐capped Ag nanoparticles inside TiO2 film show an increase in the total absorbance of the electrode. Moreover, EIS measurements confirm that MPA‐capped Ag nanoparticles inhibit the charge recombination and improve the stability of nanoparticles in I3−/I− electrolyte. The DSSC assembled with optimal content of MPA‐capped Ag nanoparticles demonstrated an enhanced power conversion efficiency (8.82% ± 0.07%) compared with the pure TiO2 (7.30% ± 0.05%). The increase in cell efficiency was attributed to the enhanced dye light absorption in strength and spectral range due to the surface plasmon resonance of MPA‐capped Ag nanoparticles in the photoanode.

Ultrahigh density plasmonic hot spots with ultrahigh electromagnetic field for improved photocatalytic activities

Plasmonic hot spots located among closely dispersed plasmonic nanoparticles (NPs) are intensively studied for efficient conversion of solar to chemical or electrical energy applications. Here, a successful method to synthesize high-density unaggregated plasmonic Ag or Au NPs (AgNPs or AuNPs) onto nanostructured semiconductors with 3D densely organized NPs is demonstrated. The densely dispersed plasmonic AgNPs or AuNPs are assembled chemically on the entire surface of the ZnO nanorods through bifunctional thioctic acid bridging linkers. The fabricated NPs possessing small interparticle gaps generate numerous plasmonic hot spots which boost catalytic activities of the photocatalysts. As depicted by exact 3D finite-difference time domain simulations, the electromagnetic fields are magnified exponentially among interparticle gaps, hot spots, due to the plasmonic coupling effects of the neighboring AgNPs. The electromagnetic fields are strengthened by decreasing the interparticle spacing of coupled AgNPs. It is consistent with the result that the photocatalytic reaction rate increases non-linearly with the Ag content under full-spectrum light irradiation. Using the spectral characterizations and electromagnetic field simulations, we unambiguously confirm the enhancement of photoactivity due to coupling of plasmonic hot spot effect to nanostructured semiconductors. Moreover, diverse heterostructures based on the plasmonic NPs on various ZnO nanostructures (films, nanorod arrays, branched nanowires, and mesoporous structures) or functional materials (CuInGaSe2 absorber films, multiferroic BiFeO3 films, visible-light photoactive Cu2S and CdS nanorods) are successfully fabricated using the present synthesis methodology.

High spatial resolution mapping of individual and collective localized surface plasmon resonance modes of silver nanoparticle aggregates: correlation to optical measurements.

Non-isolated nanoparticles show a plasmonic response that is governed by the localized surface plasmon resonance (LSPR) collective modes created by the nanoparticle aggregates. The individual and collective LSPR modes of silver nanoparticle aggregated by covalent binding by means of bifunctional molecular linkers are described in this study. Individual contributions to the collective modes are investigated at nanometer scale by means of energy-filtering transmission electron microscopy and compared to ultraviolet–visible spectroscopy. It is found that the aspect ratio and the shape of the clusters are the two main contributors to the low-energy collective modes.

Antibody-Drug Conjugates for Tumor Targeting-Novel Conjugation Chemistries and the Promise of non-IgG Binding Proteins.

Antibody-drug conjugates (ADCs) have emerged as a promising class of anticancer agents, combining the specificity of antibodies for tumor targeting and the destructive potential of highly potent drugs as payload. An essential component of these immunoconjugates is a bifunctional linker capable of reacting with the antibody and the payload to assemble a functional entity. Linker design is fundamental, as it must provide high stability in the circulation to prevent premature drug release, but be capable of releasing the active drug inside the target cell upon receptor-mediated endocytosis. Although ADCs have demonstrated an increased therapeutic window, compared to conventional chemotherapy in recent clinical trials, therapeutic success rates are still far from optimal. To explore other regimes of half-life variation and drug conjugation stoichiometries, it is necessary to investigate additional binding proteins which offer access to a wide range of formats, all with molecularly defined drug conjugation. Here, we delineate recent progress with site-specific and biorthogonal conjugation chemistries, and discuss alternative, biophysically more stable protein scaffolds like Designed Ankyrin Repeat Proteins (DARPins), which may provide such additional engineering opportunities for drug conjugates with improved pharmacological performance.

Microporous Metal-Organic Framework Based on a Bifunctional Linker for Selective Sorption of CO2 over N2 and CH4.

A bifunctional organic linker 4-(4-carboxyphenyl)-1,2,4-triazole (HCPT), incorporating both carboxylate and triazole groups, has been successfully used in the construction of a 2-fold interpenetrated dynamic metal-organic framework (MOF), {[Cu3(CPT)4(μ3-OH)]·NO3·7H2O·EtOH}n (1) based on a triangular Cu(II)-hydroxo cluster as secondary building unit (SBU). Upon solvation/desolvation and temperature, the crystal cell parameters of 1 could be fine-tuned. More importantly, a transformation from disordered phase to a more ordered phase after activation was observed via a single-crystal-to-single-crystal mode. Gas sorption studies reveal that the activated 1 exhibits highly selective sorption of CO2 over N2 and CH4 at room temperature.

Fabrication and spectroscopic studies of folic acid-conjugated Fe3O4@Au core–shell for targeted drug delivery application

Gold coated magnetite core shell is a kind of nanoparticle that include magnetic iron oxide core with a thin layer nanogold. Fe3O4–gold core–shell nanostructure can be used in biomedical applications such as magnetic bioseparation, bioimaging, targeting drug delivery and cancer treatment. In this study, the synthesis and characterization of gold coated magnetite nanoparticles were discussed. Magnetite nanoparticles with an average size of 6nm in diameter were synthesized by the chemical co-precipitation method and gold-coated Fe3O4 core–shell nanostructures were produced with an average size of 11.5nm in diameter by reduction of Au3+ with citrate ion in the presence of Fe3O4. Folate-conjugated gold coated magnetite nanoparticles were synthesized to targeting folate receptor that is overexpressed on the surface of cancerous cells. For this purpose, we used l-cysteine, as a bi-functional linker for attachment to gold surface and it was linked to the gold nanoparticles surface through its thiol group. Then, we conjugated amino-terminated nanoparticles to folic acid with an amide-linkage formation. These gold magnetic nanoparticles were characterized by various techniques such as X-ray powder diffraction (XRD) analysis, Fourier transform infrared spectrometer (FT-IR), UV–visible spectroscopy, transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM), dispersive analysis of X-ray (EDAX) and vibrating sample magnetometer (VSM) analysis. The magnetic and optical properties of Fe3O4 nanostructure were changed by gold coating and attachment of l-cysteine and folic acid to Fe3O4@Au nanoparticles.

Platinum(II) as bifunctional linker in antibody-drug conjugate formation: coupling of a 4-nitrobenzo-2-oxa-1,3-diazole fluorophore to trastuzumab as a model.

The potential of platinum(II) as a bifunctional linker in the coordination of small molecules, such as imaging agents or (cytotoxic) drugs, to monoclonal antibodies (mAbs) was investigated with a 4-nitrobenzo-2-oxa-1,3-diazole (NBD) fluorophore and trastuzumab (Herceptin™) as a model antibody. The effect of ligand and reaction conditions on conjugation efficiency was explored for [Pt(en)(L-NBD)Cl](NO3 ) (en=ethylenediamine), with L=N-heteroaromatic, N-alkyl amine, or thioether. Conjugation proceeded most efficiently at pH 8.0 in the presence of NaClO4 or Na2 SO4 in tricine or HEPES buffer. Reaction of N-coordinated complexes (20 equiv) with trastuzumab at 37 °C for 2 h, followed by removal of weakly bound complexes with excess thiourea, afforded conjugates with an NBD/mAb ratio of 1.5-2.9 that were stable in phosphate-buffered saline at room temperature for at least 48 h. In contrast, thioether-coordinated complexes afforded unstable conjugates. Finally, surface plasmon resonance analysis showed no loss in binding affinity of trastuzumab after conjugation.