Elastin-calmodulin scaffold for protein microarray fabrication

In this work, we report a new method to reversibly immobilize proteins to a surface in a functionally active orientation directly from cell lysate by employing a fusion protein consisting of a thermal-responsive elastin (ELP) domain as the surface anchor and a calcium-responsive calmodulin (CalM) domain for protein capturing. Incorporation of an M13 tag into recombinant proteins enables not only easy surface immobilization but also direct purification from cell lysates. The feasibility of concept was demonstrated using the M13-tagged yellow fluorescent protein (M13-YFP). The ELP-CalM functionalized surfaces were shown to capture M13-YFP directly from cell lysate through the specific calmodulin-M13 association in a calcium-dependent manner. We also demonstrated that immobilization is reversible; the bound proteins were released from the surface in the presence of EDTA.     

Simple surface functionalization of magnetic nanoparticles with methotrexate-conjugated bovine serum albumin as a biocompatible drug delivery vehicle

Magnetic nanoparticles (MNPs) functionalized with methotrexate (MTX)-conjugated bovine serum albumin (BSA) as a biocompatible drug delivery vehicle were synthesized using a facile method. Characterization of the functionalized MNPs (Fe₃O₄@BSA-MTX NPs) was performed using various techniques including UV–visible spectroscopy, dynamic light scattering, vibrating sample magnetometry and X-ray diffraction. The particle size and zeta potential of Fe₃O₄@BSA-MTX NPs were 105.7 ± 3.81 nm (mean ± SD) and −18.2 mV, respectively. MTX release from Fe₃O₄@BSA-MTX NPs showed an enzyme-dependent release pattern. Hemo-biocompatibility of Fe₃O₄@BSA-MTX NPs was confirmed using hemolysis test. In addition, the cytotoxicity of functionalized MNPs and free MTX against MCF-7 cell line was investigated using MTT assay. The results of experiments revealed that the Fe₃O₄@BSA-MTX NPs as a biocompatible carrier could improve the therapeutic effect of MTX.     

Chelating ligands for nanocrystals' surface functionalization

A new family of ligands for the surface functionalization of CdSe nanocrystals is proposed, namely alkyl or aryl derivatives of carbodithioic acids (R-C(S)SH). The main advantages of these new ligands are as follows: they nearly quantitatively exchange the initial surface ligands (TOPO) in very mild conditions; they significantly improve the resistance of nanocrystals against photooxidation because of their ability of strong chelate-type binding to metal atoms; their relatively simple preparation via Grignard intermediates facilitates the development of new bifunctional ligands containing, in addition to the anchoring carbodithioate group, a second function, which enables the grafting of molecules or macromolecules of interest on the nanocrystal surface. To give an example of this approach, we report, for the first time, the grafting of an electroactive oligomer from the polyaniline family-aniline tetramer-on CdSe nanocrystals after their functionalization with 4-formyldithiobenzoic acid. The grafting proceeds via a condensation reaction between the aldehyde group of the ligand and the terminal primary amine group of the tetramer. The resulting organic/inorganic hybrid exhibits complete extinction of the fluorescence of its constituents, indicating efficient charge or energy transfer between the organic and the inorganic semiconductors.     

Optimizing the formation of biocompatible gold nanorods for cancer research: functionalization, stabilization and purification

We have investigated the most efficient way of preparing biocompatible gold nanorods (GNR) used as tool for cancer imaging and therapy. The surface of cetyltrimethylammonium bromide-stabilized gold nanorods (GNR-CTAB) was functionalized with various thio-polyethylene glycols of the general formula HS-PEGmX (m=356-10,000; X=-OMe, -NH(2)). The influence of several parameters such as PEG chain length, reaction conditions and purification method on long-term stability, morphology and optical properties of the produced GNR-S-PEGmX was studied, demonstrating the existence of a threshold HS-PEGmX chain length (with molecular weight m≥2000) for efficient steric stabilization of GNR. Several purification techniques were compared: dialysis, centrifugation and a rarely used technique in this field, size exclusion chromatography. While a very weak efficiency of dialysis was evidenced, both centrifugation and size exclusion chromatography were found to provide pure GNRs, though the latter method yielded nanoparticles with a significantly higher stability. Finally, the long-term stability of the produced GNRs was investigated in various media: water, PBS buffer and serum.     

Microfluidic fabrication of monodisperse biocompatible and biodegradable polymersomes with controlled permeability

We describe a versatile technique for fabricating monodisperse polymersomes with biocompatible and biodegradable diblock copolymers for efficient encapsulation of actives. We use double emulsion as a template for the assembly of amphiphilic diblock copolymers into vesicle structures. These polymersomes can be used to encapsulate small hydrophilic solutes. When triggered by an osmotic shock, the polymersomes break and release the solutes, providing a simple and effective release mechanism. The technique can also be applied to diblock copolymers with different hydrophilic-to-hydrophobic block ratios, or mixtures of diblock copolymers and hydrophobic homopolymers. The ability to make polymer vesicles with copolymers of different block ratios and to incorporate different homopolymers into the polymersomes will allow the tuning of polymersome properties for specific technological applications.     

A general strategy for one-step fabrication of biocompatible microcapsules with controlled active release

Fabrication of biocompatible core-shell microcapsules in a controllable and scalable manner remains an important but challenging task.Here,we develop a one-step microfluidic approach for the highthroughput production of biocompatible microcapsules,which utilizes single emulsions as templates and controls the precipitation of biocompatible polymer at the water/oil interface.The facile method enables the loading of various oils in the core and the enhancement of polymer shell strength by polyelectrolyte coating.The resulting microcapsules have the advantages of controllability,scalability,biocompatibility,high encapsulation efficiency and high loading capacity.The core-shell microcapsules are ideal delivery vehicles for programmable active release and various controlled release mechanisms are demonstrated,including burst release by vigorous shaking,pH-triggered release for targeted intestinal release and sustained release of perfume over a long period of time.The utility of our technique paves the way for practical applications of core-shell microcapsules.     

A renewable, chemoselective, and quantitative ligand density microarray for the study of biospecific interactions

Novel renewable microarray technology has been developed to immobilize and release carbohydrates and proteins from self-assembled monolayers (SAMs) of electroactive quinone-terminated alkanethiolates on gold surfaces. This method may be applied to a variety of research fields for use in biosensor technology and the generation of renewable and tailored microarrays for biospecific cell-based assays.     

Electrochemical template synthesis of multisegment nanowires: fabrication and protein functionalization

Multisegment nanowires represent a unique platform for engineering multifunctional nanoparticles for a wide range of applications. For example, the optical and magnetic properties of nanowires can be tailored by modifying the size, shape, and composition of each segment. Similarly, surface modification can be used to tailor chemical and biological properties. In this article, we report on recent work on electrochemical template synthesis of nanogap electrodes, the fabrication of multisegment nanowires with embedded catalysts, and the selective functionalization of multisegment nanowires with proteins.     

Regio- and chemoselective multiple functionalization of pyrimidine derivatives by selective magnesiations using TMPMgCl.LiCl

Successive regio- and chemoselective magnesiations of pyrimidines using TMPMgCl.LiCl furnish, after trapping with various electrophiles, highly functionalized derivatives in good to excellent yields. Applications to the synthesis of antiviral and anti-inflammatory agents such as p38 and sPLA2 kinase inhibitors are reported.     

Simple fabrication of a smart microarray of polystyrene microbeads for immunoassay

We describe a simple method to fabricate an array of polystyrene microbeads (PS μbeads) conjugated with an elastin-like polypeptide (ELP) on a glass surface using a removable polymer template (RPT). A thin layer of adhesive was spun-cast on glass and cured by UV radiation. Micropatterns of an RPT were then transferred onto the surface by microcontact printing. The adhesion of PS μbeads on the surface depended on the adhesion performance of the adhesive layer, which could be adjusted by irradiation time. An array of PS μbeads conjugated with ELP was used for a smart immunoassay of prostate-specific antigen (PSA), a cancer marker. By controlling the phase transition of ELP molecules, PSA molecules were selectively adhered or released from the bead surface. The selective and reversible binding of PSA molecules on the bead surface was characterized with fluorescence microscopy.     

Direct method for surface silyl functionalization of mesoporous silica

A direct method of surface silyl modification and simultaneous surfactant removal of mesoporous silica is investigated in its physicochemical details. Twelve different silanes of various functionalities are studied. The method employs an alcohol solution of silanes to allow the simultaneous surfactant/silyl exchange process, which results in a more uniform monolayer coverage of the surface and a higher amount of surface attachments of silane. We vary the solution concentration of silanes to study the effect on loadings. It is found that the variation of the surface loading of the silyl group follows a Langmuir adsorption model closely. The method gives one a well-controlled monolayer coverage of the surface. The loadings are determined by the exchange equilibrium. Fittings of the loading data to Langmuir adsorption isotherms give one the adsorption equilibrium constants and maximum surface loadings. We categorize the silanes into three different groups according to the values of the equilibrium constants and discuss them with respect to molecular structures. We also report on the extensive characterizations of the surface-functionalized mesoporous materials, such as nitrogen adsorptions, X-ray diffraction, 29Si magic-angle spinning NMR, 13C magic-angle spinning NMR, and IR spectroscopy. The method provides one with a convenient and highly controllable approach to the surface functionalization of mesoporous silica.     

Thioacylation reactions for the surface functionalization of phosphorus-containing dendrimers

The functionalization of phosphorus-containing dendrimers was easily achieved through thioacylation reactions involving new dendrimers capped with dithioester end groups and various functionalized amines. These reactions were successfully applied to the first generation (12 end groups) and the third generation of the dendrimer (48 end groups) and allowed their functionalization by various primary or secondary amines, alcohols, glycols, and azides. [reaction: see text]     

Dielectrophoresis-based particle exchanger for the manipulation and surface functionalization of particles

We present a microfluidic device where micro- and nanoparticles can be continuously functionalized in flow. This device relies on an element called "particle exchanger", which allows for particles to be taken from one medium and exposed to some reagent while minimizing mixing of the two liquids. In the exchanger, two liquids are brought in contact and particles are pushed from one to the other by the application of a dielectrophoretic force. We determined the maximum flow velocity at which all the particles are exchanged for a range of particle sizes. We also present a simple theory that accounts for the behaviour of the device when the particle size is scaled. Diffusion mixing in the exchanger is also evaluated. Finally, we demonstrate particle functionalization within the microfluidic device by coupling a fluorescent tag to avidin-modified 880 nm particles. The concept presented in this paper has been developed for synthesis of modified particles but is also applicable to on-chip bead-based chemistry or cellular biology.     

Enzyme-encapsulated silica monolayers for rapid functionalization of a gold surface

We report a simple and rapid method for the deposition of amorphous silica onto a gold surface. The method is based on the ability of lysozyme to mediate the formation of silica nanoparticles. A monolayer of lysozyme is deposited via non-specific binding to gold. The lysozyme then mediates the self-assembled formation of a silica monolayer. The silica formation described herein occurs on a surface plasmon resonance (SPR) gold surface and is characterized by SPR spectroscopy. The silica layer significantly increases the surface area compared to the gold substrate and is directly compatible with a detection system. The maximum surface concentration of lysozyme was found to be a monolayer of 2.6 ng/mm(2) which allowed the deposition of a silica layer of a further 2 ng/mm(2). For additional surface functionalization, the silica was also demonstrated to be a suitable matrix for immobilization of biomolecules. The encapsulation of organophosphate hydrolase (OPH) was demonstrated as a model system. The silica forms at ambient conditions in a reaction that allows the encapsulation of enzymes directly during silica formation. OPH was successfully encapsulated within the silica particles and a detection limit for the substrate, paraoxon, using the surface-encapsulated enzyme was found to be 20 microM.     

Efficient preparation of amine-modified oligodeoxynucleotide using modified H-phosphonate chemistry for DNA microarray fabrication

Amine-modified oligodeoxynucleotides (AMO) are commonly used probe oligodeoxynucleotides for DNA microarray preparation. Two methods are currently used for AMO preparation—use of amine phosphoramidites protected by acid-labile monomethoxytrityl (MMT) groups or alkali-labile trifluoroacetyl (TFA) groups. Because conventional AMO preparation procedures have defects, for example stringent acidic conditions are required for deprotection of MMT and hydrophobic purification cannot be used for TFA-protected amino groups, conventional preparation of AMO is unlikely to result in the expected outcome. In this paper a method of AMO synthesis using modified H-phosphonate chemistry is suggested. An aliphatic diamine is coupled with a phosphonate group forming a phosphoramidate linkage to the last internucleotide phosphate of oligodeoxynucleotides. In this method dimethoxytrityl (DMT) purification steps are used and stringent acid deprotection is not required to obtain the AMO. Although the method could lead to formation of AMO diastereomers, melting-temperature and CD analysis showed for two AMO that DNA duplex formation was the same as when normal oligodeoxynucleotides were used. Also, when these AMO were used as probes for DNA microarrays the immobilization efficiency was similar to that for AMO probes prepared by conventional means using an amino-modifier unit. The hybridization performance of these AMO was better than for those prepared conventionally. The procedures suggested would be useful for preparation of efficient AMO for fabrication of DNA microarrays and DNA-based nanoparticle systems. Nagendra Kumar Kamisetty and Seung Pil Pack have equally contributed to this work.     

Shotgun proteomic analytical approach for studying proteins adsorbed onto liposome surface

The knowledge about the interaction between plasma proteins and nanocarriers employed for in vivo delivery is fundamental to understand their biodistribution. Protein adsorption onto nanoparticle surface (protein corona) is strongly affected by vector surface characteristics. In general, the primary interaction is thought to be electrostatic, thus surface charge of carrier is supposed to play a central role in protein adsorption. Because protein corona composition can be critical in modifying the interactive surface that is recognized by cells, characterizing its formation onto lipid particles may serve as a fundamental predictive model for the in vivo efficiency of a lipidic vector. In the present work, protein coronas adsorbed onto three differently charged cationic liposome formulations were compared by a shotgun proteomic approach based on nano-liquid chromatography–high-resolution mass spectrometry. About 130 proteins were identified in each corona, with only small differences between the different cationic liposome formulations. However, this study could be useful for the future controlled design of colloidal drug carriers and possibly in the controlled creation of biocompatible surfaces of other devices that come into contact with proteins into body fluids.     

Characterizing the surface quality and droplet interface shape for microarray plates

The variation in the surface quality of microarray plates was examined by measuring the contact angles of 480 droplets on five microarray plates. It was found that the measured contact angle did not accurately predict the droplet shape for moderate Bond numbers (~0.5 ≤ N(B) ≤ 5). By defining an apparent contact angle using the ratio of the contact radius to the height, the variance in the predicted interface shape decreased by greater than a factor of 3 for both local and globally averaged characteristics. The error in the predicted droplet height was also reduced by 3 orders of magnitude.     

Maleimide-activated aryl diazonium salts for electrode surface functionalization with biological and redox-active molecules

A versatile and simple method is introduced for formation of maleimide-functionalized surfaces using maleimide-activated aryl diazonium salts. We show for the first time electrodeposition of N-(4-diazophenyl)maleimide tetrafluoroborate on gold and carbon electrodes which was characterized via voltammetry, grazing angle FTIR, and ellipsometry. Electrodeposition conditions were used to control film thickness and yielded submonolayer-to-multilayer grafting. The resulting phenylmaleimide surfaces served as effective coupling agents for electrode functionalization with ferrocene and the redox-active protein cytochrome c. The utility of phenylmaleimide diazonium toward formation of a diazonium-activated conjugate, followed by direct electrodeposition of the diazonium-modified DNA onto the electrode surface, was also demonstrated. Effective electron transfer was obtained between immobilized molecules and the electrodes. This novel application of N-phenylmaleimide diazonium may facilitate the development of bioelectronic devices including biofuel cells, biosensors, and DNA and protein microarrays.     

Cellulose nanocrystal surface functionalization for the controlled sorption of water and organic vapours

The surface grafting of cellulose nanocrystals (CNC) is a valuable tool to increase opportunities for their application. This work had several goals designed to improve CNC: reduction of hornification, increased re-dispersibility after CNC drying, and tuning of the surface graft to enhance the adsorption of particular molecules. To achieve this, the CNC surfaces were modified chemically with aromatic surface grafts using widely employed methods: the creation of urethane linkages, silylation and esterification. Even a low degree of grafting sufficed to increase water contact angles to as much as 96°. The analysis of water sorption isotherms showed that at high water activities, capillary condensation could be suppressed and hysteresis was decreased. This indicates that hornification was significantly suppressed. However, although the contact angles increased, the water sorption isotherms were changed only slightly because of reduced hysteresis. The grafts were not able to shield the surface from water vapour sorption. A comparison of the sorption isotherms of anisole and cyclohexane, sorbates with a similar surface area, showed that the sorption of anisole was three times higher than that of cyclohexane. The specific sorption of aromatic molecules was achieved and the most efficient methodology was the esterification of CNC with carboxylic acids containing a flexible linker between the aromatic moiety and ester bond.