A covalent method of gentamicin bonding to silica supports

Results of a novel method of covalent bonding of an antibiotic (gentamicin) to silica bead supports are shown. Gentamicin was immobilized to four types of matrix: silica gel and porous glass beads activated by either silanization (APTES) or by adhesively bound keratin (with immobilization yield ranging from 36.5 to 91%). Gentamicin was immobilized to the supports after opening its carbohydrate ring in the molecule. This method of gentamicin activation before the immobilization process did not inhibit its antibiotic activity. The four gentamicin-containing immobilized preparations were stable, meaning that they did not release the antibiotic into the solution during the 30 days of incubation, not even during shaking experiments.     

Micropatterning Polydiacetylene Supramolecular Vesicles on Glass Substrates using a Pre‐Patterned Hydrophobic Thin Film

A micropatterning method for depositing polydiacetylene (PDA) supramolecular vesicles on glass substrates, employing a pre‐patterned hydrophobic thin film, is presented. The pre‐patterned hydrophobic thin film is used to define surface‐exposed regions on hydrophilic glass substrates where selective immobilization of PDA vesicles takes place. The observation that fluorescent patterns are generated by heat treatment of the deposited PDAs demonstrates that the vesicles are successfully immobilized in the form of micropatterns. Finally, the possibility that this strategy may be used to design a vesicle‐based sensor chip system is demonstrated by the observation that fluorescence patterns are generated when the immobilized PDA vesicles interact with cyclodextrin.     

Desiccation Kinetics and Biothermodynamics of Glass Forming Trehalose Solutions in Thin Films

In this study, the desiccation kinetics of aqueous trehalose solutions were investigated numerically by solving the coupled heat and mass transfer problem with a moving interface using the finite element method. The free volume models for vapor pressure and mutual diffusion coefficient were incorporated into the model to account for the effect of glass transition on the heat and mass transport process that ultimately determines the desiccation kinetics. It was found that the temperature in the film could drop significantly upon the initiation of drying due to the absorption of latent heat associated with water evaporation although the spatial distribution of temperature in the solution is very homogeneous. On the contrary, the spatial distribution of water content in the solution is non-homogeneous, particularly at the solution–vapor interface where an extremely thin layer of skin with extremely low molecular mobility usually forms during drying. The solution film can be dried to ∼6–10 wt.% residual water within minutes for thin films; but drying times depends strongly on the initial film thickness, initial solution concentration, temperature, and convective coefficient. Desiccation to below 6 wt.% residual water is very slow due to the retarded water mobility in the extremely thin skin where the solution is in the glassy state. Since the water mobility in a trehalose solution or glass with 6–10% residual water is still high enough to allow degradative reactions to occur in a relatively short time at room temperature, it is important that the samples should be kept at a temperature around 0 °C or lower for storage after drying. Furthermore, approaches that might enable further quick reduction of the residual water to less than 6–10 wt.% are also proposed so that a sample could be preserved at super-zero or even room temperature. The established models and the reported results will be useful for the development of effective protocols for lyopreservation of biomaterials including living cells using trehalose as the excipient.     

Conjugation of fluorescein isothiocyanate to antibodies: I. Experiments on the conditions of conjugation

In the present study experiments concerning the conjugation of fluorescein isothiocyanate (FITC) to antibodies, which were previously performed by Goldstein, Slizys and Chase (1961), McKinney, Spillane and Pearce (1964) and Wood, Thompson and Goldstein (1965) are compiled, reproduced, extended and discussed.

The results of the experiments indicate that if a relatively pure IgG, obtained by DEAE Sephadex chromatography, is conjugated with FITC of high quality, a maximal molecular fluorescein/protein (F/P) ratio is reached in a short time if reaction temperature, pH and protein concentration are high. Maximal labelling was obtained in 30–60 minutes at room temperature, pH 9.5 and an initial protein concentration of 25 mg/ml. The separation of optimally labelled antibodies from under- and over-labelled proteins may be achieved by gradient DEAE Sephadex chromatography. Electrophoretically distinct IgG molecules proved to have about the same affinity for FITC. A correlation between the activity of antibodies in fluorescent and precipitation techniques was found.

     

Cyclo-(DfKRG) peptide grafting onto Ti-6Al-4V: physical characterization and interest towards human osteoprogenitor cells adhesion

In the present paper, specific interest has been devoted to the design of new hybrid materials associating Ti-6Al-4V alloy and osteoprogenitor cells through the grafting of two RGD containing peptides displaying a different conformation (linear RGD and cyclo-DfKRG) onto titanium surface. Biomimetic modification was performed by means of a three-step reaction procedure: silanization with APTES, cross-linking with SMP and finally immobilization of peptides thanks to thiol bonding. The whole process was performed in anhydrous conditions to ensure homogeneous biomolecules layout as well as to guarantee a sufficient amount of biomolecules grafted onto surfaces. The efficiency of this new route for biomimetic modification of titanium surface was demonstrated by measuring the adhesion between 1 and 24 h of osteoprogenitor cells isolated from HBMSC. Benefits of the as-proposed method were related to the high concentration of peptides grafted onto the surface (around 20 pmol/mm(2)) as well as to the capacity of cyclo-DfKRG peptide to interact with integrin receptors. Moreover, High Resolution beta-imager (using [(35)S]-Cys) has exhibited the stability of peptides grafted onto the surface when treated in harsh conditions.     

Reactive magnetic poly(divinylbenzene-co-glycidyl methacrylate) colloidal particles for specific antigen detection using microcontact printing technique

Epoxy-functionalized magnetic poly(divinylbenzene-co-glycidyl methacrylate) colloidal particles (mPDGs) were prepared by co-polymerization of 1,4-divinylbenzene and glycidyl methacrylate monomers. The reaction was conducted by batch emulsion polymerization in the presence of an oil in water magnetic emulsion as a seed. The chemical composition, morphology, iron oxide content, magnetic properties, particle size and colloidal stability of the prepared magnetic polymer particles were characterized using Fourier transform infrared spectroscopy, transmission electron microscopy, thermal gravimetric analysis, vibrating sample magnetometry, dynamic light scattering, and zeta potential determination, respectively. The prepared mPDGs were immobilized on a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES)/octadecyltrichlorosilane (OTS), which were patterned on glass using microcontact printing technique, forming mPDGs–APTES/OTS reactive surface. This construction (mPDGs–APTES/OTS) was used as a solid support for immunoassay. The immobilized magnetic particles were bioconjugated with monoclonal anti-human IL-10 antibody to provide specific and selective recognition sites for the recombinant human IL-10 protein (antigen). Fluorescence microscopic examination was carried out to follow this immunoassay using fluorescently labeled anti-human IL-10 antibody. The results obtained proved the successful use of mPDGs–APTES/OTS microcontact printed surfaces in an immunoassay, which can be exploited and integrated into microsystems in order to elaborate medical devices (e.g. biosensors) which could provide rapid analysis at high sensitivity with low volumes of analyte.     

Micropatterned assembly of silica nanoparticles for a protein microarray with enhanced detection sensitivity

We used an assembly of silica nanoparticles (SNPs) as a three-dimensional template for protein immobilization to prepare a protein microarray with enhanced protein loading capacity and detection sensitivity. SNPs were first modified with 3-aminopropyltriethoxysilane (APTES) for covalent immobilization of protein and micropatterned on poly(ethylene glycol)(PEG)-coated glass slides using elastomeric membranes with an array of holes. Proteins were selectively immobilized only on the SNP region, while the PEG regions served as an effective barrier to protein adsorption. Because of multi-layered SNPs that had curved surface, protein loading in the SNP micropattern was about six times greater than on a planar surface, as observed by fluorescence microscopy, which consequently improved the protein activity and reaction rate. GOX-catalyzed glucose oxidation and the molecular recognition mediated, specific binding between biotin and streptavidin were both successfully assayed using SNP microarrays, with better fluorescence signal and sensitivity than corresponding planar microarrays.     

Surface functionalization of bioactive glasses

Different cleaning and silanization methods have been applied to bioactive glasses with the aim of covalently bonding bone morphogenetic proteins (BMP-2) to the surface. Several glasses, with different bioactivity index, were cleaned with acidic, basic, or neutral aqueous media to investigate the role of pH in the formation of silanols on glass surfaces of different reactivity. The cleaned glasses were then functionalized using 3-aminopropyl-triethoxysilane (APTS). After the optimization of the silanization procedure, proteins of different complexity were immobilized on the functionalized glasses. To optimize the protein immobilization, a model protein (carnosine) was first used, and the procedure was then used to bind human BMP-2. The glass surfaces were characterized during each step of the treatment by water contact angles and X-ray photoelectron analyses. The APTS functionalization was then used to immobilize bone morphogenetic protein on the bioactive glasses. This result suggested that such a treatment could be successfully used as an efficient alternative to systemic administration of transforming growth factors for the development of local delivery vehicle implants.     

Characterization of the Crosslinking Kinetics of a Thin Polymeric Layer by Real‐Time Dielectric Relaxation Spectroscopy

The crosslinking kinetic of a thin polymeric layer based on a prepolymer of a phthalic aciddiallylester was studied by real‐time dielectric spectroscopy in the frequency range from 10^–1 to 10⁵ Hz. With increasing reaction time the real part of the dielectric function ε ′ decreases. The time dependence of ε ′ can be described by a stretched exponential function with a stretching exponent of 0.5. This means that the influence of the chemical reaction on ε ′ cannot be described by a first order kinetic. From the temperature dependence of the characteristic time constant an activation energy of 71 kJ/mol could be estimated for the reaction. From the dielectric loss data the change of the relaxation rate of the dynamic glass transition f_pα with the reaction time is obtained. After a temperature dependent induction period f_pα decreases very strongly. No plateau value which corresponds to a glass transition in the crosslinked system is obtained for long reaction times.     

Chemoselective fabrication of high density peptide microarray by hetero-bifunctional tetra(ethylene glycol) linker for click chemistry conjugation

A hetero-bifunctional tetra(ethylene glycol) molecule with silane and azide termini was synthesized, and this molecule was used to prepare azide-derivatized glass surface in one step. The resulting glass surface was available for fabricating peptide microarray by the conjugation with alkyne-containing peptide using click chemistry, which proceeded to the completion at low temperature and in aqueous solution. A high density of peptide on the surface was achieved due to concise overall procedure and highly efficient conjugation reaction. Immobilized peptides were highly bio-functional on the surface, as demonstrated by the ability to detect protease activity. Due to the biologically orthogonal manner of conjugation, peptide conjugated by site-specific immobilization was more accessible by protease than that conjugated by random amide conjugation. This site-specific and high efficient immobilization technique could be expanded to large scale development of biocompatible peptide and protein arrays for use in various applications.     

质粒DNA荧光修饰方法的研究

目的 研究一种质粒DNA荧光标记的简便方法,以便进行DNA递送的示踪.方法 质粒经 溴激活后,于不同温度下存放不同时间,再与1,10-二氨基葵烷形成氨基衍生物.氨基修饰质粒与荧光素异硫氰酸酯( FITC)反应,制备出荧光标记的质粒,纯化并收集产物,计算其标记效率,评估溴激活质粒在不同温度下存放不同时长对标记反应的影响;观察荧光标记对质粒转染效率的影响,并用激光共聚焦显微镜观察和流式细胞仪检测示踪效果.结果 实验数据显示溴活化的质粒随存放时间的延长标记效率反而下降,存放4℃较室温(25℃)的标记效率更高,提示溴活化质粒不适宜存放;而与没有标记的质粒相比,荧光标记的质粒对转染效率几乎没有影响.荧光标记的质粒应用于流式细胞仪检测和激光共聚焦显微观察都取得了较好的示踪效果.结论 本研究建立了一种荧光标记质粒DNA的简便方法,并且在基因递送的示踪实验中获得了较好的效果.     

Localized electrical stimulation to C2C12 myotubes cultured on a porous membrane-based substrate

We report a porous membrane-based cell culture device that can conduct localized electrical stimulation of a cell monolayer. The device’s cell culture substrate is a microporous alumina membrane with an underlying thin poly(dimethylsiloxane) (PDMS) film spotted with holes. When electric current is generated between the device’s Pt ring electrodes—one of which is placed above the cells and the other below the PDMS layer—the current density condenses at the holes in the PDMS film, and cells located above the holes can be electrically stimulated. C2C12 cells were confluently cultured on the substrate and were differentiated to myotubes. To control the stimulated area in the substrate, we attempted to seal and reopen the holes of the PDMS film by using an air bubble. Since the current pulse could be effectively blocked at the sealed holes, fluorescent Ca²⁺ transients, indicative of cellular excitation, were observed from the myotubes located above holes in the open state. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Covalent immobilization of proteins for the biosensor based on imaging ellipsometry

In the development of biosensors, the immobilization of biomolecules at interfaces played a crucial role. The feasibility of using 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde (Glu) to modify silicon surface to immobilize covalently protein for immunoassay with the biosensor based on imaging ellipsometry was investigated. The higher density and stability of human IgG layer could be obtained on the silicon surface modified with APTES and Glu than that on the silicon surface modified with dichlorodimethylsilane (DDS). The human IgG molecules immobilized covalently on APTES-Glu surface bound more anti-IgG molecules than that on DDS surface, which indicated that the human IgG molecules could maintain higher binding capability on APTES-Glu surface. Tween 20 was able to block the undesirable adsorption on APTES-Glu surface, and also enhanced the recognition between human IgG and its antibody on both APTES-Glu and DDS surfaces. The combination of this protein covalent immobilization and the biosensor has the potential to be developed into a fast, simple immunoassay technique.     

Modified fabrication process of protein chips using a short-chain self-assembled monolayer

In previous work a short chain SAM, 4,4-Dithiodibutyric Acid (DTBA) was found to be a thin monolayer in protein chips. However, obtaining uniform fluorescent intensity remains difficult because water-soluble carbodiimides (EDC) in an aqueous system cause the hydrolysis of N-hydroxysuccinimide ester (NHS esters). The hydrolysis of NHS esters reduces coupling yields and therefore reduces the fluorescent intensity of protein chips. The NHS can increase the stability of active intermediate resulting from the reaction of EDC and NHS, but the ratio of the concentration of EDC to that of NHS strongly affects this stability. The effects of the solvents used in the washing step are studied to solve this problem. The results reveal that PBST (PBS + 5% Tween20) is more effective in reducing the hydrolysis of NHS esters than deionized water. Additionally, the effects of 3:1 and 5:2 EDC/NHS ratios on the chips are examined. The 3:1 EDC/NHS ratio yields a higher fluorescent intensity than the 5:2 ratio. The effects on the chips of dissolving EDC in DI water, DI water + 0.1 M MES and alcohol are also investigated. The results show that alcohol provides higher fluorescent intensity than other solvents and the reaction time of 4 h yields a high fluorescent intensity with 3:1 EDC/NHS ratio. A modified fabrication process of protein chips using 4,4-DTBA is developed. In this work, 160 mM 4,4-DTBA is used as a self-assembled monolayer in the fabrication of protein chips. Experiments to characterize 4,4-DTBA are performed by contact angle goniometry and Fourier transform infrared spectroscopy (FTIR). Furthermore, the immobilized protein A-FITC (fluorescein isothiocyanate) is adopted in fluorescent assays.     

Study of two different thin film coating methods in transmission laser micro-joining of thin Ti-film coated glass and polyimide for biomedical applications

Biomedical devices and implants require precision joining for hermetic sealing which can be achieved with low power lasers. The effect of two different thin metal film coating methods was studied in transmission laser micro-joints of titanium-coated glass and polyimide. The coating methods were cathodic arc physical vapor deposition (CA-PVD) and electron beam evaporation (EB-PVD). Titanium-coated glass joined to polyimide film can have neural electrode application. The improvement of the joint quality will be essential for robust performance of the device. Low power fiber laser (wave length = 1100 nm) was used for transmission laser micro-joining of thin titanium (Ti) film (～200 nm) coated Pyrex borosilicate 7740 glass wafer (0.5 mm thick) and polyimide (Imidex) film (0.2 mm thick). Ti film acts as the coupling agent in the joining process. The Ti film deposition rate in the CA-PVD was 5–10 Å /s and in the EB-PVD 1.5 Å /s. The laser joint strength was measured by a lap shear test, the Ti film surfaces were analyzed by atomic force microscopy (AFM) and the lap shear tested joints were analyzed by optical microscopy and scanning electron microscopy (SEM). The film properties and the failure modes of the joints were correlated to joint strength. The CA-PVD produced around 4 times stronger laser joints than EB-PVD. The adhesion of the Ti film on glass by CA-PVD is better than that of the EB-PVD method. This is likely to be due to a higher film deposition rate and consequently higher adhesion or sticking coefficient for the CA-PVD particles arriving on the substrate compared to that of the EB-PVD film. EB-PVD shows poor laser bonding properties due to the development of thermal hotspots which occurs from film decohesion.     

Charge binding of rhodamine derivative to OH- stabilized nanomaghemite: universal nanocarrier for construction of magnetofluorescent biosensors

Superparamagnetic nanoparticles (20-40 nm) of maghemite, γ-Fe(2)O(3), with well-defined stoichiometric structure, are synthesized by the borohydride reduction of ferric chloride at an elevated temperature (100°C) followed by thermal treatment of the reaction product. Prepared maghemite nanoparticles reveal excellent colloidal stability for a long time without the necessity for any additional surface modification. These colloidal features are due to surface stabilizing OH(-) groups, which act as charge barriers preventing a particle aggregation and enabling a reversible binding of various oppositely charged organic substances. Such binding with rhodamine B isothiocyanate results in the fluorescent magnetic nanocarrier providing, at the same time, a spacer arm for covalent immobilization of other biosubstances including enzymes. In this work, we exploit this general applicability of the developed nanocarrier for covalent immobilization of glucose oxidase. This is the first reported example of magnetically drivable fluorescent nanocatalyst. The immobilized enzyme creates a 3-5 nm thick layer on the nanoparticle surface as proved by high-resolution transmission electron microscopy. This layer corresponds to 10 enzyme molecules, which are bound to the nanoparticle surface as found by the fluorimetric determination of flavin adenine dinucleotide. The developed magnetic fluorescent nanocatalyst, showing a rate constant of 32.7s(-1) toward glucose oxidation, can be used as a biosensor in various biochemical, biotechnological, and food chemistry applications. The presence of the nanocatalyst can be simply monitored by its fluorescence; moreover, it can be easily separated from the solution by an external magnetic field and repeatedly used without a loss of catalytic efficiency.     

Using hydroxyapatite nanoparticles and decreased crystallinity to promote osteoblast adhesion similar to functionalizing with RGD

Better materials are needed to promote bone growth. For this reason, the present study created nanometer crystalline hydroxyapatite (HA) and amorphous calcium phosphate compacts functionalized with the arginine-glycine-aspartic acid (RGD) peptide sequence. Crystalline HA and amorphous calcium phosphate nanoparticles were synthesized by a wet chemical process followed by a hydrothermal treatment for 2 h at 200 degrees C and 70 degrees C, respectively. Resulting particles were then pressed into compacts. For the preparation of conventional HA particles (or those with micron diameters), the aforementioned pressed compacts were sintered at 1,100 degrees C for 2 h. Peptide functionalization was conducted by means of a three step reaction procedure: silanization with 3-aminopropyltriethoxysilane (APTES), cross-linking with N-succinimidyl-3-maleimido propionate (SMP), and finally peptide immobilization. The three step reaction procedure was characterized by a novel 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) fluorescence technique. For all materials, results showed that the immobilization of the cell adhesive RGD sequence increased osteoblast (bone-forming cell) adhesion compared to those non-functionalized and those functionalized with the noncell adhesive control peptide (RGE) after 4 h. However, surprisingly, results also showed that the adhesion of osteoblasts on non-functionalized amorphous nanoparticulate calcium phosphate was similar to conventional HA functionalized with RGD. Osteoblast adhesion on nanocrystalline HA (unfunctionalized and functionalized with RGD) was below that of the respective functionalized amorphous calcium phosphate but above that of the respective functionalized conventional HA. In this manner, results of this study suggest that decreasing the particulate size into the nanometer regime and reducing crystallinity of calcium phosphate based materials may promote osteoblast adhesion to the same degree as the well-established techniques of functionalizing conventional HA with RGD.     

Two amino acid-based superlow fouling polymers: Poly(lysine methacrylamide) and poly(ornithine methacrylamide)

We developed and investigated two new antifouling zwitterionic polymers, poly(lysine methacrylamide) (pLysAA) and poly(ornithine methacrylamide) (pOrnAA), both derived from natural amino acids – lysine and ornithine, respectively. The pLysAA and pOrnAA brushes were grafted on gold via the surface-initiated photoiniferter-mediated polymerization, with the polymer film thickness controlled by the UV-irradiation time. Nonspecific adsorption from human blood serum and plasma was investigated by surface plasmon resonance. Results show that the adsorption level decreased with the increasing film thickness. With the thin films of ∼14.5 nm, the minimal adsorption on pLysAA was 3.9 ng cm⁻² from serum and 5.4 ng cm⁻² from plasma, whereas the lowest adsorption on pOrnAA was 1.8 and 3.2 ng cm⁻², from serum and plasma, respectively. Such protein resistance is comparable to other widely reported antifouling surfaces such as poly(sulfobetaine methacrylate) and polyacrylamide, with a much thinner polymer film thickness. Both pLysAA and pOrnAA showed better protein resistance than the previously reported serine-based poly(serine methacrylate), whereas the pOrnAA is the best among three. The pLysAA- and pOrnAA-grafted surfaces also highly resisted the endothelial cell attachment and Escherichia coli K12 bacterial adhesion. Nanogels made of pLysAA and pOrnAA were found to be ultrastable in undiluted serum, with no aggregation observed after culturing for 24 h. Dextran labeled with fluorescein isothiocyanate (FITC–dextran) was encapsulated in nanogels as a model drug. The encapsulated FITC–dextran exhibited controlled release from the pOrnAA nanogels. The superlow fouling, biomimetic and multifunctional properties of pLysAA and pOrnAA make them promising materials for a wide range of applications, such as implant coating, drug delivery and biosensing.     

Localized surface plasmon resonance biosensor integrated with microfluidic chip

A sensitive and low-cost microfluidic integrated biosensor is developed based on the localized surface plasmon resonance (LSPR) properties of gold nanoparticles, which allows label-free monitoring of biomolecular interactions in real-time. A novel quadrant detection scheme is introduced which continuously measures the change of the light transmitted through the nanoparticle-coated sensor surface. Using a green light emitting diode (LED) as a light source in combination with the quadrant detection scheme, a resolution of 10⁻⁴ in refractive index units (RIU) is determined. This performance is comparable to conventional LSPR-based biosensors. The biological sensing is demonstrated using an antigen/antibody (biotin/anti-biotin) system with an optimized gold nanoparticle film. The immobilization of biotin on a thiol-based self-assembled monolayer (SAM) and the subsequent affinity binding of anti-biotin are quantitatively detected by the microfluidic integrated biosensor and a detection limit of 270 ng/mL of anti-biotin was achieved. The microfluidic chip is capable of transporting a precise amount of biological samples to the detection areas to achieve highly sensitive and specific biosensing with decreased reaction time and less reagent consumption. The obtained results are compared with those measured by a surface plasmon resonance (SPR)-based Biacore system for the same binding event. This study demonstrates the feasibility of the integration of LSPR-based biosensing with microfluidic technologies, resulting in a low-cost and portable biosensor candidate compared to the larger and more expensive commercial instruments.     

Oligo-microarray based on oligonucleotide immobilization on glass surface modified with activated acrylic acid-co-acrylamide copolymer

Oligo-microarray can be fabricated on modified surface of glass slides involving immobilization of oligonucleotides. In this study, glass slides were modified with acrylic acid-co-acrylamide copolymer and EDC (1-ethyl-3(3-dimethylaminopropyl)-carbodiimide)/NHS (N-hydroxysuccinimide) by covalent linkage method, and covalent immobilization of unmodified oligonucleotide on the glass surface was achieved. The platform with binding of stable and sensitive oligonucleotides was prepared for oligo-microarray fabrication. An optimum concentration of 0.5 g/L was used for spotting. The lengths of 26 to 70 mer oligonucleotides were immobilized on the surface efficiently. The spots were approximately 160 nm in diameter and their mean value of fluorescent intensity was measured in the range of 3.2 x 10(4) to 7.3 x 10(4) after hybridization. The modified glass surface was scanned by SEM and the dendritic structure was observed. Results showed that the process of preparation of the modified glass surface was simple and cost effective, and the modified surface can be used for the oligo-microarray fabrication and also attachment of protein, PNA etc.