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PubMedDate not supplied

Self-assembled extracellular matrix protein networks by microcontact printing.

Physiological patterns of the extracellular matrix protein, laminin-1, were obtained on glass substrates by physisorption-assisted microcontact printing. Besides the well-retained antigenicity confirmed by indirect immunofluorescence assays, we investigated the supramolecular organization of the proteins by atomic force microscopy. We found the characteristic protein self-assembling in polygonal networks with well-defined sub-100 nm quaternary structures of laminin. The formation of these physiological mesh-like protein matrices was obtained by means of one-step soft lithography without any preliminary functionalization of glass, which can be exploited for many possible applications for cell cultures and biomolecular devices.

Coated Materials, Biocompatible↗
PubMed2005

An inverted microcontact printing method on topographically structured polystyrene chips for arrayed micro-3-D culturing of single cells.

With the goal to investigate the relation of shape and function of single cells or clusters of cells in a 3-dimensional (3-D) microenvironment, we present a novel platform technology to create arrays of microwells on polystyrene (PS) chips for hosting cells in a local microenvironment characterized by controlled shape and surface chemistry. The micro-3-D cell culturing combines 2-dimensional chemical patterning with topographical microstructuring presenting to the cells a local 3-D host structure. Microwells of controlled dimensions were produced by a two-step replication process, based on standard microfabrication of Si, replica molding into poly(dimethylsiloxane), and hot embossing of PS. This allowed the production of large numbers of microstructured surfaces with high reproducibility and fidelity of replication. Using inverted micro contact printing, the plateau surface between the microwells was successfully passivated to block adsorption of proteins and prevent cell attachment by transfer of a graft-copolymer, poly(l-lysine)-g-poly(ethylene glycol). The surface inside the microwells was subsequently modified by spontaneous adsorption of proteins or functionalized PLL-g-PEG/PEG-X (X=biotin or specific, cell-interactive peptide) to elicit specific responses inside the wells. Preliminary cell experiments demonstrated the functionality of such a device to host single epithelial cells (MDCK II) inside the functionalized microwells and thus to control their 3-D shape. This novel platform is useful for fundamental cell-biological studies and applications in the area of cell-based sensing.

Adsorption↗
PubMed2005

Novel hepatocyte culture system developed using microfabrication and collagen/polyethylene glycol microcontact printing.

The better understanding of cell biology and cell communication requires novel culture systems that better represent the natural cell environment in tissues and organs. We developed a spherical organoid (spheroid) microarray culture system using a combination of microfabrication and microcontact printing. The system consisted of a chip that had cylindrical cavities of 300 microm diameter at a density of 700 cavities/cm2. The bottom faces of these cavities were defined as two different regions that either supported or inhibited cell adhesion. In the cell adhesion region, the center of the bottom face of a 100 microm diameter in a cavity was modified with collagen (Col), and in the non-adhesion region, the entire region around the cavity, except the Col spots, was modified with polyethylene glycol. Primary hepatocytes spontaneously formed spheroids with a uniform diameter at the center of each cavity on the chip. Hepatocytes forming spheroids had a cuboidal cell shape, similar to hepatocytes in vivo, and stably maintained liver-specific phenotypes, such as liver-enriched transcriptional factors, albumin secretion, urea cycle enzymes, and intercellular adhesion molecules. This novel culture system may be applicable as a cellular platform for fundamental studies in cell biology and tissue engineering applications.

Animals↗
PubMed2005

Deinking of laser printed copy paper with a mediated free radical system.

This work investigated the effect of a chelator-mediated bio-mimetic free radical treatment on repulping and flotation operations during the deinking of laser printed copy paper. A chelator-mediated free radical treatment was carried out at two different chemical levels and a two-step repulping method, which combined conventional alkaline repulping as well as free radical treatment, was also developed. Flotation trials were performed on each of these treated samples to separate ink particles. Results from image analysis and paper physical properties testing are presented, and the deinking efficiency for these different treatments is also compared. Results indicate that under properly controlled conditions, free radical treatment can perform better than conventional chemical deinking methods.

Chelating Agents↗
PubMed2006

Performance of combined process of anoxic baffled reactor-biological contact oxidation treating printing and dyeing wastewater.

A study of the treatment of printing and dyeing wastewater was carried out using the combined process of anoxic baffled reactor-biological contact oxidation. The results showed the pH ascended continuously and the oxidation-reduction potential dropped gradually from compartment 1-6 in ABR. When hydraulic retention time was 12h, color removal efficiency was 92% and the color of effluent of ABR could satisfy the professional emission standard (grade-1) of textile and dyeing industry of China. The total COD removal efficiency of the combined process was 86.6% and the COD of effluent could satisfy the professional emission standard (grade-2) of textile and dyeing industry of China.

Bacteria, Anaerobic↗
PubMed2006

Enzymatic deinking of laser printed office waste papers: some governing parameters on deinking efficiency.

The protocol for the enzymatic deinking of laser printed waste papers on a laboratory scale using cellulase (C) and hemicellulase (H) of Aspergillus niger (Amano) was developed as an effective method for paper recycling. A maximum deinking efficiency of almost 73% by the enzyme combination of C:H was obtained using the deinking conditions of pulping consistency of 1.0% (w/v) with the pulping time of 1.0min, temperature of 50 degrees C, pH=3.5, agitation rate of 60rpm, pulp concentration of 4% (w/v), concentration of each enzyme of 2.5U/g air dried pulp and the enzyme ratio of 1:1. The deinking efficiency was further enhanced to 95% using the optimized flotation system consisting of pH=6.0, Tween 80 of concentration 0.5% (w/w), working air flow rate of 10.0L/min and temperature of 45 degrees C. The deinked papers were found to exhibit properties comparable to the commercial papers suggesting the effectiveness of the enzymatic process developed.

Aspergillus↗
PubMed2004

Construction of a glucose sensor based on a screen-printed electrode and a novel mediator pyocyanin from Pseudomonas aeruginosa.

Pyocyanin is the blue phenazine pigment produced by Pseudomonas aeruginosa. Pyocyanin production using immobilized cells was investigated. The maximum production of pyocyanin was obtained using cells immobilized in kappa-carrageenan. Moreover, 0.01% PO4(3-), 0.2% Mg(2+), 0.001% Fe(2+), 1% glycerine, 0.8% leucine and 0.8% dl-alanine were also essential for pyocyanin production. Pyocyanin was purified by chloroform extraction and silica gel column chromatography. An amperometric biosensor system using a screen-printed electrode and pyocyanin as mediator were also developed for a more accurate determination of glucose concentration. Pyocyanin, which exists in the oxidated form, was reduced by the reaction between glucose oxidase and glucose. The reduced form was then converted back to the oxidized form by an oxidative reaction on the electrode. There was a linear relation ship between sensor output currents and glucose concentrations ranging from 1 to 20mM under the following conditions: -200 mV of the applied potential, pH 5.0, and 10 U of the immobilized enzyme. The coefficient of variation was below 3% (n = 5) for the glucose sensor.

Biosensing Techniques↗
PubMed2004

Optimisation of the composition of a screen-printed acrylate polymer enzyme layer with respect to an improved selectivity and stability of enzyme electrodes.

Glucose oxidase (GOD) was immobilized on screen-printed platinum electrodes by entrapment in a screen printable paste polymerized by irradiation with UV-light. The influences of different additives, in particular polymers and graphite, on the sensitivity and stability of the sensor and the permeability of the enzyme layer for a possible electrochemical interferent were investigated. The chosen additives were Gafquat 755N, poly-L-lysine, bovine serum albumin (BSA), sodium dodecylsulfate (SDS), polyethylene glycol (PEG), Nafion and graphite. All additives led to increases of glucose signals, i.e. improved the sensitivity of glucose detection with Gafquat 755N, poly-L-lysine, SDS and graphite showing the strongest influences with increases by a factor 4, 6.5, 5 and 10, respectively. Ascorbic acid was used as a model interferent showing the influence of the enzyme layer composition on the selectivity of the sensor. The addition of Gafquat 755N or poly-L-lysine led to higher signals not only for glucose, but also for ascorbic acid. SDS addition already reduced the influence of ascorbic acid, which was almost completely eliminated when Nafion (5%) and PEG (10%) were added. A comparable beneficial effect on the selectivity of the sensors was also observed for the addition of 0.5% graphite. Thus, the enzyme electrodes with PEG, Nafion or graphite as additives in the enzyme layer were applied to glucose determinations in food samples and samples obtained from E. coli cultivations.

Acrylates↗
PubMed2005

A separation-free amperometric immunosensor for vitellogenin based on screen-printed carbon arrays modified with a conductive polymer.

A disposable amperometric immunosensor was studied for the rapid detection of carp (Carassius auratus) Vitellogenin (Vtg). The sensor was fabricated based on screen-printed carbon arrays (SPCAs) containing eight carbon working and an integrated carbon counter electrodes. To construct the sensor, a conducting polymer (poly-terthiophene carboxylic acid) was electropolymerized on the surface of working electrodes and the polymer-coated SPCAs was characterized by SEM. Horseradish peroxidase (HRP) and a monoclonal antibody (anti-Vtg) specific to carp Vtg were covalently attached onto the polymer modified SPCAs. The immobilization of HRP and anti-Vtg onto the polymer-coated SPCAs was examined using cyclic voltammetry and quartz crystal microbalance studies. In order to detect the amount of Vtg, glucose oxidase (GOx)-labelled Vtg bound to the sensor surface under competition with the Vtg analyte was quantified amperometrically using glucose as a substrate. The performance of the eight sensors in arrays was evaluated by obtaining the calibration plots for Vtg. The sensor arrays exhibit a linear range of the Vtg concentration from 0.25 to 7.8 ng/ml and the detection limit was determined to be 0.09 ng/ml. Furthermore, the performance of the immunosensor for the determination of Vtg was evaluated by a standard addition method performed in fish serum samples.

Animals↗
PubMed2005

Enzyme-based impedimetric detection of PCR products using oligonucleotide-modified screen-printed gold electrodes.

This paper describes the optimisation and the analytical performances of an enzyme-based electrochemical genosensor, developed using disposable oligonucleotide-modified screen-printed gold electrodes. The immobilisation of a thiol-tethered probe was qualitatively investigated by means of faradic impedance spectroscopy. Impedance spectra confirmed that the thiol moiety unambiguously drives the immobilisation of the oligonucleotide probe. Furthermore, both probe surface densities and hybridisation efficiencies were quantified through chronocoulometric measurements. Electrochemical transduction of the hybridisation process was also performed by means of faradic impedance spectroscopy, after coupling of a streptavidin-alkaline phosphatase conjugate and bio-catalysed precipitation of an insoluble and insulating product onto the sensing interface. Chronocoulometric results allowed discussion of the magnitude of hybridisation signals in terms of probe surface densities and their corresponding hybridisation efficiency. The genosensor response varied linearly (r2 = 0.9998) with the oligonucleotide target concentration over three orders of magnitude, between 12 pmol/L and 12 nmol/L. The estimated detection limit was 1.2 pmol/L (i.e., 7.2 x 10(6) target molecules in 10 microL of sample solution). The analytical usefulness of the impedimetric genosensor was finally demonstrated analysing amplified samples obtained from the pBI121 plasmid and soy and maize powders containing 1 and 5% of genetically modified product. Sensing of such unmodified amplicons was achieved via sandwich hybridisation with a biotinylated signaling probe. The electrochemical enzyme-amplified assay allowed unambiguous identification of all genetically modified samples, while no significant non-specific signal was detected in the case of all negative controls.

Biosensing Techniques↗
PubMed2005

A multi-biosensor based on immobilized Photosystem II on screen-printed electrodes for the detection of herbicides in river water.

A multi-biosensor for detection of herbicides and pollutants was constructed using various photosynthetic preparations as biosensing elements. The photosynthetic thylakoid from Spinacia oleracea L., Senecio vulgaris and its mutant resistant to atrazine were immobilized with (BSA-GA) on the surface of screen-printed sensors composed of a graphite-working electrode and Ag/AgCl reference electrode deposited on a polymeric substrate. The biosensor was composed of four flow cells with independent illumination of 650 nm to activate electron transfer in Photosystem II. The principle of the detection was based on the fact that herbicides selectively block electron transport activity in a concentration-dependent manner and that the four PSII biomediators show differential recognition activity toward herbicides. Changes of the activity were registered amperometrically as rate of photoreduction of the artificial electron acceptor DQ. The setup resulted in a reusable herbicide multibiosensor with a good stability (half-life of 16.7 h for spinach thylakoids) and limit of detection of about 10(-8) M for herbicides recovered in spring in river.

Adsorption↗
PubMed2005

Immunosensor for Mycobacterium tuberculosis on screen-printed carbon electrodes.

In this work, two methods have been compared to produce enzymatic voltammetric immunosensors for the determination of Mycobacterium tuberculosis antigens (Ag360 and Ag231), using a pre-oxidised screen-printed carbon electrode (SPCE) as a signal transduction element. The enzyme alkaline phosphatase (AP) was used in combination with the substrate 3-indoxyl phosphate (3-IP). In one design, the immune complexes between M. tuberculosis antigens and monoclonal antibodies against M. tuberculosis were formed out of the electrode surface. Then, the immune complexes were captured by biotinylated rabbit anti-M. tuberculosis antibodies, immobilised on the streptavidin modified SPCEs through the streptavidin:biotin reaction. Finally, an alkaline phosphatase (AP) labelled rabbit IgG anti-mouse immunoglobulin G was used as a detector antibody. In the other design, the M. tuberculosis antigens were captured by monoclonal antibodies against M. tuberculosis, which were immobilised on the electrode surface through the reaction with rabbit IgG passively adsorbed on the SPCEs. The biotinylated rabbit anti-M. tuberculosis antibodies were used with an alkaline phosphatase labelled streptavidin as detector antibodies. The best results for M. tuberculosis antigen determination were obtained using the immunosensor on the streptavidin modified SPCEs and the immune complexes between antigen Ag231 and monoclonal antibodies MabF184-3, with a detection limit of 1.0 ng/ml. The immunosensor was also applied to Ag231 spiked proteic matrices.

Alkaline Phosphatase↗
PubMed2004

Glucose biosensor based on multi-wall carbon nanotubes and screen printed carbon electrodes.

This paper describes a disposable electrochemical biosensor for glucose monitoring. The sensor was based on multi-wall carbon nanotubes (MWCNTs) immobilized with glucose oxidase and upon screen printed carbon electrode. The effect of MWCNTs on the response of amperometric glucose oxidase electrode for glucose was examined. Results obtained, of interest for basic and applied biochemistry, represent a first step in construction of a MWCNT-enzyme electrode biosensor with potentialities for a successful application in the biosensor area.

Adsorption↗
PubMed2005

One-step screen-printed electrode modified in its bulk with HRP based on direct electron transfer for hydrogen peroxide detection in flow injection mode.

The preparation and performances of screen-printed carbon electrodes modified in their bulk with HRP (HRP-SPCE) is reported. The resulting modified HRP-SPCE was prepared in a one-step procedure, and then was optimised as an amperometric biosensor operating at [0-100] mV versus Ag/AgCl in flow injection mode for hydrogen peroxide. The amperometric response was due to direct electron transfer (DET) between HRP and SPCE surface. Factors such as chemical modification of the enzyme or the nature and rate of the binder were investigated regards to their influence on the sensitivity, linear range and operational stability. The best performing HRP-SPCE in terms of sensitivity and operational stability was obtained when graphite powder was modified with HRP previously oxidised by periodate ion (IO(4)(-)).

Adsorption↗
PubMed2006

Comparison of amperometric biosensors fabricated by palladium sputtering, palladium electrodeposition and Nafion/carbon nanotube casting on screen-printed carbon electrodes.

Different strategies, including palladium electrodeposition (Pd(CV)), Pd sputtering (Pd(S)) and Nafion-solubilized carbon nanotube casting (Nafion/CNT), were used to modify screen-printed carbon electrodes (SPCEs) for the fabrication of amperometric enzyme biosensors. The electrochemical properties of the bare and modified SPCEs and the optimal conditions for surface modification were determined. The electrochemical response of the bare SPCE to H(2)O(2) under the potential of 0.3 V could be improved about 100-fold by Pd modification by electrodeposition or sputtering. By contrast, the electrochemical response of the bare SPCE was enhanced by only about 11-fold by Nafion/CNT casting. Moreover, the Pd(CV)-SPCEs exhibited better reproducibility of electrochemical response (a relative standard deviation (R.S.D.)<6.0%) than freshly prepared Pd(S)-SPCEs (R.S.D.>10%). The glucose biosensor fabricated from Pd-modified electrodes could be stored for up to 108 days without loosing significant activity. The Pd(CV)-SPCE also showed very reliable signal characteristics upon 50 consecutively repeated measurements of ascorbic acid. The electrocatalytic detection of the Pd-SPCE was combined with additional advantages of resistance to surface fouling and hence good stability. In conclusion, this study demonstrated that deposition of Pd thin film on SPCEs by electrodeposition or sputtering provided superior enhancement of electrochemical properties compared to Nafion/CNT-SPCEs. Despite their high electrochemical response, Pd(S)-SPCEs required an activation process to improve stability and Pd(CV)-SPCEs suffered from poor between electrode reproducibility.

Biosensing Techniques↗
PubMed2006

Direct electrochemical addressing of immunoglobulins: immuno-chip on screen-printed microarray.

An original immobilisation technology is presented for the development of chemiluminescent protein biochips, suitable for measurement in complex matrices. The immobilisation strategy involved is based on diazotated aniline derivatives, which could be electro-addressed, thus creating a covalent linkage with a conducting material surface. The present electrochemical system is a cost effective and mass-produced carbon paste screen-printed (SP) microarray composed of eight 0.2 mm2 working electrodes, one carbon pseudo-reference electrode and one auxiliary electrode. Rabbit immunoglobulins (IgG) were chemically modified with an aniline derivative (4-carboxymethylaniline) in order to be easily electro-grafted to the SP microarray surface. The possibility of successively electro-address the eight sensing layers of a particular array, with a good reproducibility (more than 80%) and without loss of reactivity was demonstrated. Moreover, these immobilised proteins were subsequently used as a capture agent for the determination of rheumatoid factor (RF) in human sera. The absence of non-specific signal or interference problem enabled the detection of RF values in complex samples in the 5.3-485 IU/ml range with a good correlation with the standard Auraflex ELISA test method.

Animals↗
PubMed2006

Anodic cell-protein deposition on inverse inkjet printed micro structured gold surfaces.

The transformation of fibrinogen into fibrin is biologically activated in a complex multi-step process known as the coagulation cascade. This transformation can also be triggered by anodic surfaces. It has been suggested that this mechanism is a result of an electron transfer from the anode to the fibrinogen molecule resulting in the formation of fibrin. In this study we used this pathway to simultaneously deposit vital cells (fibroblasts and keratinocytes) and fibrin on micro structured gold electrodes. The electrodes were produced using a novel inverse inkjet-printing technology in combination with subsequent gold-sputtering, resulting in minimal structure-sizes of 35 microm (+/-6 microm). Cell deposition and fibrin-coagulation were found to occur on the anode only, following exactly the micro structured electrode surface. Successful deposition was limited by the minimal voltage (0.8 V) needed for the formation of fibrin and the maximum voltage (1.85 V) resulting in the deterioration of the Au-electrodes due to electrolysis and possible damaging of the deposited cells due to the formation of molecular chlorine. Furthermore, it was demonstrated that this technique is suitable to co-cultivate different cell types in a layered fashion. Subsequent to the electrically mediated anodic cell-protein deposition, cells were cultivated for up to 4 days and then characterized by vital fluorescence staining, methyl violet-staining and scanning electron microscopy. Cell-vitality was found to be dependent on the experimental setup; in this study non-vital cells were only observed, when sequentially depositing two different cell types. Finally, the coagulation mechanism was studied using HPLC, SDS-gel-chromatography and ATR/FTIR.

Animals↗
PubMed2006

Screen-printed biosensors for glucose determination in grape juice.

An approach to the glucose determination by amperometric biosensing in wine industry applications is presented. Integrated screen-printed biosensors based on horseradish peroxidase (HRP) and glucose oxidase (GOx) have been developed. The experimental design methodology has been used to find the optimum conditions of the experimental variables, in such a way that a chronoamperometric response specific for glucose was recorded. Under these conditions, repeatability and reproducibility of the modified electrodes have been analyzed. The detection limit for glucose has been calculated taking into account the probability of false positive (alpha) and negative (beta), reaching a medium value of 4.37+/-0.21 micromol dm-3 (alpha=beta=0.05, and a replicate n=4). The biosensor was applied to the determination of glucose in white wine samples.

Beverages↗