Surface modification using a novel type I hydrophobin HGFI

Surface wettability conversion with hydrophobins is important for its applications in biodevices. In this work, the application of a type I hydrophobin HGFI in surface wettability conversion on mica, glass, and poly(dimethylsiloxane) (PDMS) was investigated. X-ray photoelectron spectroscopy (XPS) and water-contact-angle (WCA) measurements indicated that HGFI modification could efficiently change the surface wettability. Data also showed that self-assembled HGFI had better stability than type II hydrophobin HFBI. Protein patterning and the following immunoassay illustrated that surface modification with HGFI should be a feasible strategy for biosensor device fabrication. Figure A hydrophobin HGFI has been applied into surface wettability conversion for protein immobilization Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Incubation type Si-based planar ion channel biosensor

A new planar-type ion channel biosensor with the function of cell culture has been fabricated using silicon on an insulator substrate as the sensor chip material. Coating of the sensor chip with fibronectin was essentially important for cell incubation on the chip. Although the seal resistance was quite low (∼7 MΩ) compared with the pipette patch-clamp gigaohm seal, the whole-cell channel current of the transient receptor potential vanilloid type 1 (TRPV1) channel expressing HEK293 cells was successfully observed, with a good signal-to-noise ratio, using capsaicin as a ligand molecule. Figure A new planer type ion channel biosensor with function of cell culture is fabricated using the silicon on insulator substrate as the sensor chip material. The coating of the sensor chip by the fibronectin was essentially important for the cell incubation on the chip. Whole cell channel current of TRPV1 channel was successfully observed using capsaicin as a ligand molecule with good signal to noise.     

Microcontact printing of Concanavalin A and its effect on mammalian cell morphology

In this study a major lectin called Concanavalin A (ConA) has been micropatterned on a glass substrate by microcontact printing and the patterns have been characterized with fluorescent and atomic force microscope for their uniformity. Interaction of the patterns with mammalian cells has been investigated by culturing L929 mouse fibroblast cells on the ConA printed glass surface. Cell culture results obtained from the microcontact printed patterns have also been compared and benchmarked with another patterning technique named micromolding in capillaries (MIMIC). It has been revealed that in spite of molecular level heterogeneity and agglomeration of protein molecules in microcontact printed form, they can still interact with cell surface glycoproteins, impede the mobility of membrane receptor which results in altered morphology of the fibroblast cells.     

High-throughput evaluation of quiescent hematopoietic progenitor cells using a micro-multiwell plate

Conventional assays for hematopoietic progenitor cells (HPCs) require long-term culturing, a labor-intensive procedure, and technique proficiency. We aimed to develop a high-throughput method to determine frequency of quiescent primitive HPCs by a combination of the micro-multiwell plate and 5-fluorouracil (5-FU) treatment. The micro-multiwell plate was made of a silicone sheet with a 6 × 6 array of 1-mm diameter holes and a glass substrate. To enrich primitive HPCs in a CD34 population, CD34 cells and stromal cells were applied to micro-multiwells and cultured in the presence of 5-FU for 2 days. The quiescent primitive HPCs that survived after 5-FU treatment were then expanded with cytokines in the absence of 5-FU for a further 10 days. After culturing, cells were immunostained and the number of primitive HPCs in inoculated CD34 cells was estimated from fluorescent intensity for each well under a stereoscopic fluorescent microscope. The frequencies of primitive HPCs correlated well with frequencies of cobblestone area-forming cells for two CD34 cell lots. Our method allows high-throughput screening for primitive HPCs in CD34 cells. Figure Representative image of a micro-multiwell plate fordetecting primitive hematopoietic stem cells     

The use of electrochemical impedance spectroscopy for biosensing

This review introduces the basic concepts and terms associated with impedance and techniques of measuring impedance. The focus of this review is on the application of this transduction method for sensing purposes. Examples of its use in combination with enzymes, antibodies, DNA and with cells will be described. Important fields of application include immune and nucleic acid analysis. Special attention is devoted to the various electrode design and amplification schemes developed for sensitivity enhancement. Electrolyte insulator semiconductor (EIS) structures will be treated separately. Figure An alternating current which is forced to pass an interface is sensitive to surface changes and will detect impedance changes due to biomolecule immobilisation or formation of a recognition complex. This can be used for the construction of biosensor electrodes     

Biosensor incorporating cell barrier architectures for detecting <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> alpha toxin

Alpha toxin is a common virulent factor of Staphylococcus aureus and is believed to play crucial roles in pathogenicity induced by S. aureus. Alpha toxin is also known to induce permeability to endothelial cell monolayers in vitro due to the formation of interendothelial gaps. The present study is directed towards measuring alpha toxin using a whole-cell-based biosensor. The biosensor, consisting of a confluent monolayer of human umbilical vein endothelial cells (HUVECs) on a potassium ion-selective electrode, takes advantage of cell permeability dysfunction to detect the presence of small quantities of alpha toxin. When a confluent monolayer of cells was formed on the membrane surface, the response of the electrode toward the marker ion, potassium, was inhibited. Upon exposing this sensor to varying concentrations of alpha toxin for 20 min, an increase in sensor response to potassium was observed. The response thus obtained was indirectly related to the concentration of alpha toxin. The detection limit of this sensor for alpha toxin was found to be 0.1 ng/ml. Cell monolayers were stained with silver nitrate to quantify the formation of intercellular gaps as well as to study the effect of this toxin on HUVECs morphology. A strong positive correlation was observed between the response obtained from the biosensor and the area of the intercellular gaps. Silver staining also revealed the tendency of cells to round up upon being exposed to alpha toxin. Figure Measuring alpha toxin using a whole-cell-based biosensor     

Patterning of neural stem cells on poly(lactic-co-glycolic acid) film modified by hydrophobin

Patterning of neural stem cells (NSCs) is of great importance for its potential applications in the therapy of nerve injuries. Due to the critical requirements and the great difficulty in NSCs cultivation, developing new methods for NSCs patterning is very challenging and has progressed slowly in recent years. In this study, we reported a new method for patterning NSCs on a hydrophobin II (HFBI) modified poly(lactic-co-glycolic acid) (PLGA) film by using microcontact printing (μCP) technique. HFBI modification converted the PLGA surface from hydrophobic to hydrophilic, which should facilitate the absorption of serum on it. Serum was transferred onto the modified PLGA film by microcontact printing (μCP) to promote NSCs adhesion on the PLGA surface. Since the serum-coated PLGA surface promoted NSCs adhesion and the serum-free PLGA surface inhibited NSCs adhesion, micro-patterns of NSCs were obtained by directly culturing NSCs on the PLGA surface patterned with serum. This method allows the precise control of NSCs adhesion on the PLGA film without using the conventional cell-repellent species, which is anticipated to make great contribution in the fields of therapy of nerve injuries.     

Enzyme-functionalized mesoporous silica for bioanalytical applications

The unique properties of mesoporous silica materials (MPs) have attracted substantial interest for use as enzyme-immobilization matrices. These features include high surface area, chemical, thermal, and mechanical stability, highly uniform pore distribution and tunable pore size, high adsorption capacity, and an ordered porous network for free diffusion of substrates and reaction products. Research demonstrated that enzymes encapsulated or entrapped in MPs retain their biocatalytic activity and are more stable than enzymes in solution. This review discusses recent advances in the study and use of mesoporous silica for enzyme immobilization and application in biosensor technology. Different types of MPs, their morphological and structural characteristics, and strategies used for their functionalization with enzymes are discussed. Finally, prospective and potential benefits of these materials for bioanalytical applications and biosensor technology are also presented. Figure Enzyme-functionalized mesoporous silica fibers and their integration in a biosensor design. The immobilization process takes place essentially in the silica micropores.     

Comparison of protein immobilisation methods onto oxidised and native carbon nanofibres for optimum biosensor development

The properties of native and oxidised graphene layered carbon nanofibres are compared, and their utilisation in enzyme biosensor systems using different immobilisation methods are evaluated. The efficient oxidation of carbon nanofibres with concentrated H₂SO₄/HNO₃ is confirmed by Raman spectroscopy while the introduction of carboxylic acid groups on the surface of the fibres by titration studies. The oxidised fibres show enhanced oxidation efficiency to hydrogen peroxide, while at the same time they exhibit a more efficient and selective interaction with enzymes. The analytical characteristics of biosensor systems based on the adsorption or covalent immobilisation of the enzyme glucose oxidase on carbon nanofibres are compared. The study reveals that carbon nanofibres are excellent substrates for enzyme immobilisation allowing the development of highly stable biosensor systems. Figure Immobilization of proteins on carbon nanofibres     

Electrodeposition polymers as immobilization matrices in amperometric biosensors: improved polymer synthesis and biosensor fabrication

Electrodeposition polymers can be precipitated on electrode surfaces upon electrochemical-induced modulations of the pH value in the diffusion zone in front of the electrode. The formed polymer films can be used as immobilization matrices in amperometric biosensors. In order to rationally control the thus obtained biosensor properties, it is indispensable to develop strategies for the reproducible synthesis of electrodeposition polymers as well as methods for the non-manual and reproducible sensor fabrication. Based on instrumental developments such as a specifically designed parallel synthesizer with improved stirring and temperature control, an automatic pipetting robot for the preparation of the monomer mixtures and controlled removal of polymerization inhibitors, the reproducible synthesis of libraries of electrodeposition polymers was achieved. Moreover, the polymerization process could be monitored using in-line thermocouples, and it could be shown that the chosen strategies led to reproducible polymerization reactions. By adaptation of an electrochemical robotic system integrating a Au microtiter plate and automatic electrode cleaning by means of a polishing wheel reproducible biosensor fabrication using glucose oxidase as a model enzyme could be demonstrated. These results open the route for the rational development of biosensors and control of the sensor properties by choosing specifically designed electrodeposition polymers.      

Development of an efficient amine-functionalized glass platform by additional silanization treatment with alkylsilane

Aminosilane-treated molecular layers on glass surfaces are frequently used as functional platforms for biosensor preparation. All the amino groups present on the surface are not available in reactive forms, because surface amino groups interact with remaining unreacted surface silanol groups. Such nonspecific interactions might reduce the efficiency of chemical immobilization of biomolecules such as DNA, enzymes, antibodies, etc., in biosensor fabrication. To improve immobilization efficiency we have used additional surface silanization with alkylsilane (capping) to convert the remaining silanol groups into Si–O–Si linkages, thereby liberating the amino groups from nonspecific interaction with the silanol groups. We prepared different types of capped amine surface and evaluated the effect of capping on immobilization efficiency by investigating the fluorescence intensity of Cy3-NHS (N-hydroxysuccinimide) dye that reacted with amino groups. The results indicate that most of the capped amine surfaces resulted in enhanced efficiency of immobilization of Cy3-NHS compared with the untreated control amine surface. We found a trend that trialkoxysilanes had greater capping effects on immobilization efficiency than monoalkoxysilanes. It was also found that the aliphatic chain of alkylsilane, which does not participate in the capping of the silanol, had an important function in enhancing immobilization efficiency. These results would be useful for preparation of an amine-modified surface platform, with enhanced immobilization efficiency, which is essential for developing many kinds of biosensors on a silica matrix. Enhancement of amine funtionality by capping with alkylsilane     

Label-free characterization of cell adhesion using reflectometric interference spectroscopy (RIfS)

Reflectometric interference spectroscopy (RIfS) is a label-free, time-resolved technique for detecting interactions of molecules immobilized on a surface with ligands in solution. Here we show that RIfS also permits the detection of the adhesion of tissue culture cells to a functionalized surface in a flow system. Interactions of T cells with other leukocytes or epithelial cells of blood vessels are crucial steps in the regulating immune response and inflammatory reactions. Jurkat T cell leukemia cells rapidly attached to a transducer functionalized with a monoclonal antibody directed against the T cell receptor (TCR)/CD3 complex, followed by activation-dependent cell spreading. RIfS curves were obtained for the Jurkat derivative JCaM 1.6 (which lacks the key signaling protein Lck), cells preincubated with cytochalasin D (an inhibitor of actin polymerization), and for surfaces functionalized with an antibody directed against the coreceptor CD28. These curves differed with respect to the maximum signal and the initial slope of the increase in optical thickness. The testing of chemical inhibitors, cell surface molecules and gene products relevant to a key event in T cell immunity illustrates the potential of label-free techniques for the analysis of activation-dependent cell-surface contacts. The first two authors contributed equally to this paper     

Phase-changing sacrificial layers in microfluidic devices: adding another dimension to separations

The use of polymers in microchip fabrication affords new opportunities for the development of powerful, miniaturized separation techniques. One method in particular, the use of phase-changing sacrificial layers, allows for simplified designs and many additional features to the now standard fabrication of microchips. With the possibility of adding a third dimension to the design of separation devices, various means of enhancing analysis now become possible. The application of phase-changing sacrificial layers in microchip analysis systems is discussed, both in terms of current uses and future possibilities. Figure Phase-changing sacrificial materials enable multilayer microfluidic device layouts     

Recent advances in capillary electrophoretic analysis of individual cells

Because variability exists within populations of cells, single-cell analysis has become increasingly important for probing complex cellular environments. Capillary electrophoresis (CE) is an excellent technique for identifying and quantifying the contents of single cells owing to its small volume requirements and fast, efficient separations with highly sensitive detection. Recent progress in both whole-cell and subcellular sampling has allowed researchers to study cellular function in the areas of neuroscience, oncology, enzymology, immunology, and gene expression.      

Biosensing applications of clay-modified electrodes: a review

Two-dimensional layered inorganic solids, such as cationic clays and layered double hydroxides (LDHs), also defined as anionic clays, have open structures which are favourable for interactions with enzymes and which intercalate redox mediators. This review aims to show the interest in clays and LDHs as suitable host matrices likely to immobilize enzymes onto electrode surfaces for biosensing applications. It is meant to provide an overview of the various types of electrochemical biosensors that have been developed with these 2D layered materials, along with significant advances over the last several years. The different biosensor configurations and their specific transduction procedures are discussed.      

Amperometric biosensor for the determination of creatine

An amperometric biosensor for the determination of creatine was developed. The carbon rod electrode surface was coated with sarcosine oxidase (SOX) and creatine amidinohydrolase by cross-linking under glutaraldehyde vapour. The SOX from Arthrobacter sp. 1–1 N was purified and previously used for creation of a creatine biosensor. The natural SOX electron acceptor, oxygen, was replaced by an redox mediating system, which allowed amperometric detection of an analytical signal at +400-mV potential. The response time of the biosensor was less than 1 min. The biosensor showed a linear dependence of the signal vs. creatine concentration at physiological creatine concentration levels. The optimal pH in 0.1 M tris(hydroxymethyl)aminomethane (Tris)–HCl buffer was found to be at pH 8.0. The half-life of the biosensor was 8 days in 0.1 M Tris–HCl buffer (pH 8.0) at 20 °C. Principal scheme of consecutively followed catalytic reactions used to design a biosensor for the determination of creatine     

TOF-S-SIMS molecular depth profiling of organic bilayers using mechanical wear test methodology

Recent publications on static secondary ion mass spectrometry (S-SIMS) focus on molecular depth profiling by using polyatomic or ultra-low energy monoatomic projectiles. Since their applicability depends on the relationship between the ion yield and the depth, which is hard to obtain without extensive studies, a combination of a wear test method with S-SIMS surface analysis was performed in the current study. Using this non-sputtering procedure, the relation between the signal intensity and the local concentration remains in principle the same as that at the surface (which is easy to determine). Mechanical erosion was successfully applied to expose sub-surface material from organic multilayers. Through surface analysis with S-SIMS on the gradually exposed deeper planes, molecular depth profiles could be obtained. The study was conducted on a model system relevant to offset printing, consisting of two polymer layers, containing dyes and a surfactant, cast on an Al substrate. Figure Concept of mechanical erosion followed by S-SIMS surface analysis to obtain molecular depth profiles     

Detecting proteins complex formation using steady-state diffusion in a nanochannel

In this work, we present theoretical and experimental studies of nanofluidic channels as a potential biosensor for measuring rapid protein complex formation. Using the specific properties offered by nanofluidics, such as the decrease of effective diffusion of biomolecules in confined spaces, we are able to monitor the binding affinity of two proteins. We propose a theoretical model describing the concentration profile of proteins in a nanoslit and show that a complex composed by two bound biomolecules induces a wider diffusion profile than a single protein when driven through a nanochannel. To validate this model experimentally, we measured the increase of the fluorescent diffusion profile when specific biotinylated dextran was added to fluorescent streptavidin. We report here a direct and relatively simple technique to measure the affinity between proteins. Figure We present theoretical and experimental studies of nanofluidic channels as potential biosensors for rapidly measuring protein complex formation. Our system is based on steady-state diffusion effects which are observed inside a nanoslit.     

Interpretation of laser desorption mass spectra of unexpected inorganic species found in a cosmetic sample of forensic interest: fingernail polish

Monitoring of cell cultures in microbioreactors is a crucial task in cell bioassays and toxicological tests. In this work a novel tool based on a miniaturized sensor array fabricated using low-temperature cofired ceramics (LTCC) technology is presented. The developed device is applied to the monitoring of cell-culture media change, detection of the growth of various species, and in toxicological studies performed with the use of cells. Noninvasive monitoring performed with the LTCC microelectrode array can be applied for future cell-engineering purposes. Figure Microelectrode array for monitoring of cell cultures     

Nanostructured electrochemical DNA biosensors for detection of the effect of berberine on DNA from cancer cells

Multi walled carbon nanotubes (MWNT) in dimethylformamide (DMF) or aqueous sodium dodecyl sulfate (SDS) solution, colloidal gold nanoparticles (GNP) in phosphate buffer solution (PBS), and a GNP–MWNT mixture in aqueous SDS solution have been investigated for chemical modification of a screen-printed carbon electrode used as the signal transducer of a dsDNA-based biosensor. Differential pulse voltammetry of the DNA redox marker and the guanine moiety anodic oxidation and cyclic voltammetry with K₃[Fe(CN)₆] as indicator revealed substantial enhancement of the response of the biosensor, particularly when MWNT in SDS solution was used. The biosensor was used in testing of berberine, an isoquinoline plant alkaloid with significant antimicrobial and anticancer activity. Berberine had a very strong, concentration-dependent, effect on the structural stability of DNA from the human cancer cells (U937 cells) whereas non-cancer cells were changed only when berberine concentrations were relatively high 75 and 50 μg mL⁻¹. Figure Schematic illustration of preparation of the nanostructured films: (a) layer-to-layer coverage (DNA/nanomaterial/SPE); (b) mixed coverage (DNA-nanomaterial/SPE)