A label-free potentiometric sensor principle for the detection of antibody-antigen interactions.

We report here on a new potentiometric biosensing principle for the detection of antibody-antigen interactions at the sensing membrane surface without the need to add a label or a reporter ion to the sample solution. This is accomplished by establishing a steady-state outward flux of a marker ion from the membrane into the contacting solution. The immunobinding event at the sensing surface retards the marker ion, which results in its accumulation at the membrane surface and hence in a potential response. The ion-selective membranes were surface-modified with an antibody against respiratory syncytial virus using click chemistry between biotin molecules functionalized with a triple bond and an azide group on the modified poly (vinyl chloride) group of the membrane. The bioassay sensor was then built up with streptavidin and subsequent biotinylated antibody. A quaternary ammonium ion served as the marker ion. The observed potential was found to be modulated by the presence of respiratory syncytial virus bound on the membrane surface. The sensing architecture was confirmed with quartz crystal microbalance studies, and stir effects confirmed the kinetic nature of the marker release from the membrane. The sensitivity of the model sensor was compared to that of a commercially available point-of-care test, with promising results.

Novel antibiotics targeting respiratory ATP synthesis in Gram-positive pathogenic bacteria.

Emergence of drug-resistant bacteria represents a high, unmet medical need, and discovery of new antibacterials acting on new bacterial targets is strongly needed. ATP synthase has been validated as an antibacterial target in Mycobacterium tuberculosis, where its activity can be specifically blocked by the diarylquinoline TMC207. However, potency of TMC207 is restricted to mycobacteria with little or no effect on the growth of other Gram-positive or Gram-negative bacteria. Here, we identify diarylquinolines with activity against key Gram-positive pathogens, significantly extending the antibacterial spectrum of the diarylquinoline class of drugs. These compounds inhibited growth of Staphylococcus aureus in planktonic state as well as in metabolically resting bacteria grown in a biofilm culture. Furthermore, time-kill experiments showed that the selected hits are rapidly bactericidal. Drug-resistant mutations were mapped to the ATP synthase enzyme, and biochemical analysis as well as drug-target interaction studies reveal ATP synthase as a target for these compounds. Moreover, knockdown of the ATP synthase expression strongly suppressed growth of S. aureus, revealing a crucial role of this target in bacterial growth and metabolism. Our data represent a proof of principle for using the diarylquinoline class of antibacterials in key Gram-positive pathogens. Our results suggest that broadening the antibacterial spectrum for this chemical class is possible without drifting off from the target. Development of the diarylquinolines class may represent a promising strategy for combating Gram-positive pathogens.

Impact of Stigma on People Living with Chronic Hepatitis B.

BACKGROUND: People with chronic infectious diseases such as hepatitis B can face stigma, which can influence everyday life as well as willingness to engage with medical professionals or disclose disease status. A systematic literature review was performed to characterize the level and type of stigma experienced by people infected with hepatitis B virus (HBV) as well as to identify instruments used to measure it. METHODS: A literature review was performed using the PubMed, Embase and Cochrane Library databases to identify studies describing HBV-related stigma. For inclusion, articles were required to be published in full-text form, in English and report quantitative or qualitative data on HBV-related stigma that could be extracted. RESULTS: A total of 23 (17 quantitative and 6 qualitative) articles examined HBV-related stigma. The scope of the review was global but nearly all identified studies were conducted in countries in the WHO Southeast Asia or Western Pacific regions or within immigrant communities in North America. Several quantitative studies utilized tools specifically designed to assess aspects of stigma. Qualitative studies were primarily conducted via patient interviews. Internalized and social stigma were common among people living with chronic HBV . Some people also perceived structural/institutional stigma, with up to 20% believing that they may be denied healthcare and up to 30% stating they may experience workplace discrimination due to HBV. CONCLUSION: HBV-related stigma is common, particularly in some countries in Southeast Asia and the Western Pacific region and among Asian immigrant communities, but is poorly characterized in non-Asian populations. Initiatives are needed to document and combat stigma (particularly in settings/jurisdictions where it is poorly described) as well as its clinical and socioeconomic consequences.

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.

Antibody Fragments as Probe in Biosensor Development.

Today's proteomic analyses are generating increasing numbers of biomarkers, making it essential to possess highly specific probes able to recognize those targets. Antibodies are considered to be the first choice as molecular recognition units due to their target specificity and affinity, which make them excellent probes in biosensor development. However several problems such as difficult directional immobilization, unstable behavior, loss of specificity and steric hindrance, may arise from using these large molecules. Luckily, protein engineering techniques offer designed antibody formats suitable for biomarker analysis. Minimization strategies of antibodies into Fab fragments, scFv or even single-domain antibody fragments like VH, VL or VHHs are reviewed. Not only the size of the probe but also other issues like choice of immobilization tag, type of solid support and probe stability are of critical importance in assay development for biosensing. In this respect, multiple approaches to specifically orient and couple antibody fragments in a generic one-step procedure directly on a biosensor substrate are discussed.

Surface modification of gamma-Fe2O3@SiO2 magnetic nanoparticles for the controlled interaction with biomolecules.

Modifying the surface of magnetic nanoparticles (MNPs) to allow for controlled interaction with biomolecules enables their implementation in biomedical applications such as contrast agents for magnetic resonance imaging, labels in magnetic biosensing or media for magnetically assisted bioseparation. In this paper, self-assembly of trialkoxysilanes is used to chemically functionalize the surface of gamma-Fe2O3@SiO2 core-shell particles. First, the silane deposition procedure was optimized using infrared analysis in order to obtain maximum packing density of the silanes on the particles. The surface coverage was determined to be approximately 8 x 10(14) molecules/cm2. It was shown that the magnetic, crystalline, and morphological properties of the MNPs were not altered by deposition of a thin silane coating. The optimized procedure was transferred for the deposition of aldehyde and poly(ethylene glycol) (PEG) presenting silanes. The presence of both silanes on the particle surface was confirmed using XPS and FTIR. The interaction of proteins with silane-modified MNPs was monitored using a Bradford protein assay. Our results demonstrate that, by introducing aldehyde functions, the MNPs are capable of covalently binding human IgG while retaining their specific binding capacity. Maximum surface coverage occurs at 46 microg antibodies per mg particle, which corresponds to 35 antibodies bound to an average sized MNP (54 nm in diameter). The human IgG functionalized MNPs exhibit a high degree of specificity (approximately 90%) and retained a binding capacity of 32%. Using the same approach, streptavidin was coupled onto the MNPs and the biotin binding capacity was determined using biotinylated fluorescein. At maximum surface coverage, a biotin binding capacity of 1500 pmol/mg was obtained, corresponding to a streptavidin activity of 76%. On the other hand, by introducing PEG functions the non-specific adsorption of serum proteins could be significantly suppressed down to approximately 3 microg/mg. We conclude that self-assembly of silane films creates a generic platform for the controlled interactions of MNPs with biomolecules.

Comparison of random and oriented immobilisation of antibody fragments on mixed self-assembled monolayers.

The sensitivity of immunosensors is strongly dependent on the amount of immobilised antibodies and their remaining antigen binding properties. The use of smaller and well-oriented antibody fragments as bioreceptor molecules influences the final immunosensor signal. The aim of this study was to compare the immunosensor responses of different immobilised antibody fragments, such as F(ab')2 and Fab', with their parental IgG. In addition, we evaluated the oriented versus the random covalent immobilisation method of the Fab' fragments. First, an optimisation of cleavage protocol to generate these F(ab')2 and Fab' fragments was performed. Subsequently, we pursued a study with limited denaturation effects during immobilisation of the bioreceptor molecules and with reduced steric hindrance during antigen binding using mixed self-assembled monolayers (SAM) of thiols as the chemical linking layer. The Surface Plasmon Resonance technique was used to evaluate the degree of immobilisation of the antibody fragments and their parental IgGs on the mixed SAMs and the binding signals of their specific antigens. In this study, we demonstrate that for a particular antibody/antigen system (anti-hIgG/hIgG), the optimised fragmentation protocol in combination with an oriented immobilisation of Fab' fragments on mixed SAMs leads to a >2-fold increase of the antigen binding signals compared to randomly covalent immobilised full-length antibodies.

Reduced nonspecific adsorption on covalently immobilized protein surfaces using poly(ethylene oxide) containing blocking agents.

In a number of applications, e.g. DNA/protein micro-array technology, enzyme-linked immunosorbent assay (ELISA) technology or surface plasmon resonance (SPR) technology, the covalent coupling of proteins to surfaces is required. Following the covalent coupling of proteins, the remaining reactive groups should be blocked in order to avoid covalent binding of the analyte to the reactive surface. To this end, preferably blocking agents containing groups that avoid nonspecific adsorption should be used. These blocking agents are typically ethanolamine and cysteine for protein coupling via amino groups and thiol groups, respectively. This report presents novel blocking agents containing poly(ethylene oxide) (PEO) groups. These blocking agents show enhanced qualities to avoid nonspecific adsorption and can therefore have advantages in versatile protein-surface technologies.

Stability of mixed PEO-thiol SAMs for biosensing applications.

The secret of a successful affinity biosensor partially hides in the chemical interface layer between the transducer system and the biological receptor molecules. Over the past decade, several methodologies for the construction of such interface layers have been developed on the basis of the deposition of self-assembled monolayers (SAMs) of alkanethiols on gold. Moreover, mixed SAMs of polyethylene oxide (PEO) containing thiols have been applied for the immobilization of biological receptors. Despite the intense research in the field of thiol SAMs, relatively little is known about their biosensing properties in correlation with their long-term stability. Especially the impact of the storage conditions on their biosensing characteristics has not been reported before to our knowledge. To address these issues, we prepared mixed PEO SAMs and tested their stability and biosensing performance in several storage conditions, i.e., air, N2, ethanol, phosphate buffer, and H2O. The quality of the SAMs was monitored as a function of time using various characterization techniques such as cyclic voltammetry, contact angle, grazing angle Fourier-transform infrared spectroscopy, and X-ray photoelectron spectroscopy. In addition, the impact of the different storage conditions on the biosensor properties was investigated using surface plasmon resonance. Via the latter technique, the receptor immobilization, the analyte recognition, and the nonspecific binding were extensively studied using the prostate specific antigen as a model system. Our experiments showed that very small structural differences in the SAM can have a great impact in their final biosensing properties. In addition it was shown that the mixed SAMs stored in air or N2 are very stable and retain their biosensor properties for at least 30 days, while ethanol appeared to be the worst storage medium due to partial oxidation of the thiol headgroup. In conclusion, care must be taken to avoid SAM degradation during storage to retain typical SAM characteristics, which is very important for their general use in many proposed applications.

Formation of dense self-assembled monolayers of (n-decyl)trichlorosilanes on Ta/Ta2O5.

Tantalum pentoxide (Ta2O5) is a promising material for the realization of biological interfaces because of its high dielectric constant, its high chemical stability, and its excellent passivating properties. Nevertheless, the deposition of highly organized silane SAMs to realize well-defined and tailored Ta2O5-based (bio)interfaces, has not been studied in great detail as of yet. In this work, we have investigated the formation of a highly ordered, dense monolayer of trichlorosilanes on Ta2O5 surfaces. Specifically, two different cleaning procedures for Ta2O5 were compared and (n-decyl)trichlorosilane (DTS) was used to study the effect of both cleaning methods on the silanization of Ta2O5. Both types of cleaning allowed the formation of complete and crystalline DTS monolayers on Ta2O5, in contrast with the incomplete, disordered silane layer assembled on uncleaned Ta2O5. The deposited self-assembled monolayers were studied by means of contact angle goniometry, Brewster angle FTIR, X-ray photoelectron spectroscopy, cyclic voltammetry, and ellipsometry. Infrared analysis exhibited a highly ordered DTS silane film on Ta2O5 and indicated a larger tilt angle of the alkyl chains on this substrate by comparison to DTS on SiO2. Furthermore, with use of ellipsometry and XPS, the silane film thickness on Ta2O5 was determined to be substantially smaller than that reported in the literature for DTS on SiO2, supporting the observations of an increased tilt angle (approximately 45 degrees ) on Ta2O5 than on SiO2 (approximately 10 degrees ). By means of cyclic voltammetry, the formation of a dense, essentially pinhole-free, silane film was observed on the cleaned samples. In conclusion, the fully characterized and optimized procedure for the silanization of Ta2O5 surfaces with trichlorosilanes will allow the formation of well-defined, reproducible, and controllable chemical interfaces on Ta2O5.

Surface plasmon resonance-based interaction studies reveal competition of Streptomyces lividans type I signal peptidases for binding preproteins.

Type I signal peptidases (SPases) are responsible for the cleavage of signal peptides from secretory proteins. Streptomyces lividans contains four different SPases, denoted SipW, SipX, SipY and SipZ, having at least some differences in their substrate specificity. In this report in vitro preprotein binding/processing and protein secretion in single SPase mutants was determined to gain more insight into the substrate specificity of the different SPases and the underlying molecular basis. Results indicated that preproteins do not preferentially bind to a particular SPase, suggesting SPase competition for binding preproteins. This observation, together with the fact that each SPase could process each preprotein tested with a similar efficiency in an in vitro assay, suggested that there is no real specificity in substrate binding and processing, and that they are all actively involved in preprotein processing in vivo. Although this seems to be the case for some proteins tested, high-level secretion of others was clearly dependent on only one particular SPase demonstrating clear differences in substrate preference at the in vivo processing level. Hence, these results strongly suggest that there are additional factors other than the cleavage requirements of the enzymes that strongly affect the substrate preference of SPases in vivo.

Realization and characterization of porous gold for increased protein coverage on acoustic sensors.

Immunosensors show great potential for the direct detection of biological molecules. The sensitivity of these affinity-based biosensors is dictated by the amount of receptor molecules immobilized on the sensor surface. An enlargement of the sensor area would allow for an increase of the binding capacity, hence a larger amount of immobilized receptor molecules. To this end, we use electrochemically deposited "gold black" as a porous sensor surface for the immobilization of proteins. In this paper, we have analyzed the different parameters that define the electrochemical growth of porous gold, starting from flat gold surfaces, using different characterization techniques. Applied potentials of -0.5 V versus a reference electrode were found to constitute the most adequate conditions to grow porous gold surfaces. Using cyclic voltammetry, a 16 times increase of the surface area was observed under these electrochemical deposition conditions. In addition, we have assessed the immobilization degree of alkanethiols and of proteins on these different porous surfaces. The optimized deposition conditions for realizing porous gold substrates lead to a 11.4-fold increase of thiol adsorption and a 3.3-fold increase of protein adsorption, using the quartz crystal microbalance (QCM-D) as a biological transducer system. Hence, it follows that the high specific area of the porous gold can amplify the final sensitivity of the original flat surface device.