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Kalograiaki I.,CSIC - Institute of Physical Chemistry "Rocasolano" | Kalograiaki I.,CIBER ISCIII | Euba B.,CIBER ISCIII | Euba B.,Institute Agrobiotecnologia | And 8 more authors.
Analytical Chemistry | Year: 2016

Recognition of bacterial surface epitopes by host receptors plays an important role in the infectious process and is intimately associated with bacterial virulence. Delineation of bacteria-host interactions commonly relies on the detection of binding events between purified bacteria- and host-target molecules. In this work, we describe a combined microarray and quartz crystal microbalance (QCM) approach for the analysis of carbohydrate-mediated interactions directly on the bacterial surface, thus preserving the native environment of the bacterial targets. Nontypeable Haemophilus influenzae (NTHi) was selected as a model pathogenic species not displaying a polysaccharide capsule or O-antigen-containing lipopolysaccharide, a trait commonly found in several important respiratory pathogens. Here, we demonstrate the usefulness of NTHi microarrays for exploring the presence of carbohydrate structures on the bacterial surface. Furthermore, the microarray approach is shown to be efficient for detecting strain-selective binding of three innate immune lectins, namely, surfactant protein D, human galectin-8, and Siglec-14, to different NTHi clinical isolates. In parallel, QCM bacteria-chips were developed for the analysis of lectin-binding kinetics and affinity. This novel QCM approach involves capture of NTHi on lectin-derivatized chips followed by formaldehyde fixation, rendering the bacteria an integrated part of the sensor chip, and subsequent binding assays with label-free lectins. The binding parameters obtained for selected NTHi-lectin pairs provide further insights into the interactions occurring at the bacterial surface. © 2016 American Chemical Society.


Li X.,Northwest University, China | Song S.,Northwest University, China | Pei Y.,Northwest University, China | Dong H.,Huazhong University of Science and Technology | And 2 more authors.
Sensors and Actuators, B: Chemical | Year: 2016

A two-dimensional (2D) and a three-dimensional (3D) His-tag capture surfaces were fabricated for oriented and reversible immobilization of His-tagged proteins on quartz crystal microbalance (QCM) biosensor surfaces, which can be used for label-free and real-time detection of the interactions between His-tagged protein and its interacting protein (analyte). His-tagged proteins immobilized on the 2D His-tag capture surface maintained a higher binding activity than those immobilized on a 2D carboxyl surface via amine coupling. The 3D His-tag capture surface has about twice the amount of immobilization capacity as the 2D His-tag capture surface, which enables a higher sensitivity for detection. His-tag capture surface can be optionally regenerated to remove the His-tagged protein as well as the analyte for the next cycle of His-tagged protein immobilization, or to only selectively remove the analyte, leaving the His-tagged protein on the surface for the next cycle of analyte binding. Furthermore, the kinetic and affinity studies of the interactions between the His-tagged protein and its interacting protein were performed. This study provides an efficient way to study protein-protein interactions by oriented and reversible immobilization of His-tagged proteins on QCM biosensor surfaces. © 2015 Elsevier B.V. All rights reserved.


Pei Z.,Northwest University, China | Anderson H.,Attana | Anderson H.,Uppsala University | Myrskog A.,Attana | And 4 more authors.
Analytical Biochemistry | Year: 2010

The performance of immunosensors is highly dependent on the amount of immobilized antibodies and their remaining antigen binding capacity. In this work, a method for immobilization of antibodies on a two-dimensional carboxyl surface has been optimized using quartz crystal microbalance biosensors. We show that successful immobilization is highly dependent on surface pKa, antibody pI, and pH of immobilization buffer. By the use of EDC/sulfo-NHS (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysulfosuccinimide) activation reagents, the effect of the intrinsic surface pKa is avoided and immobilization at very low pH is therefore possible, and this is important for immobilization of acidic proteins. Antigen binding capacity as a function of immobilization pH was studied. In most cases, the antigen binding capacity followed the immobilization response. However, the antigen-to-antibody binding ratio differed between the antibodies investigated, and for one of the antibodies the antigen binding capacity was significantly lower than expected from immobilization in a certain pH range. Tests with anti-Fc and anti-Fab2 antibodies on different antibody surfaces indicated that the orientation of the antibodies on the surface had a profound effect on the antigen binding capacity of the immobilized antibodies. © 2009 Elsevier Inc.


PubMed | Cambridge College, Northwest Agriculture and Forestry University and Attana
Type: | Journal: Scientific reports | Year: 2015

A novel approach to the study of binding thermodynamics and kinetics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a quartz crystal microbalance (QCM) biosensor was developed, in which binding events take place at the cell surface, more closely mimicking a biologically relevant environment. In this study, colon adenocarcinoma cells (KM-12) and ovary adenocarcinoma cells (SKOV-3) grew on the optimized polystyrene-coated biosensor chip without fixation. The association and dissociation between the cell surface carbohydrates and a range of lectins, including WGA, Con A, UEA-I, GS-II, PNA and SBA, were monitored in real time and without label for evaluation of cell surface glycosylation. Furthermore, the thermodynamic and kinetic parameters of the interaction between lectins and cell surface glycan were studied, providing detailed information about the interactions, such as the association rate constant, dissociation rate constant, affinity constant, as well as the changes of entropy, enthalpy and Gibbs free energy. This application provides an insight into the cell surface glycosylation and the complex molecular recognition on the intact cell surface, which may have impacts on disease diagnosis and drug discovery.


PubMed | Attana, CSIC - Institute of Physical Chemistry "Rocasolano" and CIBER ISCIII
Type: Journal Article | Journal: Analytical chemistry | Year: 2016

Recognition of bacterial surface epitopes by host receptors plays an important role in the infectious process and is intimately associated with bacterial virulence. Delineation of bacteria-host interactions commonly relies on the detection of binding events between purified bacteria- and host-target molecules. In this work, we describe a combined microarray and quartz crystal microbalance (QCM) approach for the analysis of carbohydrate-mediated interactions directly on the bacterial surface, thus preserving the native environment of the bacterial targets. Nontypeable Haemophilus influenzae (NTHi) was selected as a model pathogenic species not displaying a polysaccharide capsule or O-antigen-containing lipopolysaccharide, a trait commonly found in several important respiratory pathogens. Here, we demonstrate the usefulness of NTHi microarrays for exploring the presence of carbohydrate structures on the bacterial surface. Furthermore, the microarray approach is shown to be efficient for detecting strain-selective binding of three innate immune lectins, namely, surfactant protein D, human galectin-8, and Siglec-14, to different NTHi clinical isolates. In parallel, QCM bacteria-chips were developed for the analysis of lectin-binding kinetics and affinity. This novel QCM approach involves capture of NTHi on lectin-derivatized chips followed by formaldehyde fixation, rendering the bacteria an integrated part of the sensor chip, and subsequent binding assays with label-free lectins. The binding parameters obtained for selected NTHi-lectin pairs provide further insights into the interactions occurring at the bacterial surface.

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