Abstract
Optical biosensors based on photonic crystal surface waves (PC SWs) offer a possibility to study binding interactions with living cells, overcoming the limitation of rather small evanescent field penetration depth into a sample medium that is characteristic for typical optical biosensors. Besides this, simultaneous excitation of s- and p-polarized surface waves with different penetration depths is realized here, permitting unambiguous separation of surface and volume contributions to the measured signal. PC-based biosensors do not require a bulk signal correction, compared to widely used surface plasmon resonance-based devices. We developed a chitosan-based protocol of PC chip functionalization for bacterial attachment and performed experiments on antibody binding to living bacteria measured in real time by the PCSW-based biosensor. Data analysis reveals specific binding and gives the value of the dissociation constant for monoclonal antibodies (IgG2b) against bacterial lipopolysaccharides equal to KD = 6.2 ± 3.4 nM. To our knowledge, this is a first demonstration of antibody-binding kinetics to living bacteria by a label-free optical biosensor.
Highlights
According to reviews on antimicrobial resistance, each year 700,000 people die of infections caused by drug-resistant bacteria
We describe an experimental protocol for the binding experiment, data handling and a binding model, and discuss the experimental data on antibody binding kinetics and further data analysis including calculation of the dissociation constant
K-12 strain belongs to Gram-negative bacteria, which to living bacterial coli DH5a to Gram-negative bacteria, have a cell wall formed by a peptidoglycan layer covered by the membrane composed of which have a cell wall formed by a peptidoglycan layer covered by the membrane composed of lipopolysaccharides (LPS)
Summary
According to reviews on antimicrobial resistance, each year 700,000 people die of infections caused by drug-resistant bacteria. Key drug properties can be understood from kinetics measurements of its binding to a target under conditions which are as close as possible to a native cell environment. There is a need for an efficient method that enables fast time-resolved measurement of binding interactions. Label-free optical biosensors based on various transducers are actively entering this field [3,4]. Attempts were made to measure binding kinetics on living bacteria by optical surface plasmon resonance (SPR)-based biosensors [5]. The experiments were not fully successful because of an insufficient penetration depth of the sensing evanescent field into living bacteria
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