Electra Gizeli

A Love plate biosensor utilising a polymer layer

Abstract A Love plate device, based on an SSBW piezoelectric substrate coated with a polymer (PMMA) layer, was used to excite a surface guiding shear horizontal wave. The surface mass sensitivity of the device was calculated as a function of the thickness of the polymer layer, usin a modelling theory derived for the polymer-Love plate. LB films were used to assess experimentally the surface mass sensitivity and results were compared with theoretical predictions. Investigation of protein interaction with the PMMA surface was performed by following the adsorption of IgG in the range 1–400 μg/ml. Finally, the deposition of a protein multilayer, consisted of IgG/anti-IgG and protein A, was clearly detected using the polymer-coated Love plate device.

A novel Love-plate acoustic sensor utilizing polymer overlayers

A Love-plate sensor, consisting of a surface skimming bulk wave (SSBW) device coated with a polymer layer, was found to increase the acoustic signal through coupling of the SSBW wave to a Love wave. Insertion loss, phase and frequency measurements were used to assess the optimum thickness of the polymer layer and the sensitivity of the device to mass-loading and viscous coupling.<<ETX>>

Sensitivity of the acoustic waveguide biosensor to protein binding as a function of the waveguide properties

The aim of this work is to study the effect of operating frequency, piezoelectric substrate and waveguide layer thickness on the sensitivity of the acoustic waveguide sensor during the specific binding of an antibody by a protein. Shear horizontal (SH) wave devices consisting of (a) a LiTaO3 substrate operating at 104 MHz, (b) a quartz substrate operating at 108 MHz and (c) a quartz substrate operating at 155 MHz were coated with a photoresist polymer layer in order to produce acoustic waveguide devices supporting a Love wave. The effect of the thickness of the polymer layer on the Love wave was assessed by measuring the amplitude and phase of the wave before and after coating. The sensitivity of the above three biosensors was compared during the detection of the specific binding of different concentrations of Immunoglobulin G in the range of 0.7-667 nM to a protein A modified surface. Results indicate that the thickness of the polymer guiding layer is critical for obtaining the maximum sensitivity for a given geometry but a trade-off has to be made between the theoretically determined optimum thickness for waveguiding and the device insertion loss. It was also found that increasing the frequency of operation results in a further increase in the device sensitivity to protein detection.

Quantitative Determination of Size and Shape of Surface-Bound DNA Using an Acoustic Wave Sensor

DNA bending plays a significant role in many biological processes, such as gene regulation, DNA replication, and chromosomal packing. Understanding how such processes take place and how they can, in turn, be regulated by artificial agents for individual oriented therapies is of importance to both biology and medicine. In this work, we describe the application of an acoustic wave device for characterizing the conformation of DNA molecules tethered to the device surface via a biotin-neutravidin interaction. The acoustic energy dissipation per unit mass observed upon DNA binding is directly related to DNA intrinsic viscosity, providing quantitative information on the size and shape of the tethered molecules. The validity of the above approach was verified by showing that the predesigned geometries of model double-stranded and triple-helix DNA molecules could be quantitatively distinguished: the resolution of the acoustic measurements is sufficient to allow discrimination between same size DNA carrying a bent at different positions along the chain. Furthermore, the significance of this analysis to the study of biologically relevant systems is shown during the evaluation of DNA conformational change upon protein (histone) binding.

Design considerations for the acoustic waveguide biosensor

Love waveguide devices utilize shear-horizontal waves which propagate on the top layer of a coated SSBW acoustic device. Initially, attention was focused on the selection of a material which would effectively guide the Love wave. Silica and polymethyl methacrylate were used as guiding layers and the mass sensitivity of the corresponding sensors was tested in air. Low-shear-acoustic-velocity polymer overlayers were found to guide the Love wave most effectively with a maximum sensitivity of . The polymer waveguide sensor was further used to detect protein adsorption on the polymer surface from IgG solutions within the concentration range of 1 - . Radiolabelled IgG was applied to the device surface in order to calibrate the wave response to protein surface mass density. Finally, the effect of the acoustoelectric interaction on liquid-based applications was studied by utilizing a three-layer waveguide geometry. It was found that the evaporation of a 50 nm gold layer on the polymer overlayer can be used to eliminate acoustoelectric interactions without interfering with the Love wave propagation. After activation with protein A and IgG, the above system was used successfully to detect the direct binding of 400 ppb atrazine.

Acoustic sensors as a biophysical tool for probing cell attachment and cell/surface interactions

Acoustic biosensors offer the possibility to analyse cell attachment and spreading. This is due to the offered speed of detection, the real-time non-invasive approach and their high sensitivity not only to mass coupling, but also to viscoelastic changes occurring close to the sensor surface. Quartz crystal microbalance (QCM) and surface acoustic wave (Love-wave) systems have been used to monitor the adhesion of animal cells to various surfaces and record the behaviour of cell layers under various conditions. The sensors detect cells mostly via their sensitivity in viscoelasticity and mechanical properties. Particularly, the QCM sensor detects cytoskeletal rearrangements caused by specific drugs affecting either actin microfilaments or microtubules. The Love-wave sensor directly measures cell/substrate bonds via acoustic damping and provides 2D kinetic and affinity parameters. Other studies have applied the QCM sensor as a diagnostic tool for leukaemia and, potentially, for chemotherapeutic agents. Acoustic sensors have also been used in the evaluation of the cytocompatibility of artificial surfaces and, in general, they have the potential to become powerful tools for even more diverse cellular analysis.

Pulse mode shear horizontal-surface acoustic wave (SH-SAW) system for liquid based sensing applications

In this work, we describe a novel pulse mode shear horizontal-surface acoustic wave (SH-SAW) polymer coated biosensor that monitors rapid changes in both amplitude and phase. The SH-SAW sensors were fabricated on 36 degrees rotated Y-cut X propagating lithium tantalate (36 YX.LT). The sensitivity of the device to both mass loading and visco-elastic effects may be increased by using a thin guiding layer of cross-linked polymer. Two acoustic modes are excited by the electrodes in this crystalline direction. Metallisation of the propagation path of the 36 YX.LT devices allows the two modes to be discriminated. Successive polymer coatings resulted in the observation of resonant conditions in both modes as the layer thickness was increased. Using the 36 YX.LT devices, we have investigated the application of a novel pulse mode system by sensing a sequence of deposition and removal of a biological layer consisting of vesicles of the phospholipid POPC. A continuous wave system was used to verify the accuracy of the pulse mode system by sensing a series of poly(ethylene glycol) (PEG) solutions. The data clearly demonstrates the ability of the 36 YX.LT pulse mode system to provide rapid measurements of both amplitude and phase for biosensing applications.

Mechanisms of α-Defensin Bactericidal Action: Comparative Membrane Disruption by Cryptdin-4 and Its Disulfide-Null Analogue†

Mammalian alpha-defensins all have a conserved triple-stranded beta-sheet structure that is constrained by an invariant tridisulfide array, and the peptides exert bactericidal effects by permeabilizing the target cell envelope. Curiously, the disordered, disulfide-null variant of mouse alpha-defensin cryptdin-4 (Crp4), termed (6C/A)-Crp4, has bactericidal activity equal to or greater than that of the native peptide, providing a rationale for comparing the mechanisms by which the peptides interact with and disrupt phospholipid vesicles of defined composition. For both live Escherichia coli ML35 cells and model membranes, disordered (6C/A)-Crp4 induced leakage in a manner similar to that of Crp4 but had less overall membrane permeabilizing activity. Crp4 induction of the leakage of the fluorophore from electronegative liposomes was strongly dependent on vesicle lipid charge and composition, and the incorporation of cardiolipin into liposomes of low electronegative charge to mimic bacterial membrane composition conferred sensitivity to Crp4- and (6C/A)-Crp4-mediated vesicle lysis. Membrane perturbation studies using biomimetic lipid/polydiacetylene vesicles showed that Crp4 inserts more pronouncedly into membranes containing a high fraction of electronegative lipids or cardiolipin than (6C/A)-Crp4 does, correlating directly with measurements of induced leakage. Fluorescence resonance energy transfer experiments provided evidence that Crp4 translocates across highly charged or cardiolipin-containing membranes, in a process coupled with membrane permeabilization, but (6C/A)-Crp4 did not translocate across lipid bilayers and consistently displayed membrane surface association. Thus, despite the greater in vitro bactericidal activity of (6C/A)-Crp4, native, beta-sheet-containing Crp4 induces membrane permeabilization more effectively than disulfide-null Crp4 by translocating and forming transient membrane defects. (6C/A)-Crp4, on the other hand, appears to induce greater membrane disintegration.

Construction of three-dimensional biomolecule structures employing femtosecond lasers

The authors demonstrate here a method for three-dimensional patterning of proteins and other biological molecules. The method employs femtosecond-laser-induced three-photon polymerization, a technique which enables the construction of arbitrary two- and three-dimensional structures of submicron resolution. Biotin is subsequently attached to the three-dimensional (3D) structures via UV-activated cross-linking. The integrity of the photolytically immobilized biotin is confirmed by detecting the binding of fluorescently labeled avidin via fluorescence microscopy and via a surface acoustic sensor technique. In all, the technique opens the way for the fabrication of structures with a wide range of biomaterials as well as studying their dynamics within complex 3D structures.

Modelling of the mass sensitivity of the Love wave device in the presence of a viscous liquid

This paper describes a new theoretical model for the analysis of the mass sensitivity of the Love wave device. We use this model along with calculations from perturbation theory to calculate sensitivity. The model is based on Love wave propagation in an isotropic, non-piezoelectric quartz substrate over-layered by a silica waveguiding layer and immersed in a viscous liquid. The analysis considers power flow in the three-layered system, comprising the quartz substrate, the silica over-layer and the viscous liquid. The model fully accounts for the first time for the power that flows through the liquid phase and assesses its effect on mass sensitivity. Mass sensitivity values were obtained by determining the displacement field throughout the viscous liquid and the Love waveguide device. The resulting velocity field was used to generate a number of trial functions that had solutions assuming stress and strain continuity at the boundaries, and the complex dispersion relation was evaluated numerically. The resulting wavevector k and propagation constant β values were used to calculate the power in both the Love wave device and the liquid layer and the velocity amplitude at the surface. The model predicts that the mass sensitivity of the Love wave device will increase in the presence of a viscous solution due to power flow into the liquid.