Fredrik Höök

Quartz crystal microbalance setup for frequency and Q‐factor measurements in gaseous and liquid environments

An experimental setup has been constructed for simultaneous measurements of the frequency, the absolute Q factor, and the amplitude of oscillation of a quartz crystal microbalance (QCM). The technical solution allows operation in vacuum, air, or liquid. The crystal is driven at its resonant frequency by an oscillator that can be intermittently disconnected causing the crystal oscillation amplitude to decay exponentially. From the recorded decay curve the absolute Q factor (calculated from the decay time constant), the frequency of the freely oscillating crystal, and the amplitude of oscillation are obtained. All measurements are fully automated. One electrode of the QCM in our setup was connected to true ground which makes possible simultaneous electrochemistry. The performance is illustrated by experiments in fluids of varying viscosity (gas and liquid) and by proteinadsorptionin situ. We found, in addition to the above results, that the amplitude of oscillation is not always directly proportional to the Q factor, as the commonly used theory states. This puts limitations on the customary use of the amplitude of oscillation as a measure of the Q factor.

A comparative study of protein adsorption on titanium oxide surfaces using in situ ellipsometry, optical waveguide lightmode spectroscopy, and quartz crystal microbalance/dissipation

Abstract The adsorption kinetics of three model proteins—human serum albumin, fibrinogen and hemoglobin—has been measured and compared using three different experimental techniques: optical waveguide lightmode spectroscopy (OWLS), ellipsometry (ELM) and quartz crystal microbalance (QCM-D). The studies were complemented by also monitoring the corresponding antibody interactions with the pre-adsorbed protein layer. All measurements were performed with identically prepared titanium oxide coated substrates. All three techniques are suitable to follow in-situ kinetics of protein–surface and protein–antibody interactions, and provide quantitative values of the adsorbed adlayer mass. The results have, however, different physical contents. The optical techniques OWLS and ELM provide in most cases consistent and comparable results, which can be straightforwardly converted to adsorbed protein molar (‘dry’) mass. QCM-D, on the other hand, produces measured values that are generally higher in terms of mass. This, in turn, provides valuable, complementary information in two respects: (i) the mass calculated from the resonance frequency shift includes both protein mass and water that binds or hydrodynamically couples to the protein adlayer; and (ii) analysis of the energy dissipation in the adlayer and its magnitude in relation to the frequency shift (c.f. adsorbed mass) provides insight about the mechanical/structural properties such as viscoelasticity.

Simultaneous frequency and dissipation factor QCM measurements of biomolecular adsorption and cell adhesion

We have measured the energy dissipation of the quartz crystal microbalance (QCM), operating in the liquid phase, when mono- or multi-layers of biomolecules and biofilms form on the QCM electrode (with a time resolution of ca. 1 s). Examples are taken from protein adsorption, lipid vesicle adsorption and cell adhesion studies. Our results show that even very thin (a few nm) biofilms dissipate a significant amount of energy owing to the QCM oscillation. Various mechanisms for this energy dissipation are discussed. Three main contributions to the measured increase in energy dissipation are considered. (i) A viscoelastic porous structure (the biofilm) that is strained during oscillation, (ii) trapped liquid that moves between or in and out of the pores due to the deformation of the film and (iii) the load from the bulk liquid which increases the strain of the film. These mechanisms are, in reality, not entirely separable, rather, they constitute an effective viscoelastic load. The biofilms can therefore not be considered rigidly coupled to the QCM oscillation. It is further shown theoretically that viscoelastic layers with thicknesses comparable to the biofilms studied in this work can induce energy dissipation of the same magnitude as the measured ones.

QCM Operation in Liquids: An Explanation of Measured Variations in Frequency and Q Factor with Liquid Conductivity.

Recently, several reports have shown that when one side of a quartz crystal microbalance (QCM) is exposed to a liquid, the parallel (but not the series) resonant frequency is influenced by the conductivity and dielectric constant of the liquid. The effect is still controversial and constitutes a serious complication in many applications of the QCM in liquid environments. One suggestion has been that acoustically induced surface charges couple to charged species in the conducting liquid. To explore this effect, we have measured the parallel and the series mode resonance frequencies, and the corresponding Q factors, for a QCM with one side facing a liquid. These four quantities have all been measured versus liquid conductivity, using a recently developed experimental setup. It allows the simultaneous measurement of the resonant frequency and the Q factor of an oscillating quartz crystal, intermittently disconnected from the driving circuit. Based on these results, a simple model together with an equivalent circuit for a quartz crystal exposed to a liquid is presented. The analysis shows that it is not necessary to infer the existence of surface charges (or other microscopic phenomena such as electrical double layers) to account for the influence of the liquids electrical properties on the resonant frequency. Our results show that the contacting conductive liquid, in effect, enlarges the electrode area on the liquid side and thereby changes the parallel resonant frequency. By proper design of the QCM measurement, perturbing effects due to the liquids electrical properties can be circumvented.

Quartz crystal microbalance with dissipation monitoring of supported lipid bilayers on various substrates.

Supported lipid bilayers (SLBs) mimic biological membranes and are a versatile platform for a wide range of biophysical research fields including lipid–protein interactions, protein–protein interactions and membrane-based biosensors. The quartz crystal microbalance with dissipation monitoring (QCM-D) has had a pivotal role in understanding SLB formation on various substrates. As shown by its real-time kinetic monitoring of SLB formation, QCM-D can probe the dynamics of biomacromolecular interactions. We present a protocol for constructing zwitterionic SLBs supported on silicon oxide and titanium oxide, and discuss technical issues that need to be considered when working with charged lipid compositions. Furthermore, we explain a recently developed strategy that uses an amphipathic, α-helical (AH) peptide to form SLBs on gold and titanium oxide substrates. The protocols can be completed in less than 3 h.

Label-free plasmonic detection of biomolecular binding by a single gold nanorod.

We report the use of individual gold nanorods as plasmonic transducers to detect the binding of streptavidin to individual biotin-conjugated nanorods in real time on a surface. Label-free detection at the single-nanorod level was performed by tracking the wavelength shift of the nanorod-localized surface plasmon resonant scattering spectrum using a dark-field microspectroscopy system. The lowest streptavidin concentration that was experimentally measured was 1 nM, which is a factor of 1000-fold lower than the previously reported detection limit for streptavidin binding by biotinylated single plasmonic nanostructures. We believe that the current optical setup is able to reliably measure wavelength shifts as small as 0.3 nm. Binding of streptavidin at 1 nM concentration induces a mean resonant wavelength shift of 0.59 nm suggesting that we are currently operating at close to the limit of detection of the system.

Vesicle adsorption on SiO2 and TiO2: Dependence on vesicle size

Kinetics of vesicle adsorption on SiO2 and TiO2 surfaces was investigated for vesicle sizes from 25 to 200 nm. On SiO2, nonruptured vesicles are adsorbed up to a critical coverage, Θc, after which spontaneous rupture and bilayer formation occurs in qualitatively the same manner for all vesicle sizes. On TiO2, adsorption of nonruptured vesicles continues up to saturation. A comparison between the kinetics at low coverages on SiO2 and TiO2, where intact vesicles adsorb on both surfaces, reveals that the vesicles on SiO2 are more flattened than nonruptured vesicles adsorbed on TiO2, indicating a stronger vesicle-surface interaction on SiO2.

A Method Improving the Accuracy of Fluorescence Recovery after Photobleaching Analysis

Fluorescence recovery after photobleaching has been an established technique of quantifying the mobility of molecular species in cells and cell membranes for more than 30 years. However, under nonideal experimental conditions, the current methods of analysis still suffer from occasional problems; for example, when the signal/noise ratio is low, when there are temporal fluctuations in the illumination, or when there is bleaching during the recovery process. We here present a method of analysis that overcomes these problems, yielding accurate results even under nonideal experimental conditions. The method is based on circular averaging of each image, followed by spatial frequency analysis of the averaged radial data, and requires no prior knowledge of the shape of the bleached area. The method was validated using both simulated and experimental fluorescence recovery after photobleaching data, illustrating that the diffusion coefficient of a single diffusing component can be determined to within approximately 1%, even for small signal levels (100 photon counts), and that at typical signal levels (5000 photon counts) a system with two diffusion coefficients can be analyzed with <10% error.

Comparison of surface plasmon resonance and quartz crystal microbalance in the study of whole blood and plasma coagulation

The coagulation of blood plasma and whole blood was studied with a surface plasmon resonance (SPR) based device and a quartz crystal microbalance instrument with energy dissipation detection (QCM-D). The SPR and QCM-D response signals were similar in shape but differing in time scales, reflecting differences in detection mechanisms. The QCM-D response time was longer than SPR, as a physical coupling of the sample to the substrate is required for molecules to be detected by the QCM-method. Change of sample properties within the evanescent field is sufficient for detection with SPR. Both the SPR signals and the QCM-D frequency and dissipation shifts showed dependency on concentrations of coagulation activator and sensitivity to heparin additions. The ratio of dissipation to frequency shifts, commonly considered to reflect viscoelastic properties of the sample, varied with the concentration of activator in blood plasma but not in whole blood. Additions of heparin to the thromboplastin activated whole blood sample, however, made the ratio variation reoccur. Implications of these observations for the understanding of the blood coagulation processes as well as the potential of the two methods in the clinic and in research are discussed.

Patterns of DNA-Labeled and scFv-Antibody-Carrying Lipid Vesicles Directed by Material-Specific Immobilization of DNA and Supported Lipid Bilayer Formation on an Au/SiO2 Template

Much effort is currently concentrated on research devoted to biofunctional patterned surfaces, which constitute the fundament for the development of microarrays for high-throughput gene and protein analyses. DNA microarrays have proved very successful, and the concept is in the process of being applied to protein arrays. However, in contrast to DNA fragments, proteins are easily denatured in contact with solid supports, and robotic printing of proteins onto chemically reactive glass slides will not necessarily be applicable as a generic protocol for the preparation of protein arrays. Supported phosphatidylcholine lipid bilayers have emerged as interesting candidate substrates for protein chips, since they efficiently reduce nonspecific protein adsorption 5] and, at the same time, allow different strategies for protein immobilization with biospecific water, desalted with a NAP5 column (Amersham Pharmacia, USA) according to manufacturers protocols, and stored as working stock solutions at 20 C until use. Epoxy-derivatized slides were prepared from plain glass slides (Sigma, USA) as previously described. Nhydroxysuccinimide slides were also used to spot the proteins but consistently gave inferior results. Proteins were prepared in NaHCO3 buffer (0.1M, pH 9) and arrayed on epoxy slides with a spacing of 180 m between the spots by using an statistical microarray analysis arrayer (Engineering Services Inc. , Ontario, Canada). After a 2-hour incubation period the slides were either used immediately, or stored for future use at 4 C. The slides, if stored, were typically used within 48 h of printing.