Quartz Crystal Microbalance with Dissipation Monitoring: Enabling Real-Time Characterization of Biological Materials and Their Interactions

In recent years, there has been a rapid growth in the number of scientific reports in which the quartz crystal microbalance (QCM) technique has played a key role in elucidating various aspects of biological materials and their interactions. This article illustrates some key advances in the development of a special variation of this technique called quartz crystal microbalance with dissipation monitoring (QCM-D). The main feature and advantage of QCM-D, compared with the conventional QCM, is that it in addition to measuring changes in resonant frequency (Δf), a simultaneous parameter related to the energy loss or dissipation (ΔD) of the system is also measured. Δf essentially measures changes in the mass attached to the sensor surface, while ΔD measures properties related to the viscoelastic properties of the adlayer. Thus, QCM-D measures two totally independent properties of the adlayer. The focus of this review is an overview of the QCM-D technology and highlights of recent applications. Specifically, recent applications dealing with DNA, proteins, lipids, and cells will be detailed. This is not intended as a comprehensive review of all possible applications of the QCM-D technology, but rather a glimpse into a few highlighted application areas in the biomolecular field that were published in 2007.     

Probing fundamental film parameters of immobilized enzymes--towards enhanced biosensor performance. Part I--QCM-D mass and rheological measurements

Enzyme immobilization is an ever-growing research-area for both analytical and industrial applications. Of critical importance in this area are the effects of immobilization procedures upon the functionality of the immobilized biomolecules. Both beneficial and detrimental effects can be conferred through the selection and tuning of the immobilization procedure. Quartz-crystal microbalance with dissipation (QCM-D) has been previously used to great effect in tracking alterations to thin films of biomolecules immobilized onto quartz transducers.In this study, we investigate the ability of QCM-D to track and monitor film parameters of a monolayer of laccase immobilized on a series of self-assembled monolayers (SAMs), differing in lateral density of binding residues on the SAM and height of the SAM from the quartz surface. Both mass gains and rheological parameters for these varying surfaces were measured and trends later compared to the apparent enzyme kinetics of the immobilized laccase films, assessed electroanalytically (Paper II in this two part study). For covalent attachment of proteins, both shear and viscosity were increased relative to physically adsorbed proteins. An increase in lateral density of protein-binding surface of the SAM components was shown to increase the shear/viscosity of the resultant film while an increase in distance from the electrode (through incorporation of lysine linkers) was shown to decrease the shear/viscosity while simultaneously increasing the wet mass gain of the films. Shear and viscosity may be indicative of both enzyme denaturation and increased lateral protein packing within the film structure hence it is assumed that less distortion occurs with the inclusion of linkers which allow for more optimal protein immobilization.     

Competitive protein adsorption on micro patterned polymeric biomaterials, and viscoelastic properties of tailor made extracellular matrices

Cell adhesion on biomaterial surfaces and the vitality of anchorage dependent cells is affected by several parameters of an adsorbate layer which is intentionally or spontaneously formed. Surface pre-treatments and several conditioning steps prior and during to the cell/biomaterial contact affect the composition, orientation, quantity and viscoelasticity of the interfacing layer between cells and biomaterial. This work was performed to elucidate the response of cells on two modified biomaterial surfaces based on protein or carbohydrate adsorbates: (a) Masked UV irradiations opened a simple route to obtain chemically patterned substrates controlling serum protein adsorption and cell adhesion. It is possible to achieve structures of subcellular size and to produce immobilized gradients. In order to examine the protein matrix deposited on these substrates we applied a quartz microbalance technique (QCM-D) capable to extract viscoelastic data in addition to the mass uptake during plasma protein deposition. It was found that the quantity and viscosity of surface bound albumin is lowered when the surface is modified (patterned) by UV exposure. Hence, the UV modification promotes the competitive adsorption of cell adhesion proteins from the media or upon secretion by the cells and yields to the observed cell patterns. (b) Another tissue engineering technique, using immobilized, modified and/or cross linked hyaluronic acid (HA), an important extra cellular matrix component in vivo, is also examined by QCM-D. Our data demonstrate that HA can be modified by an activation with a carbodiimide, followed by the application of an alpha,omega-bisamino polyethyleneglycol. The QCM-D data can be interpreted as a stiffening of the HA layer combined with the release of hydration water. Further, the hydration state and the viscoelastic behaviour of surface bound ultrathin HA hydrogels was examined. Quantification of viscoelastic parameters of thin films of ECM by QCM-D is valuable for the interpretation of durotaxis, describing effects of mechanical substrate parameters on the adhesion and motility of cells.     

Quartz crystal microbalance studies of multilayer glucagon fibrillation at the solid-liquid interface

We have used a quartz crystal microbalance with dissipation (QCM-D) to monitor the changes in layer thickness and viscoelastic properties accompanying multilayer amyloid deposition in situ for the first time. By means of atomic force microscope imaging, an unequivocal correlation is established between the interfacial nucleation and growth of glucagon fibrils and the QCM-D response. The combination of the two techniques allows us to study the temporal evolution of the interfacial fibrillation process. We have modeled the QCM-D data using an extension to the Kelvin-Voigt viscoelastic model. Three phases were observed in the fibrillation process: 1), a rigid multilayer of glucagon monomers forms and slowly rearranges; 2), this multilayer subsequently evolves into a dramatically more viscoelastic layer, containing a polymorphic network of micrometer-long fibrils growing from multiple nucleation sites; and 3), the fibrillar formation effectively stops as a result of the depletion of bulk-phase monomers, although the process can be continued without a lag phase by subsequent addition of fresh monomers. The robustness of the QCM-D technique, consolidated by complementary atomic force microscope studies, should make it possible to combine different components thought to be involved in the plaque formation process and thus build up realistic models of amyloid plaque formation in vitro.     

Comparison of surface plasmon resonance spectroscopy and quartz crystal microbalance techniques for studying DNA assembly and hybridization

In this study we evaluate the strengths and weaknesses of surface plasmon resonance (SPR) spectroscopy and quartz crystal microbalance (QCM) technique for studying DNA assembly and hybridization reactions. Specifically, we apply in parallel an SPR instrument and a 5 MHz QCM device with dissipation monitoring (QCM-D) to monitor the assembly of biotinylated DNA (biotin-DNA) on a streptavidin-modified surface and the subsequent target DNA hybridization. Through the parallel measurements, we demonstrate that SPR is more suitable for quantitative analysis of DNA binding amount, which is essential for interfacial DNA probe density control and for the analysis of its effect on hybridization efficiency and kinetics. Although the QCM is not quantitative to the same extent as SPR (QCM measures the total mass of the bound DNA molecules together with the associated water), the dissipation factor of the QCM provides a qualitative measure of the viscoelastic properties of DNA films and the conformation of the bound DNA molecules. The complexity in mass measurement does not impair QCM's potential for a kinetic evaluation of the hybridization processes. For quantification of target DNA, the biotin-DNA modified SPR and QCM sensors are exposed to target DNA with increasing concentration. The plots of SPR/QCM signals versus target DNA concentration show that water entrapment between DNA strands make the QCM sensitivity for the hybridization assay well comparable with that of the SPR, although the intrinsic mass sensitivity of the 5 MHz QCM is approximately 20 times lower.     

DNA immobilization/hybridization on plasma-polymerized pyrrole

The present work describes the fabrication and characterization of the conducting polymer coatings prepared by the continuous wave plasma polymerization and the applications as adhesion layers for studying DNA immobilization/hybridization. The stability of plasma polymerized pyrrole (ppPY) in the aqueous solution was characterized by ellipsometry. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy were used to investigate polymer matrix properties and oligonucleotide/DNA binding interaction. The successful DNA immobilization on ppPY surfaces was found to depend on the macromolecular architecture of plasma polymerized films. The plasma polymers with similar thickness deposited at different input powers showed various comparable immobilization properties. The plasma-polymerized films prepared at the low input power showed a lower sensitivity toward DNA binding than those films deposited at the high input power. This result will be important to study plasma polymerized films as potential DNA biosensors in the future.     

Quartz crystal microbalance-with dissipation monitoring (QCM-D) for real time measurements of blood coagulation density and immune complement activation on artificial surfaces

A recently developed variant of quartz crystal microbalance (QCM) called QCM-with dissipation monitoring (QCM-D) allows simultaneous and simple measurements of changes in adsorbed mass as well as the viscoelastic property (D-factor) of deposited protein layers on the sensor surface. We have taken the QCM-D technology a step further and demonstrated its advantages in the study of protein assembly as a consequence of surface induced immune complement activation, or contact activated blood coagulation. In the present study we have continued our QCM-D investigations of surface assembly of fibrin clot formation and complement activation and incubated differently modified quartz sensor surfaces in blood plasma and sera. Polymer surfaces used were spin-coated polyethylene, poly(ethylene terephtalate), poly(methylmetacrylate) and poly(dimethylsiloxane). Also used were sputtered titanium and heparin grafted surfaces. In this investigation we found that we could describe the surface induced coagulation with four independent parameters: (1) Time of onset of coagulation, (2) fibrin deposition rate, (3) total frequency shift at stable plateau, and (4) fibrin clot density. The most important finding was that the blood plasma clot density can be assessed with the use of D determinations and that the clot density varied significantly with the chemical composition of the surface. However, the D-factor did not give any new analytical information about the possible complement activation mechanisms. Nevertheless, the QCM-D was found to be a reliable tool for the analysis of surface induced complement activation. We also compared the QCM-D technique with traditional enzyme immuno assay (EIA) measurements of soluble products from the surface activation of the complement and coagulation systems. We found that the results from EIA and QCM-D measurements corresponded well for the complement activation but not for the coagulation, probably due to the biological complexity of the coagulation system.     

QCM-D sensitivity to protein adsorption reversibility

Using a quartz crystal microbalance with dissipative monitoring (QCM-D) we have determined the adsorption reversibility and viscoelastic properties of ribonuclease A adsorbed to hydrophobic self-assembled monolayers. Consistent with previous work with proteins unfolding on hydrophobic surfaces, high protein solution concentrations, reduced adsorption times, and low ammonium sulfate concentrations lead to increased adsorption reversibility. Measured rigidity of the protein layers normalized for adsorbed protein amounts, a quantity we term specific dissipation, correlated with adsorption reversibility of ribonuclease A. These results suggest that specific dissipation may be correlated with changes in structure of adsorbed proteins.     

Real-time monitoring of the development and stability of biofilms of Streptococcus mutans using the quartz crystal microbalance with dissipation monitoring

Quartz crystal microbalance with dissipation monitoring (QCM-D) was used for continuous in-situ monitoring of cell attachment and growth of Streptococcus mutans as biofilms. Cell attachment and proliferation were monitored within an overnight period of 20 h. Biofilms generated using a 'continuous flow' method had a greater mass and were more dissipative (more viscoelastic) than those established using an 'attach and flow' strategy. Cell numbers (as colony forming units, c.f.u.) in biofilms formed inside the QCM-D device after a 2-h attachment phase and during a 20-h growth period could be related to frequency (f) changes. The percentage surface coverage on the QCM-D crystals by bacteria was estimated using the surface analysis features of the atomic force microscope and image analysis software. Both mean percentage coverage and c.f.u increased after growth of S. mutans. The energy losses displayed by the increases in the dissipative factor (D) indicated an increase in 'softness' of the attached cells. The ratio of D/f was used to provide information of the way in which viscoelasticity changed per unit mass. Flow conditions over the cells on the surface appeared to be important in creating biofilms of a greater complexity and stability and the QCM-D enabled properties of cells during attachment and binding, proliferation and removal to be monitored continuously.     

Characterization and application of a surface modification designed for QCM-D studies of biotinylated biomolecules

The rapid development of surface sensitive biosensor technologies, especially towards nanoscale devices, requires increasing control of surface chemistry to provide reliable and reproducible results, but also to take full advantage of the sensing opportunities. Here, we present a surface modification strategy to allow biotinylated biomolecules to be immobilized to gold coated sensor crystals for quartz crystal microbalance with dissipation monitoring (QCM-D) sensing. The unique feature of QCM-D is its sensitivity to nanomechanical (viscoelastic) properties at the sensing interface. The surface modification was based on mixed monolayers of oligo(ethylene glycol) (OEG) disulfides, with terminal -OH or biotin groups, on gold. Mixtures containing 1% of the biotin disulfide were concluded to be the most appropriate based on the performance when streptavidin was immobilized to biotinylated sensors and the subsequent biotinylated bovine serum albumin (BSA) interaction was studied. The OEG background kept the unspecific protein binding to a minimum, even when subjected to serum solutions with a high protein concentration. Based on characterization by contact angle goniometry, ellipsometry, and infrared spectroscopy, the monolayers were shown to be well-ordered, with the OEG chains predominantly adopting a helical conformation but also partly an amorphous structure. Storage stability was concluded to depend mainly on light exposure while almost all streptavidin binding activity was retained when storing the sensors cold and dark for 8 weeks. The surface modification was also tested for repeated antibody-antigen interactions between BSA and anti-BSA (immobilized to biotinylated protein A) in QCM-D measurements lasting for >10h with intermediate basic regeneration. This proved an excellent stability of the coating and good reproducibility was obtained for 5 interaction cycles. With this kind of generic surface modification QCM-D can be used in a variety of biosensing applications to provide not only mass but also relevant information of the structural properties of adlayers.     

Localized dynamic light scattering: a new approach to dynamic measurements in optical microscopy.

We present a new approach to probing single-particle dynamics that uses dynamic light scattering from a localized region. By scattering a focused laser beam from a micron-size particle, we measure its spatial fluctuations via the temporal autocorrelation of the scattered intensity. We demonstrate the applicability of this approach by measuring the three-dimensional force constants of a single bead and a pair of beads trapped by laser tweezers. The scattering equations that relate the scattered intensity autocorrelation to the particle position correlation function are derived. This technique has potential applications for measurement of biomolecular force constants and probing viscoelastic properties of complex media.     

A streptavidin surface on planar glass substrates for the detection of biomolecular interaction

Based on the requirements of biomolecular interaction analysis on direct optical transducers, a streptavidin surface is examined. A general protocol was developed allowing the immobilization of biotinylated compounds using the rife biotin-streptavidin system. This type of surface modification can be applied to all biosensors using glass surfaces as sensor devices. Reflectometric interference spectroscopy (RIfS), a label-free, direct optical method was used to demonstrate the quality of the transducer surfaces. The surface modification is based on an aminofunctionalized polyethylene glycol layer covalently bound to the silica surface of the transducer and shows very little nonspecific binding. Biotin molecules can be easily coupled on such layers. Streptavidin followed by a biotinylated estrone derivative was immobilized by incubation of the biotinylated transducer surface. For the streptavidin layer we obtained interference signals corresponding to a protein monolayer. Finally, using a surface prepared as described above, biomolecular interaction experiments with an antibody against estrone were carried out to show the quality of the transducer surface. With RIfS all of the affinity-based surface modifications can be detected online and time resolved.     

Characterizing the microenvironment surrounding protein sites.

Sites are microenvironments within a biomolecular structure, distinguished by their structural or functional role. A site can be defined by a three-dimensional location and a local neighborhood around this location in which the structure or function exists. We have developed a computer system to facilitate structural analysis (both qualitative and quantitative) of biomolecular sites. Our system automatically examines the spatial distributions of biophysical and biochemical properties, and reports those regions within a site where the distribution of these properties differs significantly from control nonsites. The properties range from simple atom-based characteristics such as charge to polypeptide-based characteristics such as type of secondary structure. Our analysis of sites uses non-sites as controls, providing a baseline for the quantitative assessment of the significance of the features that are uncovered. In this paper, we use radial distributions of properties to study three well-known sites (the binding sites for calcium, the milieu of disulfide bridges, and the serine protease active site). We demonstrate that the system automatically finds many of the previously described features of these sites and augments these features with some new details. In some cases, we cannot confirm the statistical significance of previously reported features. Our results demonstrate that analysis of protein structure is sensitive to assumptions about background distributions, and that these distributions should be considered explicitly during structural analyses.     

Immobilization of Bacillus licheniformis α-amylase onto reactive polymer films

Alpha-amylase was covalently immobilized onto maleic anhydride copolymer films preserving activity. The initial activity of the immobilized layers strongly depended on the immobilization solution, and on the physicochemical properties of the copolymer film. Higher enzyme loading (quantified by amino acid analysis using HPLC) and activity (measured by following starch hydrolysis) were attainable onto hydrophilic, highly swelling 3-D poly(ethylene-alt-maleic anhydride) (PEMA) copolymer films, while immobilization onto hydrophobic poly(octadecene-alt-maleic anhydride) (POMA) copolymer films resulted in low content enzyme layers and lower activity. No significant activity was lost upon dehydration/re-hydration or storage of enzyme containing PEMA copolymer layers in deionised water for up to 48 h. In contrast, α-amylase decorated POMA films suffered a significant activity loss under those conditions. The distinct behaviours may be attributed to the different intrinsic physicochemical properties of the copolymer films. The compact, hydrophobic POMA films possibly favours hydrophobic interactions between the hydrophobic moieties of the protein and the surface, which may result in conformational changes, and consequent loss of activity. Surprisingly, residual activity was found after harsh treatments of active α-amylase PEMA based layers revealing that immobilization onto the hydrophilic polymer films improved the stability of the enzyme.     

Modification of bacterial structures by a low‐temperature gas plasma and influence on packaging material

Aims: To investigate the effect of a cascaded dielectric barrier discharge (CDBD) treatment on the biological structure of a selected bacterium and on the properties of different polymer films. Methods and Results: Inactivation kinetics were measured using air as the process gas and using Bacillus atrophaeus spores and vegetative cells, which had been homogeneously distributed on a surface. The changes to the outer coats and the DNA of the endospores and cells after plasma treatment were determined using biomolecular and chemical methods. The experiments showed that damage to the DNA molecules and changes in the cell walls can be observed as a consequence of the CDBD treatment. Furthermore, the influence of the plasma treatment on the properties of various polymer films was investigated using a variety of test methods. Except the sealing strength where a slight decrease was observed (max. 20%), no negative changes of the material properties have occurred. Conclusions: CDBD treatment can affect the DNA of spores and cells, depending on the treatment time. At the same time, practically relevant inactivation rates on packaging materials were observed, without any significant changes to the material properties. Significance and Impact of the Study: Knowledge about CDBD mechanisms was acquired from a biological point of view, and the suitability of the method for treating polymer films was demonstrated.     

One step processing of aminofunctionalized gate oxides

A plasma discharge process has been developed that allows the growth of biosensor gate oxides with adapted surface properties for the direct application of biomolecular immobilization cascades. The process involves an accurate selection of processing conditions, mainly, low temperature evaporation of (3-aminopropyl)triethoxysilane (APTS) and dynamic power and flow conditions. Room temperature evaporation of APTS was achieved by designing a vessel with an internal capillary network. The initial high power (100 W) plasma conditions were replaced by milder molecular fragmentation (50 W, 25 W) in a pure Ar discharge. Under these conditions the thin SiO₂ layers presented graded properties with a denser layer at the Si (100) interface and a hybrid organic–inorganic structure at the surface. The chemistry of the films was analysed by Fourier transformed infrared spectroscopy (FTIR) and Rutherford backscattering spectroscopy combined with elastic recoil detection analysis (RBS, ERDA), which confirmed the presence of the SiO₂ and organic phases. Contact angle measurements indicate the higher contribution of the basic polar component to the surface free energy. Furthermore, the higher affinity of the surface towards biomolecular immobilization was confirmed by fluorescence microscopy. Finally, penetration of nitrobenzaldehyde was obtained by application of a molecular permeation method evaluated by UV–vis spectroscopy onto fused silica substrates.     

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.     

Aqueous two-phase systems

Biphasic systems formed by mixing of two polymers or a polymer and a salt in water can be used for separation of cells, membranes, viruses, proteins, nucleic acids, and other biomolecules. The partitioning between the two phases is dependent on the surface properties and conformation of the materials, and also on the composition of the two-phase system. The mechanism of partitioning is, however, complex and not easily predicted. Aqueous two-phase systems (ATPS) have proven to be a useful tool for analysis of biomolecular and cellular surfaces and their interactions, fractionation of cell populations, product recovery in biotechnology, and so forth. Potential for environmental remediation has also been suggested. Because ATPS are easily scalable and are also able to hold high biomass load in comparison with other separation techniques, the application that has attracted most interest so far has been the large-scale recovery of proteins from crude feedstocks. As chemicals constitute the major cost factor for large-scale systems, use of easily recyclable phase components and the phase systems generated by a single-phase chemical in water are being studied.     

Poly(vinylpyrrolidone) for bioconjugation and surface ligand immobilization

Poly(vinylpyrrolidone) (PVP), a nonionic and nontoxic polymer with antifouling properties, has been synthesized via RAFT polymerization to obtain thiol-terminated PVP. We demonstrate that when the polymer is adsorbed onto the surface of colloidal silica particles, the terminal thiol groups of PVP remain accessible for chemical modification and lend themselves to the immobilization of ligands. We show that ligand attachment onto the surface via conjugation to PVP is reversible, as the polymer can be desorbed from the surface for conjugate and surface recovery. We present the conjugation of a model peptide and an oligonucleotide to PVP via the polymer terminal thiol and demonstrate that conjugates remain functional in molecular recognition assay. The developed technique offers a novel method to functionalize low-fouling surfaces for a variety of biomedical applications and presents opportunities to use PVP as a macromolecular drug carrier.     

Immobilization of glucose oxidase in thin polypyrrole films: influence of polymerization conditions and film thickness on the activity and stability of the immobilized enzyme

Using monomers that polymerize to form electrically conducting polymers, one can control the thickness of the polymer film and the amount of enzyme that can be immobilized in the films. First, an investigation of the major variables that influence the immobilization of glucose oxidase by entrapment in polypyrrole films, prepared by electropolymerization from aqueous solutions containing the enzyme and monomer, was carried out. Then the optimized conditions were used to assess the effects of film thickness on the activity and stability of immobilized enzyme. For the films ranged in thickness from 0.1 mum to 1.6 mum, the resulting apparent activity and stability of the immobilized enzyme were found to be a strong function of the polymer film thickness. Above a thickness of 1.0 mum, the apparent activity of the immobilized enzyme increases linearly with increasing film thickness. The nonlinearity observed for films of thickness less than 1.0 mum can be attributed to the changes observed in the morphology of the resulting polypyrrole films. Furthermore, it was noted that when the glucose oxidase/polypyrrole films are stored in phosphate buffer, at 4 degrees C, the observed rate of loss in apparent activity of the immobilized enzyme is highest for the first few days, also being higher for the thinner films. However, after the loosely entrapped enzyme is leached from the polymer film, the rate of loss in activity is very low indicating that the well-entrapped enzyme, as well as the polypyrrole films, exhibit good stability. Finally, the reproducibility of the immobilization technique is excellent. (c) 1993 John Wiley & Sons, Inc.