Assembly of multilayer arrays of viral nanoparticles via biospecific recognition: a quartz crystal microbalance with dissipation monitoring study

The development of multilayered thin film assemblies containing (bio)molecules is driven by the need to miniaturize sensors, reactors, and biochips. Viral nanoparticles (VNPs) have become popular nanobuilding blocks for material fabrication, and our research has focused on the well-characterized plant virus Cowpea mosaic virus (CPMV). In a previous study, we have reported the construction of multilayer VNP assemblies. Here we extend these studies by providing further details on the formation and properties of arrays that are made by the alternating deposition of biotinylated CPMV particles and streptavidin molecules. Array formation was followed in real time by a quartz crystal microbalance with dissipation monitoring. Our data provide indications that multiple interactions between biotin and streptavidin not only promote the assembly of a multilayered structure but also generate cross-links within each layer of CPMV particles. The degree of intralayer and interlayer cross-linking and hence the mechanical properties and order of the array can be modulated by the grafting density and spacer length of the biotin moieties on the CPMV particles.     

Surface specific kinetics of lipid vesicle adsorption measured with a quartz crystal microbalance.

We have measured the kinetics of adsorption of small (12.5-nm radius) unilamellar vesicles onto SiO2, oxidized gold, and a self-assembled monolayer of methyl-terminated thiols, using a quartz crystal microbalance (QCM). Simultaneous measurements of the shift in resonant frequency and the change in energy dissipation as a function of time provide a simple way of characterizing the adsorption process. The measured parameters correspond, respectively, to adsorbed mass and to the mechanical properties of the adsorbed layer as it is formed. The adsorption kinetics are surface specific; different surfaces cause monolayer, bilayer, and intact vesicle adsorption. The formation of a lipid bilayer on SiO2 is a two-phase process in which adsorption of a layer of intact vesicles precedes the formation of the bilayer. This is, to our knowledge, the first direct evidence of intact vesicles as a precursor to bilayer formation on a planar substrate. On an oxidized gold surface, the vesicles adsorb intact. The intact adsorption of such small vesicles has not previously been demonstrated. Based on these results, we discuss the capacity of QCM measurements to provide information about the kinetics of formation and the properties of adsorbed layers.     

Real-time investigation of the muco-adhesive properties of Lactococcus lactis using a quartz crystal microbalance with dissipation monitoring

This work was devoted to probe, at the entire population level, interactions between mucins and Lactococcus lactis, using QCM-D. Real-time monitoring of adsorption on polystyrene of PGM (Pig Gastric Mucin) and subsequent adhesion of L. lactis was performed for IBB477 and MG1820 strains. Measuring simultaneously shifts in resonance frequency and dissipation on the polystyrene-coated crystal demonstrated a two-phase process for PGM adsorption. XPS analysis confirmed the presence of adsorbed mucin. The Voigt-based model was used to describe the QCM-D outputs. The predicted thickness of the PGM layer was consistent with the AFM experimental value. Adhesion of L. lactis to bare or PGM-coated polystyrene was then monitored, in combination with DAPI cell counting. Positive frequency shifts were caused by adhering bacteria. The presence of adsorbed PGM strongly reduced bacterial adhesion. However, adhesion of IBB477 to the PGM coating was greatly increased in comparison with that of MG1820. Muco-adhesion may be a highly variable and valuable phenotypic trait among L. lactis strains.     

Real-time investigation of the muco-adhesive properties of Lactococcus lactis using a quartz crystal microbalance with dissipation monitoring

This work was devoted to probe, at the entire population level, interactions between mucins and Lactococcus lactis, using QCM-D. Real-time monitoring of adsorption on polystyrene of PGM (Pig Gastric Mucin) and subsequent adhesion of L. lactis was performed for IBB477 and MG1820 strains. Measuring simultaneously shifts in resonance frequency and dissipation on the polystyrene-coated crystal demonstrated a two-phase process for PGM adsorption. XPS analysis confirmed the presence of adsorbed mucin. The Voigt-based model was used to describe the QCM-D outputs. The predicted thickness of the PGM layer was consistent with the AFM experimental value. Adhesion of L. lactis to bare or PGM-coated polystyrene was then monitored, in combination with DAPI cell counting. Positive frequency shifts were caused by adhering bacteria. The presence of adsorbed PGM strongly reduced bacterial adhesion. However, adhesion of IBB477 to the PGM coating was greatly increased in comparison with that of MG1820. Muco-adhesion may be a highly variable and valuable phenotypic trait among L. lactis strains.     

Monitoring the conformational changes of an intrinsically disordered peptide using a quartz crystal microbalance

Intrinsically disordered peptides (IDPs) have recently garnered much interest because of their role in biological processes such as molecular recognition and their ability to undergo stimulus-responsive conformational changes. The block V repeat-in-toxin motif of the Bordetella pertussis adenylate cyclase is an example of an IDP that undergoes a transition from a disordered state to an ordered beta roll conformation in the presence of calcium ions. In solution, a C-terminal capping domain is necessary for this transition to occur. To further explore the conformational behavior and folding requirements of this IDP, we have cysteine modified three previously characterized constructs, allowing for attachment to the gold surface of a quartz crystal microbalance (QCM). We demonstrate that, while immobilized, the C-terminally capped peptide exhibits similar calcium-binding properties to what have been observed in solution. In addition, immobilization on the solid surface appears to enable calcium-responsiveness in the uncapped peptides, in contrast to the behavior observed in solution. This work demonstrates the power of QCM as a tool to study the conformational changes of IDPs immobilized on surfaces and has implications for a range of potential applications where IDPs may be engineered and used including protein purification, biosensors, and other bionanotechnology applications.     

Equilibrium water contents of cellulose films determined via solvent exchange and quartz crystal microbalance with dissipation monitoring

Model cellulose surfaces have attracted increasing attention for studying interactions with cell wall matrix polymers and as substrates for enzymatic degradation studies. Quartz crystal microbalance with dissipation monitoring (QCM-D) solvent exchange studies showed that the water content of regenerated cellulose (RC) films was proportional to the film thickness (d) and was consistent with about five water molecules per anhydroglucose unit. Sulfated nanocrystalline cellulose (SNC) and desulfated nanocrystalline cellulose (DNC) films had comparable water contents and contained about five times more water than RC films. A cellulase mixture served as a probe for studies of substrate accessibility and degradation. Cellulase adsorption onto RC films was independent of d, whereas degradation times increased with d. However, adsorption onto SNC and DNC films increased with d, whereas cellulase degradation times for DNC films were independent of studied d. Enhanced access to guest molecules for SNC and DNC films revealed they are more porous than RC films.     

Human cytomegalovirus detection by a quartz crystal microbalance immunosensor

A piezoelectric affinity sensor has been developed to detect distinctive antigens of the human cytomegalovirus. Either the specific antibodies or the antigen were immobilized on the gold electrode. To develop a rapid immunoassay, various assay formats were tested in relation with the different antigen composition. First, a direct assay was carried out immobilizing the specific antibody on the crystal surface by passive adsorption. Next, Protein A, thiol/poly L-lysine mixed self-assembled monolayers were tested as methods of gold modification. A competitive format was exploited by immobilization of the antigen onto the crystal activated by SAM and poly L-lysine. This procedure yielded a preliminary calibration curve. A linear range between 2.5 and 5 μg/ml of gB epitope in solution and a detection limit of 1 μg/ml were measured.     

Real-time monitoring of the cell agglutination process with a quartz crystal microbalance

The real-time monitoring of the agglutination process of human hepatic normal cells (L-02) at the quartz crystal microbalance (QCM) gold (Au) electrode was performed. Two lectins, concanavalin A (Con A) and wheat germ agglutinin (WGA), induced the cell agglutination, resulting in the different Δf₀ and ΔR₁ responses from those caused by the normal cell attachment and growth. The cell-Con A-cell aggregates had higher affinity for the Au substrate due to the excellent adsorption ability of Con A, which was revealed by increased Δf₀ and ΔR₁ shifts and the obvious mass effect of QCM. In contrast, the lower adsorption ability of cell-WGA-cell aggregates was related to the same characteristic of WGA, presenting the decreased Δf₀ and ΔR₁ responses and the time-extended adhesion phase. Parallel microscopic observation experiments were also carried out and exhibited comparable results. The Δf₀ responses during the processes of cell growth and cell agglutination were analyzed using the equations Δf₀=a₀+a₁e^-t/τ₁+a₂e^-t/τ₂+a₃e^-t/τ₃ and Δf₀=a₀+a₁e^-t/τ₁+a₂e^-t/τ₂, respectively. Furthermore, the current work proved that the QCM measurement technique based on cell agglutination was useful for discriminating hepatic normal cells (L-02) and hepatic cancer cells (Bel7402).     

Application of peptide probe for evaluating affinity properties of proteins using quartz crystal microbalance

This study proved a possibility of a peptide probe for evaluating affinity properties of proteins. We have designed and synthesized three different peptide probes, H-Ala3-(Gly-Pro5)3-Gly-OH (peptide A), H-Ala3-(Gly-Pro5)-Gly-OH (peptide B) and H-Ala3-Gly-OH (peptide C) for testing their affinities to profilin. Each peptide probe was immobilized on a quartz crystal microbalance (QCM) sensor. The QCM sensor with the peptide A showed a 93 Hz decrease of resonant frequency which indicated profilin bound to the QCM sensor in a single layer. In a successive reaction with actin, the QCM analysis resulted in a 123 Hz decrease of resonant frequency which showed actin bound to the QCM sensor. A fluorescence microscope image of the sensor surface exhibited clear fluorescence after binding a rhodamine labeled actin on the sensor surface. These results supported stepwise reactions of profilin binding to the peptide A and actin binding to profilin. In the three peptide probes, the peptide A showed the highest affinity to profilin, i.e., sequence dependent affinity was confirmed.     

A comparative study of the cytoskeleton binding drugs nocodazole and taxol with a mammalian cell quartz crystal microbalance biosensor: different dynamic responses and energy dissipation effects

The quartz crystal microbalance (QCM) was used to create piezoelectric whole-cell biosensors utilizing either living endothelial cells (ECs) or the metastatic human mammary cancer cell line MDA-MB-231 adhering to the gold QCM surface under in vitro growth conditions. We utilized the whole-cell QCM biosensors for the detection of the effects of varying concentrations of the microtubule binding drugs taxol and nocodazole by measuring changes in the QCM steady state frequency (Deltaf) and motional resistance (DeltaR), shift values. Using 0.11-50 microM nocodazole, we observed the Deltaf shift values of the biosensors, consisting of 20,000 ECs, to decrease significantly in magnitude (nearly 100%) to a limiting value, in a dose-dependent fashion, over a 5- to 6-h incubation period following drug addition. This effect is consistent with nocodazole's known disruption of intracellular microtubules. On the other hand, 10 microM taxol caused little alteration in Deltaf over the same time period, consistent with its microtubule hyperstabilization effect. When the EC QCM biosensor Deltaf shift values were normalized by the number of ECs found firmly attached to the QCM surface via trypsin removal and electronic counting, the dose curve was shifted to lower nocodazole concentrations, resulting in a more sensitive drug biosensor. The kinetics of the Deltaf decrease with increasing nocodazole concentrations measured by the EC QCM biosensor was found to be similar at all drug concentrations and was well fit by a single first-order exponential decay equation. For all nocodazole doses, t(0.5) was invariant, averaging t(0.5)=0.83+/-0.14 h. These data demonstrate that a single dynamic sensing system within the cell, the microtubules, is disrupted by the addition of nocodazole and this process is sensed by the cell QCM biosensor. This interpretation of the data was confirmed by a fluorescence light microscopy investigation of ECs undergoing treatment with increasing nocodazole doses using a fluorescent antibody to alpha-tubulin. These studies revealed a corresponding loss of the spread morphology of the cells, concomitant with a rearrangement of the extended native microtubules into increasingly large aggregates with the cells eventually lifting from the surface in significant numbers at 50 microM. At 6 microM nocodazole, partial reversibility of the EC QCM biosensor was demonstrated. These results indicate that the EC QCM biosensor can be used to detect and study EC cytoskeleton alterations and dynamics. We suggest the potential of this cellular biosensor for the real-time identification or screening of all classes of biologically active drugs or biological macromolecules that affect cellular attachment and cellular spreading, regardless of their molecular mechanism of action.     

The quartz crystal microbalance as a novel means to study cell-substrate interactions In situ

The quartz crystal microbalance (QCM) was first introduced as a mass sensor in gas phase and in vacuum. Since oscillator circuits capable of exciting shear vibrations of quartz resonators under liquid loading have been developed, the QCM became accepted as a new, powerful technique to follow adsorption processes at solid-liquid interfaces in chemical and biological research. Lately, the QCM technique has attracted considerable interest as a novel means to monitor cell-substrate interactions of mammalian cells in vitro. Because the establishment and modulation of cell-substrate contacts is important for many physiological processes, and potent techniques to measure these interactions noninvasively are rare, the present review highlights applications of the QCM technique in this field. The suitability of the QCM device to monitor attachment and spreading of mammalian cells in real time has been well established. The QCM response is dependent on the individual cell type that is examined. In order to identify the sources for these cell-type-specific results of QCM readings, and to understand the information content of the signal, attempts have been made to decompose the overall QCM response into subcellular contributions. The aforementioned subjects, together with a condensed introduction into the QCM technology, are included in this article.     

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.     

Quantitative measurements of vancomycin binding to self-assembled peptide monolayers on chips by quartz crystal microbalance

This paper describes direct binding of a small vancomycin to peptide ligands immobilized on a sensor chip using quartz crystal microbalance. In this study, the binding ligands were composed of three components: a molecular recognition element (peptide), a conformationally flexible and hydrophilic linker, and a long-chain alkanethiol. These peptide ligands were used to establish the well-packed, self-assembled monolayers on quartz chips and could be readily synthesized using conventional organic chemistry protocols. Results of quartz crystal microbalance measurements showed that vancomycin specifically associated with the d-Ala-d-Ala-containing peptide with an affinity of 3.2+/-0.3 microM and was, as expected, completely inactive to the self-assembled monolayer presenting l-Ala-l-Ala peptide. The dissociation constant obtained correlated well with values reported in literature and was further confirmed by surface plasmon resonance measurement (2.7+/-0.7 microM). The technique used in this study should be applicable to both peptidyl and nonpeptidyl ligands of greater complexity than that used here. This method is practical, it provides quantitative binding information, and complicated analysis is avoided.     

A survey of the 2010 quartz crystal microbalance literature

In 2010 there has again been an increase in the number of papers published involving piezoelectric acoustic sensors, or quartz crystal microbalances (QCM), when compared to the last period reviewed 2006‐2009. The average number of QCM publications per annum was 124 in the period 2001‐2005, 223 in the period 2006‐9, and 273 in 2010. There are trends towards increasing use of QCM in the study of protein adsorption to surfaces (93% increase), homeostasis (67% increase), protein‐protein interactions (40% increase), and carbohydrates (43% increase). New commercial systems have been released that are driving the uptake of the technology for characterisation of binding specificities, affinities, kinetics and conformational changes associated with a molecular recognition event. This article highlights theoretical and practical aspects of the principals that underpin acoustic analysis, then reviews exemplary papers in key application areas involving small molecular weight ligands, carbohydrates, proteins, nucleic acids, viruses, bacteria, cells, and membrane interfaces. Copyright © 2012 John Wiley & Sons, Ltd.     

Chiral discrimination using a quartz crystal microbalance and comparison with gas chromatographic retention data

Quartz crystal microbalances (QCMB) have been constructed using 10 MHz AT cut quartz crystals coated with heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin, heptakis(6-O-methyl-2,3-di-O-pentyl)-β-cyclodextrin, and octakis(6-O-methyl-2,3-di-O-pentyl)-γ-cyclodextrin as 50% and 20% (w/w) solutions in OV1701. The reduction in frequency seen on exposure of each coated QCMB to pure enantiomeric forms of α- and β-pinene and cis- and trans-pinane show that statistically significant (P = 0.05, n = 7) differences are observed between the enantiomeric pairs. The apparent preferential binding shown by the QCMB for enanciomers of α- and β-pinene and cis- and trans-pinane have been compared with the elution order observed on the corresponding gas chromatographic stationary phase. The magnitude of the observed separation factor (calculated as the ratio of the OV1701 normalised frequency shift) is seen to be dependent upon the chiral stationary phase concentration. These results indicate that on-line determination of enantiomeric excess and concentration of certain monoterpenes is possible at room temperature using QCMB in conjunction with chiral gas chromatographic stationary phases. Chirality 9:225–232, 1997. © 1997 Wiley-Liss, Inc.     

Recombinant antibody fragments allow repeated measurements of C-reactive protein with a quartz crystal microbalance immunosensor

C-reactive protein (CRP) is a serum marker highly upregulated in inflammation after bacterial infection. Robust, reliable and quick quantification of CRP would be a substitute for erythrocyte sedimentation rate (ESR) with superior diagnostic value. Quartz crystal microbalance (QCM) based sensors coated with specific antibodies and integrated into lab-on-chip systems are in development for rapid point of care quantification. In this study, we isolated three CRP specific single chain (sc)Fv antibody fragments using phage display from an antibody gene library. Their affinities ranged from 2.7 × 10^?8 to 1.0 × 10^?8 M when measured by surface plasmon resonance. ScFv antibody fragment LA13-IIE3 showed best affinity, high long-term stability and remarkable resistance to denaturation. This scFv antibody fragment was coupled to a QCM sensor. CRP quantification in up to 15 samples sequentially measured on the same sensor with intermitting regeneration by buffer was demonstrated.     

Kinetic characterization of small DNA-binding molecules interacting with a DNA strand on a quartz crystal microbalance

Quantitative studies of the binding of various DNA-binding antibiotics with dsDNA are useful for drug design, not only for effective antibiotics, but also for antitumor drugs. We studied the binding kinetics, association and dissociation rate constants, and association constants (k_on, k_off, and K_a, respectively) of intercalators and groove binders, including various antibiotics, to double-stranded DNA (dA₃₀·dT₃₀ and dG₃₀·dC₃₀) immobilized on a highly sensitive 27 MHz quartz-crystal microbalance (QCM) in aqueous solution. Although a simple ethidium bromide intercalator bound to both dA₃₀·dT₃₀ and dG₃₀·dC₃₀, antibiotics that are side-binding intercalators, such as daunomycin, aclacinomycin A, and actinomycin D, with sugar or peptide moieties on the intercalator parts selectively bound to dG₃₀·dC₃₀ with high K_a and small k_off values. Nogalamycin, a dumbbell-shaped penetrating intercalator, showed low k_on and k_off values owing to slow duplex unwinding during the penetration process. Groove binders (Hoechst 33258, distamycin A, and mithramycin) had high K_a values owing to the high k_on values. Kinetic parameters depended largely on molecular shapes and DNA-binding molecule binding modes.     

Use of a quartz crystal microbalance to investigate the antiadhesive potential of N-acetyl-L-cysteine

The reduction of bacterial biofilm formation on stainless steel surfaces by N-acetyl-L-cysteine (NAC) is attributed to effects on bacterial growth and polysaccharide production, as well as an increase in the wettability of steel surfaces. In this report, we show that NAC-coated stainless steel and polystyrene surfaces affect both the initial adhesion of Bacillus cereus and Bacillus subtilis and the viscoelastic properties of the interaction between the adhered bacteria and the surface. A quartz crystal microbalance with dissipation was shown to be a powerful and sensitive technique for investigating changes in the applied NAC coating for initial cell surface interactions of bacteria. The kinetics of frequency and dissipation shifts were dependent on the bacteria, the life cycle stage of the bacteria, and the surface. We found that exponentially grown cells gave rise to a positive frequency shift as long as their cell surface hydrophobicity was zero. Furthermore, when the characteristics of binding between the cell and the surface for different growth phases were compared, the rigidity increased from exponentially grown cells to starved cells. There was a trend in which an increase in the viscoelastic properties of the interaction, caused by the NAC coating on stainless steel, resulted in a reduction in irreversibly adhered cells. Interestingly, for B. cereus that adhered to polystyrene, the viscoelastic properties decreased, while there was a reduction in adhered cells, regardless of the life cycle stage. Altogether, NAC coating on surfaces was often effective and could both decrease the initial adhesion and increase the detachment of adhered cells and spores. The most effective reduction was found for B. cereus spores, for which the decrease was caused by a combination of these two parameters.     

Study of real-time lectin-carbohydrate interactions on the surface of a quartz crystal microbalance

A quartz crystal microbalance (QCM) biosensor system for lectin-carbohydrate interactions has been developed. Yeast mannan was immobilised on polystyrene-coated quartz crystals, and interactions tested with the lectin concanavalin A (Con A). The biosensor could be easily operated, where mannan immobilisation and all binding analyses were performed in real-time using a flow-through system. The apparent binding constant for yeast mannan to Con A was estimated to be 0.4 microM, well in accordance to reported literature values. In addition, the effective concentration values (EC50-values) for a series of mannose/mannoside ligands, acting as competitors to the mannan/Con A interaction, were determined to range from 0.18 to 5.3 mM, in good correlation with a related enzyme-labelled lectin assay (ELLA) protocol.