Interactions of endoglucanases with amorphous cellulose films resolved by neutron reflectometry and quartz crystal microbalance with dissipation monitoring

A study of the interaction of four endoglucanases with amorphous cellulose films by neutron reflectometry (NR) and quartz crystal microbalance with dissipation monitoring (QCM-D) is reported. The endoglucanases include a mesophilic fungal endoglucanase (Cel45A from H. insolens), a processive endoglucanase from a marine bacterium (Cel5H from S. degradans ), and two from thermophilic bacteria (Cel9A from A. acidocaldarius and Cel5A from T. maritima ). The use of amorphous cellulose is motivated by the promise of ionic liquid pretreatment as a second generation technology that disrupts the native crystalline structure of cellulose. The endoglucanases displayed highly diverse behavior. Cel45A and Cel5H, which possess carbohydrate-binding modules (CBMs), penetrated and digested within the bulk of the films to a far greater extent than Cel9A and Cel5A, which lack CBMs. While both Cel45A and Cel5H were active within the bulk of the films, striking differences were observed. With Cel45A, substantial film expansion and interfacial broadening were observed, whereas for Cel5H the film thickness decreased with little interfacial broadening. These results are consistent with Cel45A digesting within the interior of cellulose chains as a classic endoglucanase, and Cel5H digesting predominantly at chain ends consistent with its designation as a processive endoglucanase.     

Immunodetection of inactivated Francisella tularensis bacteria by using a quartz crystal microbalance with dissipation monitoring

Francisella tularensis are very small, gram-negative bacteria which are capable of infecting a number of mammals. As a highly pathogenic species, it is a potential bioterrorism agent. In this work we demonstrate a fast immunological detection system for whole F. tularensis bacteria. The technique is based on a quartz crystal microbalance with dissipation monitoring (QCMD), which uses sensor chips modified by a specific antibody. This antibody is useful as a capture molecule to capture the lipopolysaccharide structure on the surface of the bacterial cell wall. The QCMD technique is combined with a microfluidic system and allows the label-free online detection of the binding of whole bacteria to the sensor surface in a wide dynamic concentration range. A detection limit of about 4 × 10(3) colony-forming units per milliliter can be obtained. Furthermore, a rather short analysis time and a clear discrimination against other bacteria can be achieved. Additionally, we demonstrate two possibilities for specific and significant signal enhancement by using antibody-functionalized gold nanoparticles or an enzymatic precipitation reaction. These additional steps can be seen as further proof of the specificity and validity.     

Acoustic quantification of ATP using a quartz crystal microbalance with dissipation

A quartz crystal microbalance with a dissipation monitoring (QCM-D) sensor was developed for highly sensitive and specific detection of adenosine-5'-triphosphate (ATP) by using an aptamer. The binding of ATP molecules on the aptamer films could be calculated as accurate mass changes using multiple frequency and dissipation measurements. The detection is achieved by calculating the mass changes from conformational rearrangements of the sensor surface upon interaction with the target. The sensor was demonstrated to respond to changes in ATP concentrations in real time suitable for continuous monitoring applications. This sensor showed excellent selectivity toward ATP compared with other chemically similar nucleotide GTP. The feasibility of the sensor was demonstrated by analyzing ATP concentrations in cell culture media with serum. The maximum frequency change was about -2 Hz after injection of 500 μM ATP. The affinity constant of the aptamer was determined to be 49 ± 7.59 μM. The proposed sensor can extend the application of the QCM-D system in medical diagnosis, and could be adopted for the detection of other small molecules with the use of specific aptamers.     

Real-time monitoring of enzymatic cleavage of nucleic acids using a quartz crystal microbalance.

The use of quartz crystal microbalance (QCM) for monitoring in situ the enzymatic cleavage of surface-confined nucleic acids by nucleases is described. Such real-time monitoring of mass changes associated with the enzymatic digestion indicates that the activity and specificity of nucleases is preserved at the gold surface, and can be used for manipulating surface-confined DNAs and RNAs. These observations indicate great promise for using QCM for elucidating the interactions of nucleic acids with enzymes, and for enhancing the power of hybridization biosensors.     

Analysis of interpenetrating polymer networks via quartz crystal microbalance with dissipation monitoring

A quartz crystal microbalance with dissipation monitoring (QCM-D) was used to assess the physical properties of interpenetrating polymer networks (IPNs) through swelling experiments in ambient humidity and in phosphate-buffered saline (PBS), pH 7.4. The IPNs, based on acrylamide (AAm) and poly(ethylene glycol) (pEG), swell from thin, rigid films when dry (16.7 +/- 5.2 nm on Si/SiO(2)) to expanded, viscoelastic films when hydrated (107 +/- 24.2 nm on Si/SiO2). The dry IPNs could be analyzed using the Sauerbrey relationship, but for the hydrated films it was necessary to interpret QCM-D data with a Kelvin-Voigt viscoelastic model. A complex modulus |G| of 116 +/- 38.1 kPa for the swollen IPN surface on Si/SiO2 was defined by the model. The QCM-D was also employed to quantify the adsorption of human fibrinogen, a protein important in thrombus formation, onto the IPNs. Fibrinogen adsorption studies demonstrated the sensitivity of the QCM-D, as well as confirmed the nonfouling nature of the IPN surface, where less than 5 ng/cm2 of fibrinogen was adsorbed.     

Quantitative analysis of tethered vesicle assemblies by quartz crystal microbalance with dissipation monitoring: Binding dynamics and bound water content

To implement the molecular recognition properties of membrane proteins for applications including biosensors and diagnostic arrays, the construction of a biomimetic platform capable of maintaining protein structure and function is required. In this paper, we describe a tethered phospholipid vesicle assembly that overcomes the major limitations of planar supported lipid bilayers and alternative biomimetic membrane platforms and characterize it using quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence microscopy. We provide evidence of a one-step mechanism for bilayer formation and monitor the subsequent adsorption and binding of streptavidin, vesicles, and streptavidin-coated microspheres. For all three species, we identify a critical surface density above which a significant amount of coupled interstitial water contributes to the response of the quartz resonator in a phenomenon similar to dynamic coupling due to surface roughness. A Sauerbrey-type analysis is sufficient to accurately interpret the QCM-D results for streptavidin binding if water is treated as an additional inertial mass, but viscoelastic models must be invoked for vesicle and microsphere binding. Additionally, we present evidence of vesicle flattening, possibly enhanced by a biotin-mediated membrane-membrane interaction.     

Quartz crystal microbalance with dissipation monitoring of the enzymatic hydrolysis of steam-treated lignocellulosic nanofibrils

Quartz crystal microbalance with dissipation (QCM-D) monitoring was performed to investigate the impact of steam treatment (ST) on the enzymatic hydrolysis of lignocellulosic nanofibrils (LCNFs). ST at mild temperatures up to 140 °C mainly affected the hemicellulose content of LCNFs. The hemicellulose constituents in the water-soluble fraction and the residual LCNF were quantified. The impact of changes in hemicellulose by ST on enzymatic hydrolysis was monitored by QCM-D using Acremonium cellulase as a source of multicomponent enzymes including hemicellulases. LCNFs without ST showed distinctive initial changes in frequency and energy dissipation, which differed from those of pure cellulose film, whereas these changes shifted toward typical changes of enzymatic hydrolysis of pure cellulosic films with increasing ST temperature. The QCM-D results suggested that hemicellulose located around cellulose microfibrils is rapidly decomposed, thus exposing the cellulose surface shortly after initial enzymatic hydrolysis, and then the main enzymatic hydrolysis of cellulose occurs.     

A practical guide to quartz crystal microbalance with dissipation monitoring of thin polymer films

Quartz crystal microbalance with dissipation (QCM-D) monitoring is a powerful tool used to sensitively examine the real-time responses of polymer films to external responses. For example, the technique is commonly used to monitor film growth, material adsorption, thin film swelling, and ion exchange. With its rapidly expanding use, this review is intended to introduce new users to the basic principles of QCM-D, along with practical challenges and remedies specific to polymer thin films. For both new and experienced users, specific case studies are highlighted including layer-by-layer assembly, electrochemical QCM-D, swelling, sensing, and biological application. Last, the review recommends future directions for research and areas of growth.     

Characterization of adsorption of humic acid onto alumina using quartz crystal microbalance with dissipation

In this paper, a quartz crystal microbalance with dissipation monitoring (QCM-D) is used to investigate humic acid (HA) adsorption onto alumina (Al(2)O(3)). The amount of adsorption and layer structures of HA were determined by the real-time monitoring of resonance frequency and energy dissipation changes (Δf and ΔD). The effect of HA concentration, HA molecular characteristics (molecular weight and polarity), and pH on HA adsorption onto Al(2)O(3) were investigated. The mass of HA adsorption increases as the concentration of HA increases. The masses are about 24, 60, and 87 ng cm(-2) as the concentration of DOC is 1.0, 4.85, and 92.0 mg L(-1), respectively. The adsorbed layer of HA is more nonrigid, and the mass of HA adsorption is higher at weakly acidic pH values. It was 20, 80, 65, and 45 ng cm(-2) at pH values of 4.5, 5.5, 6.5, and 8.0, respectively. This reveals that efficient HA removal by coagulation at weakly acidic pH values is not just due to the hydrolysis of Al ions as previously presumed. The adsorbed layer of hydrophobic HA is more nonrigid than hydrophobic HA (fractionated by Amberlite XAD-8 resin), and the mass adsorption for the hydrophobic fraction is about four times higher than the hydrophilic fraction (120 ng cm(-2) and 30 ng cm(-2)). The method is of value in the research to establish a quantified calculation model for the coagulation process.     

Enzyme immobilization on poly(ethylene-co-acrylic acid) films studied by quartz crystal microbalance with dissipation monitoring

In this study, we use the quartz crystal microbalance with dissipation monitoring (QCM-D) to study the immobilization of the enzyme horseradish peroxidase (HRP) on poly(ethylene-co-acrylic acid) (PEAA) films. The surface polarity of spin-coated PEAA films was varied by heat treatments in air or in a 30% NaOH aqueous solution leading to COOH-depleted or COOH-enriched surfaces, respectively. Two reaction schemes, direct adsorption and amine coupling, were employed for HRP immobilization on the two surfaces. The shifts in frequency and dissipation, Deltaf and DeltaD, measured by QCM-D and the ratio DeltaD/Deltaf were used to evaluate the binding amount and the conformation of the adsorbed enzyme. It is found that HRP immobilized via covalent linkages forms rigid and little dissipative films. In contrast, directly adsorbed HRP films exhibit a highly dissipative structure. HRP-catalyzed oxidation of the 4-chloro-1-naphthol in the presence of H(2)O(2) was used to characterize the catalytic activity of the HRP films. The results show that the enzymatic activity of the covalently immobilized HRP tends to be higher.     

The interaction between model biomaterial coatings and nylon microparticles as measured with a quartz crystal microbalance with dissipation monitoring

The interaction between cells and biomaterials has been mimicked using nylon microparticles as pseudo-cells and PLMA and PIBMA as biomaterial model acrylate polymers. The shift of fundamental resonance frequencies was negative for both polymers, indicating mass-coupling to the sensor surface. The shifts of the 3rd, 5th and 7th overtone frequencies were initially positive for both polymers, indicating a particle slip or wobbling on the surface. The QCM technique could discriminate between the two different polymers, showing increased interaction between microparticle and PLMA. The dissipation shift was positive for all overtones on both polymers, but again with faster and more prominent response for PLMA.     

Lectin-carbohydrate affinity measured using a quartz crystal microbalance

The association of two molecules is described by two parameters, association equilibrium and association rate constants, which are characteristic for a given type of interaction. Usually, they are determined for interacting molecules dissolved in solution. However, for many applications one type of molecules is immobilized on a substrate, which may influence the binding kinetics. The studied complex of concanavalin A and carboxypeptidase Y belongs to the lectin-carbohydrate type of interaction involving the recognition of oligosaccharide moieties. The concanavalin A was immobilized on a gold electrode of quartz crystal, while carboxypeptidase Y was added to a buffer (Tris-buffered saline). The constants describing the association of the investigated molecules were determined on the basis of measurements performed using a quartz crystal microbalance in liquid. The obtained values were (0.59+/-0.01)x10(6) M(-1) for the association equilibrium constant and (5.6+/-0.1)x10(4) M(-1)s(-1) for the association rate constant. The saturation binding experiment gave another value of the association constant, (2.7+/-0.02)x10(6) M(-1). The comparison of obtained values with previously published ones verifies that the molecule orientation and binding site accessibility for specific ligands could influence the association equilibrium constant value. The presented measurements demonstrate the ability of a quartz crystal microbalance to detect and to evaluate the association process occurring between molecules.     

Quartz crystal microbalance with dissipation monitoring and surface plasmon resonance studies of carboxymethyl cellulose adsorption onto regenerated cellulose surfaces

Adsorption of anionic polyelectrolytes, sodium salts of carboxymethyl celluloses (CMCs) with different degrees of substitution (DS = 0.9 and 1.2), from aqueous electrolyte solutions onto regenerated cellulose surfaces was studied using quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) experiments. The influence of both calcium chloride (CaCl(2)) and sodium chloride (NaCl) on CMC adsorption was examined. The QCM-D results demonstrated that CaCl(2) (divalent cation) caused significantly greater CMC adsorption onto regenerated cellulose surfaces than NaCl (monovalent cation) at the same ionic strength. The CMC layers adsorbed onto regenerated cellulose surfaces from CaCl(2) solutions exhibited greater stability upon exposure to flowing water than layers adsorbed from NaCl solutions. Both QCM-D and SPR results showed that CMC adsorption onto regenerated cellulose surfaces from CaCl(2) solutions increased with increasing CaCl(2) concentration up to the solubility limit (10 mM). Voigt-based viscoelastic modeling of the QCM-D data indicated that the CMC layers adsorbed onto regenerated cellulose surfaces had shear viscosities of η(f) ≈ 10(-3) N·s·m(-2) and elastic shear moduli of μ(f) ≈ 10(5) N·m(-2). Furthermore, the combination of SPR spectroscopy and QCM-D showed that the CMC layers contained 90-95% water. Adsorption isotherms for CMCs in CaCl(2) solutions were also obtained from QCM-D and were fit by Freundlich isotherms. This study demonstrated that CMC adsorption from CaCl(2) solutions is useful for the modification of cellulose surfaces.     

Hydrogels from a water-soluble zwitterionic polythiophene: dynamics under pH change and biomolecular interactions observed using quartz crystal microbalance with dissipation monitoring

The water-soluble zwitterionic polythiophene, poly(3-((S)-5-amino-5-carboxyl-3-oxapentyl)-2,5-thiophene) hydrochloride (POWT), is a conjugated polyelectrolyte (CPE) with properties well suited for biochip applications. CPEs readily form hydrogels when exposed to water-based buffer solutions or biomolecule solutions. In this work, we used in situ quartz crystal microbalance with dissipation (QCM-D) monitoring to collect information on the interaction between POWT films exposed to buffers with different pH and POWT/DNA chains. Our data show that POWT swells significantly when exposed to low-pH buffers, such as pH 4 acetate, this is seen as an increase in thickness and decrease in viscosity obtained via a Voight-based modeling of combined f and D QCM-D measurements. The magnitude of thickness and viscosity change upon changing from a pH 10 carbonate buffer to pH 4 acetate is 100% increase in thickness and 50% decrease in viscosity. The response of the hydrogel under pH change is well correlated with fluorescence data from POWT films on glass. The state of the hydrogel is important during interaction with biomolecules; illustrated by the observation that a swollen CPE hydrogel adsorbs a higher amount of DNA than a compacted one. In agreement with previous results, the QCM-D data confirmed that the POWT/DNA hydrogel sense complementary DNA specifically and with negligible binding of noncomplementary DNA. These results are important for efficient constructions of biochips in water environments using this class of materials.     

Buildup of polyelectrolyte multilayers of polyethyleneimine and microfibrillated cellulose studied by in situ dual-polarization interferometry and quartz crystal microbalance with dissipation

Polyethyleneimine (PEI) and Microfibrillated cellulose (MFC) have been used to buildup polyelectrolyte multilayers (PEM) on silicone oxide and silicone oxynitride surfaces at different pH values and with different electrolyte and polyelectrolyte/colloid concentrations of the components. Consecutive adsorption on these surfaces was studied by in situ dual-polarization interferometry (DPI) and quartz crystal microbalance measurements. The adsorption data obtained from both the techniques showed a steady buildup of multilayers. High pH and electrolyte concentration of the PEI solution was found to be beneficial for achieving a high adsorbed amount of PEI, and hence of MFC, during the buildup of the multilayer. On the other hand, an increase in the electrolyte concentration of the MFC dispersion was found to inhibit the adsorption of MFC onto PEI. The adsorbed amount of MFC was independent of the bulk MFC concentration in the investigated concentration range (15-250 mg/L). Atomic force microscopy measurements were used to image a MFC-treated silicone oxynitride chip from DPI measurements. The surface was found to be almost fully covered by randomly oriented microfibrils after the adsorption of only one bilayer of PEI/MFC. The surface roughness expressed as the rms-roughness over 1 microm2 was calculated to be 4.6 nm (1 bilayer). The adsorbed amount of PEI and MFC and the amount of water entrapped by the individual layers in the multilayer structures were estimated by combining results from the two analytical techniques using the de Feijter formula. These results indicate a total water content of ca. 41% in the PEM.     

In situ kinetic study of zinc sulfide activation using a quartz crystal microbalance with dissipation (QCM-D)

We have studied the activation kinetics of zinc sulfide (ZnS) using silver as an activator by a quartz crystal microbalance with dissipation (QCM-D). The zinc sulfide coating on QCM-D sensor was shown to have similar crystallographic structure, composition, and surface properties as nature sphalerite through the characterization of X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and xanthate adsorption measurement using QCM-D. The activation of ZnS sensor by silver was confirmed by the mass increase in ZnS sensor coupled with subsequent xanthate adsorption during QCM-D measurement, the change of surface wettability, and the presence of Ag(2)S on the surface. Two distinct stages on the silver uptake vs. time curve were identified and fitted well by a logarithmic function for the initial stage and a parabolic law in the later stage, which agrees with the two-stage zinc-silver reaction kinetics reported previously. Argon sputtering followed by XPS measurement on the ZnS surface demonstrated the penetration of silver into the bulk ZnS after activation. The present study is the first of its kind to apply the QCM-D technique to investigate sphalerite activation, which introduces a new in situ approach to investigate surface adsorption and activation in many mineral processes and surface modifications.     

In situ investigation of dye adsorption on TiO2 films using a quartz crystal microbalance with a dissipation technique

Dye adsorption plays a crucial role in dye-sensitized solar cells. Herein, we demonstrate an in situ liquid-phase analytical technique to quantify in real time adsorption of dye and coadsorbates on flat and mesoporous TiO(2) films. For the first time, a molar ratio of co-adsorbed Y123 and chenodeoxycholic acid has been measured.     

The quantification of potassium using a quartz crystal microbalance

N,N'-dibenzyl-4,13-diaza-18-crown-6 (A) and bis[(benzo-15-crown-5)-4'-ylmethyl] pimelate (B) were tested as coatings for two piezoelectric crystals for potassium quantification. Both sensors showed stability, reversibility and sensitivity characteristics that allowed their use in quantitative analysis. However, compound A is much more sensitive to potassium than B. Compound A also shows a larger relative sensitivity for potassium with regard to sodium than B. A pharmaceutical sample of known composition was analysed both by an acoustic wave sensor with a crystal coated with compound A and by conductivity. No statistically significant difference in the median of the results was found (alpha = 0.05), although precision is superior for the conductivity methodology. Performance of the sensor in terms of frequency stability and selectivity was improved by the incorporation of PVC, a plasticizer and a lipophilic salt in the coating composition. Limits of detection found for potassium were 1.92 ppm, or 1.75 ppm for a crystal with a frequency decrease due to coating of 2.9 kHz or 3.9 kHz, respectively. Selectivity coefficients (fixed interference 80 ppm) for potassium over Na, Ca, Al, Zn, Mg, and Fe ranged from 0.103 to 0.332.