QCM露点传感器水粘性影响的抑制方法研究

为了解决基于主控温式的石英晶体微天平（Quartz Crystal Microbalance, QCM）露点测量系统中冷凝水粘弹特性影响露点识别准确性的问题,对QCM电极进行疏水处理,改善凝结特性,减小水粘性引起的频率耗散,实现液态水质量变化引起的谐振频率偏移测量。在QCM电极上制备静态水接触角为133° ± 2°的疏水层并对其进行表征,将疏水电极与未经处理的电极用于露点识别实验,并与精密露点仪获得的标准露点进行比对。实验证明,通过疏水处理电极凝结面的方法能够有效提升QCM露点传感器的露点识别精度,为主控温式露点传感器结构的优化设计提供理论和实验依据。     

Transient kinetic studies of pH-dependent hydrolyses by exo-type carboxypeptidase P on a 27-MHz quartz crystal microbalance

pH-Dependent kinetic parameters (k(on), k(off), and k(cat)) of protein (myoglobin) hydrolyses catalyzed by exo-enzyme (carboxypeptidase P, CPP) were obtained by using a protein-immobilized quartz crystal microbalance (QCM) in acidic aqueous solutions. The formation of the enzyme-substrate (ES) complex (k(on)), the decay of the ES complex (k(off)), and the formation of the product (k(cat)) could be analyzed by transient kinetics as mass changes on the QCM plate. The Kd (k(off)/k(on)) value was different from the Michaelis constant Km calculated from (k(off) + k(cat))/k(on) due to k(cat) > k(off). The rate-determining step was the binding step (k(on), and the catalytic rate k(cat) was faster than other k(on) and k(off) values. In the range of pH 2.5-5.0, values of k(on) gradually increased with decreasing pH showing a maximum at pH 3.7, values of k(off) were independent of pH, and k(cat) increased gradually with decreasing pH. As a result, the apparent rate constant (k(cat)/Km) showed a maximum at pH 3.7 and gradually increased with decreasing pH. The optimum pH at 3.7 of k(on) is explained by the optimum binding ability of CPP to the COOH terminus of the substrate with hydrogen bonds. The increase of k(cat) at the lower pH correlated with the decrease of alpha-helix contents of the myoglobin substrate on the QCM.     

Molecular-imprinted, polymer-coated quartz crystal microbalances for the detection of terpenes

A piezoelectric sensor coated with an artificial biomimetic recognition element has been developed for the determination of L-menthol in the liquid phase. A highly specific noncovalently imprinted polymer (MIP) was cast in situ on to the surface of a gold-coated quartz crystal microbalance (QCM) electrode as a thin permeable film. Selective rebinding of the target analyte was observed as a frequency shift quantified by piezoelectric microgravimetry with the QCM. The detectability of L-menthol was 200 ppb with a response range of 0-1.0 ppm. The response of the MIP-QCM to a range of monoterpenes was investigated with the sensor binding menthol in favor of analogous compounds. The sensor was able to distinguish between the D- and L-enantiomers of menthol owing to the enantioselectivity of the imprinted sites. To our knowledge, this is the first report describing enantiomeric resolution within an MIP utilizing a single monomer-functional moiety interaction. It is envisaged that this technique could be employed to determine the concentration of terpenes in the atmosphere.     

Immobilization of a human epidermal growth factor receptor 2 mimotope-derived synthetic peptide on Au and its potential application for detection of herceptin in human serum by quartz crystal microbalance

Therapeutic antibodies are antigenically similar to human antibodies and are difficult to detect in assays of human serum samples without the use of the therapeutic antibody's complementary antigen. Herein for the first time, we established a platform to detect Herceptin in solutions by using a small (<2.2 kDa), inexpensive, highly stable human epidermal growth factor receptor (HER2) mimotope-derived synthetic peptide immobilized on the surface of a Au quartz electrode. We used the HER2 mimotope as a substitute for the HER2 receptor protein in piezoimmunosensor or quartz crystal microbalance (QCM) assays to detect Herceptin in human serum. We demonstrated that assay sensitivity was dependent upon the amino acids used to tether and link the peptide to the sensor surface and the buffers used to carry out the assays. The detection limit of the piezoimmunosensor assay was 0.038 nM with a linear operating range of 0.038-0.859 nM. Little nonspecific binding to other therapeutic antibodies (Avastin and Rituxan) was observed. Levels of Herceptin in serum samples obtained from treated patients, as ascertained using the synthetic peptide-based QCM assay, were typical for those treated with Herceptin. The findings of this study are significant in that low-cost synthetic peptides could be used in a QCM assay, in lieu of native or recombinant antigens or capture antibodies, to rapidly detect a therapeutic antibody in human serum. The results suggested that a synthetic peptide bearing a particular functional sequence could be applied for developing a new generation of affinity-based immunosensors to detect a broad range of clinical biomarkers.     

Ligand exchange based paraoxon imprınted QCM sensor

In the present work, a paraoxon imprinted QCM sensor has been developed for the determination of paraoxon based on the modification of paraoxon imprinted film onto a quartz crystal combining the advantages of high selectivity of the piezoelectric microgravimetry using MIP film technique and high sensitivity of QCM detection. The paraoxon selective memories have formed on QCM electrode surface by using a new metal–chelate interaction based on pre-organized monomer and the paraoxon recognition activity of these molecular memories was investigated. Molecular imprinted polymer (MIP) film for the detection of paraoxon was developed and the analytical performance of paraoxon imprinted sensor was studied. The molecular imprinted polymer were characterized by FTIR measurements. Paraoxon imprinted sensor was characterized with AFM and ellipsometer. The study also includes the measurement of binding interaction of paraoxon imprinted quartz crystal microbalance (QCM) sensor, selectivity experiments and analytical performance of QCM electrode. The detection limit and the affinity constant (K_affinity) were found to be 0.06 μM and 2.25 × 10⁴ M^? 1 for paraoxon [MAAP–Cu(II)–paraoxon] based thin film, respectively. Also, it has been observed that the selectivity of the prepared paraoxon imprinted sensor is high compared to a similar chemical structure which is parathion.     

Effects of flow rate on sensitivity and affinity in flow injection biosensor systems studied by 55-MHz wireless quartz crystal microbalance

In this paper, we developed a 55-MHz wireless-electrodeless quartz crystal microbalance (QCM) and systematically studied the effects of flow rate on the sensitivity to the detection of proteins and on the affinity between biomolecules evaluated by the flow injection system. Brownian motion of proteins in liquid suggests a low probability of meeting, and the convection effect plays an important role in the sensitivity and the affinity in the flow cell injection system. The wireless quartz crystal was isolated in the QCM cell, and flow rates between 50 and 1000 microL/min were used for monitoring binding reactions between human immunoglobulin G and Staphylococcus aureus protein A. The sensitivity was significantly increased as the flow rate increased, while the affinity value remained unchanged. However, the affinity value was affected by the reaction time for a large-concentration analyte, indicating the need of a high-sensitivity biosensor system for accurate evaluation of affinity. The electrode effect on the QCM sensitivity was also theoretically investigated, showing that the electrode significantly deteriorates the QCM sensitivity and makes the Sauerbrey equation invalid.     

A 0-phase circuit for QCM-based measurements in highly viscous liquid environments

Currently, the series resonant frequency f/sub s/ and the motional resistance Rm of liquid loaded quartz crystal microbalance (QCM) sensors are extracted either directly, through network analyzer (NWA) impedance measurements, or from QCM-stabilized oscillator circuits. Both methods have serious drawbacks that may affect measurement accuracy, especially if the sensor is operated under highly viscous load conditions and Rm exceeds 1 k/spl Omega/. This paper presents a simple passive low-loss impedance transformation LC network which greatly reduces additional electrical loading of the QCM by the measurement system or sensor electronics and maintains a symmetric resonance and a steep 0-phase crossing at f/sub s/, even if Rm increases by several orders of magnitude as a result of liquid loading. A simple S21 transmission measurement allows direct f/sub s/ reading at the 0-phase frequency, while Rm is obtained from the circuit loss at f/sub s/. Circuit operation was verified at 9 MHz by QCM measurements in a liquid with known density and viscosity. The agreement between predicted and experimental data, which was obtained by a temperature-controlled measurement, was within 1%, even in very high viscosity ranges in which Rm exceeds 10 k/spl Omega/.     

A sensor of alcohol vapours based on thin polyaniline base film and quartz crystal microbalance

Thin films of polyaniline base, emeraldine base (EB), coating on the quartz crystal microbalance (QCM) electrode were used as a sensitive layer for the detection of a number of primary aliphatic alcohols such as ethanol, methanol, 2-propanol and 1-propanol vapours. The frequency shifts (Δf) of the QCM were increased due to the vapour adsorption into the EB film. Δf were found to be linearly correlated with the concentrations of alcohols vapour in part per million (ppm). The sensitivity of the sensor was found to be governed by the chemical structure of the alcohol. The sensor shows a good reproducibility and reversibility. The diffusions of different alcohols vapour were studied and the diffusion coefficients (D) were calculated. It is concluded that the diffusion of the vapours into the EB film follows Fickian kinetics.     

Selection of ligands for affinity chromatography using quartz crystal biosensor

This paper described a new strategy for rapid selecting ligands for application in affinity chromatography using a quartz crystal microbalance (QCM) biosensor. An aminoglycoside antibiotic drug, kanamycin (KM), was immobilized on the gold electrodes of the QCM sensor chip. The binding interactions of the immobilized KM with various proteins in solution were monitored as the variations of the resonant frequency of the modified sensor. Such a rapid screen analysis of interactions indicated clearly that KM-immobilized sensor showed strong specific interaction only with lysozyme (LZM). The resultant sensorgrams were rapidly analyzed by using a kinetic analysis software based on a genetic algorithm to derive both the kinetic rate constants (k(ass) and k(diss)) and equilibrium dissociation constants (K(D)) for LZM-KM interactions. The immobilized KM showed higher affinity to LZM with a dissociation constant on the order of 10(-5) M, which is within the range of 10(-4)-10(-8) M and suitable for an affinity ligand. Therefore, KM was demonstrated for the first time as a novel affinity ligand for purification of LZM and immobilized onto the epoxy-activated silica in the presence of a high potassium phosphate concentration. The KM immobilized affinity column has proved useful for a very convenient purification of LZM from chicken egg white. The purity of LZM obtained was higher than 90%, as determined by densitometric scanning of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified fraction. These results confirmed that the selected KM ligand is indeed a valuable affinity ligand for purification of LZM. The new screening strategy based on a QCM biosensor is expected to be a promising way for rapid selecting specific ligands for purifying other valuable proteins.     

Aptameric peptide for one-step detection of protein kinase

Protein kinases are significant regulators in the cell signal pathway, and it is difficult to achieve quick kinase detection because traditional kinase assays normally rely on a time-consuming kinase phosphorylation process. Herein, we present a novel one-step strategy to detect protein kinase by using a kinase-specific aptameric peptide-functionalized quartz crystal microbalance (QCM) electrode, in which the detection can be finished in less than 10 min. A peptide kinase inhibitor (IP(20)) was used as the aptameric peptide because of its selective and strong interaction with the target protein kinase (cyclic adenosine monophosphate-dependent protein kinase A, PKA), high stability, and ease of inexpensive synthesis, presenting a new direct recognition element for kinase. The aptameric peptide was immobilized on the Au-coated quartz electrode through dual-thiol anchoring and the binding of His-tagged peptide with a nitrilotriacetic acid/Ni(II) complex, fabricating a highly specific and stable detection platform. The interaction of aptameric peptide with kinase was monitored with the QCM in real time, and the concentration of protein kinase was sensitively measured by the frequency response of the QCM with the low detection limit for PKA at 0.061 mU μL(-1) and a linear range from 0.64 to 22.33 mU μL(-1). This method is rapid and reagentless and does not require a phosphorylation process. The versatility of our aptameric peptide-based strategy has also been demonstrated by the application in kinase assay using electrochemical impedance spectroscopy. Moreover, this method was successfully applied to detect the forskolin/3-isobutyl-1-methylxanthine-stimulated activation of PKA in cell lysate.     

Detection of point mutation and insertion mutations in DNA using a quartz crystal microbalance and MutS, a mismatch binding protein

MutS protein is a mismatch binding protein that recognizes mispaired and unpaired base(s) in DNA. In this study, we incorporate the MutS protein-based mutation recognition into quartz crystal microbalance (QCM) measurements for DNA single-base substitution mutation and 1-4 base(s) insertion (or deletion) mutation detection. The method involves the immobilization of single-stranded probe DNA on a QCM surface, the hybridization of target DNA to form homoduplex or heteroduplex DNA, and finally the application of MutS protein for the mutation recognition. By measuring the MutS binding signal, DNA containing a T:G mismatch or unpaired base(s) is(are) discriminated against perfectly matched DNA at target concentrations ranging from 1nM to 5 microM. Furthermore, the QCM damping behavior upon MutS-DNA complex formation is studied using a Network Analyzer. The measured motional resistance changes per coupled MutS unit mass (deltaR/deltaf) are found to be indicative of the viscoelastic or structural properties of the bound protein, corresponding to different binding mechanisms. In addition, the deltaR/deltaf values vary remarkably when the MutS protein binds at different distances away from the QCM surface. Thus, these values can be used as a "fingerprint" for MutS mismatch recognition and also used to quantitatively locate the mutation site.     

Paraoxon imprinted biopolymer based QCM sensor

In this study, a novel quartz crystal microbalance (QCM) based on the modification of paraoxon imprinted polymer (TCM-Cd(II)-paraoxon) film onto a quartz crystal sensor has been developed for the determination of paraoxon. The sensor is based on a molecular imprinted polymer (MIP) which can be synthesized using paraoxon as a template molecule, Thiourea Modified Chitosan-Cd(II) (TCM-Cd(II)) as the metal-chelate monomer, ephychlorohydrin as a crosslinking agent. The MIP particles have been characterized by FTIR measurements and QCM sensor has characterized using AFM and ellipsometer. The performance of the paraoxon imprinted sensor has indicated that a selective and sensitive paraoxon imprinted sensor could be fabricated. The sensor is able to discriminate paraoxon in solution owing to the specific binding of the imprinted sites. The obtained paraoxon imprinted sensor has 0.02–1 μM linear range and low detection limit (0.02 μM). The selectivity studies have shown that the selectivity of prepared paraoxon imprinted sensor has found as being very high in the presence of parathion which is similar in structure with paraoxon. The paraoxon imprinted sensor has been repeatedly used for more than 7 months in many continuous experiments.     

Frequency interference between two quartz crystal microbalances

Multichannel quartz crystal microbalances (MQCM) are very attractive for biosensor applications because these devices can be miniaturized using microelectromechanical systems micromachining technology, leading to substantial reductions in cost, diagnostic time, and sample volumes. However, the miniaturization also brings about frequency interference between adjacent microbalances and the measuring accuracy will thus be affected by the extent of frequency coupling. In this paper, a theoretical analysis of the interference between two QCM on a monolithic quartz crystal is performed. It is found that both the separation between the QCM and the ratio of the electrode widths can significantly affect the frequency interference and mass sensitivity. The frequency interference is particularly strong when the two QCM have the same width or the ratio of the electrode widths is near specific values related to excited overtones. The sensitivity to mass loading decreases significantly when the geometric parameters fall within such strong interference regions. Therefore, the size and separation of the electrode structures must be carefully chosen in the design process for small quartz devices.     

Monitoring cell adhesion by piezoresonators: impact of increasing oscillation amplitudes

In recent years, the quartz crystal microbalance (QCM) has been established as a sensitive analytical tool to monitor the attachment and spreading of mammalian cells to in vitro surfaces. Due to its superior time resolution, the device is capable of reading even subtle differences in cell adhesion kinetics. However, thickness shear mode piezoresonators, which are the core component of the QCM approach, can be used not only as a sensor but also as an actuator when the oscillation amplitude of the crystal is increased so that molecular recognition at the solid-liquid interface is disturbed. In this study, we have addressed the impact of elevated lateral oscillation amplitudes on the adhesion kinetics of three mammalian cell lines. We used AT-cut piezoresonators with a fundamental resonance frequency of 5 MHz, and the analytical readout was performed by impedance analysis. Formation of stable cell-substrate contacts is retarded or entirely blocked when the lateral oscillation amplitude (in the center of the resonator) exceeds values higher than 20 nm. Shear oscillations of similar amplitude were, however, not sufficient to displace attached cells from the surface. Moreover, the experimental data prove that the normal QCM readout with oscillation amplitudes smaller than 1 nm is, indeed, non-invasive with respect to mammalian cells.     

Small mass-change detectable quartz crystal microbalance and its application to enzymatic one-base elongation on DNA

A highly sensitive 27 MHz quartz crystal microbalance instrument with an automatic flow injection system was developed to obtain realistic minimal frequency noise (±0.05 Hz) and to obtain a stable signal baseline (±1 Hz/h) by controlling the temperature of each part in the quartz crystal microbalance (QCM) system using three Peltier devices with a resolution of ±0.001 °C and by optimizing the flow system to prevent fluctuation of the internal pressure of the QCM. The improved QCM with an automatic flow injection system enabled detection of small mass changes such as binding of biotin to a streptavidin-immobilized QCM with a high signal-to-noise ratio. We also applied this device to enzyme reactions of one-base elongation by DNA polymerase (Klenow fragment, KF). We immobilized dsDNAs including the protruding end of dA, dG, dT, or dC on the QCM electrode and ran complementary dNTP monomers with KF into the QCM flow cell. We could directly detect the enzymatic one-base elongation of DNA as a small mass increase, and we found the difference in the reaction rate for each monomer.     

Quartz crystal microbalance detection of glutathione-protected nanoclusters using antibody recognition

A quartz crystal microbalance (QCM) immunosensor was developed for the quantitative detection of glutathione-protected nanoclusters. Advantages intrinsic to QCM were employed to make it an attractive alternative to other immunosensing techniques. We have addressed challenges in the area of QCM mass sensing through experimental correlation between damping resistance and frequency change for a reliable mass measurement. Electrode functionalization was optimized with the use of protein A to immobilize and present polyclonal IgG for antigen binding. This method was developed for the detection of glutathione (antigen)-protected clusters of nanometer size with high surface area and thiolate valency. Quantitation of glutathione-nanocluster binding to immobilized polyclonal antibody provides equilibrium constants (K(a) = (3.6 +/- 0.2) x 10(5) M(-1)) and kinetic rate constants (k(f) = (5.4 +/- 0.7) x 10(1) M(-1) s(-1) and k(r) = (1.5 +/- 0.4) x10(-4) s(-1)) comparable to literature reports. These observations further imply that immunoreactive nanoparticles have potential in medical diagnostics and materials assembly.     

Selective protein separation using siliceous materials with a trimethoxysilane-containing glycopolymer

A copolymer with α-D-mannose (Man) and trimethoxysilane (TMS) units was synthesized for immobilization on siliceous matrices such as a sensor cell and membrane. Immobilization of the trimethoxysilane-containing copolymer on the matrices was readily performed by incubation at high heat. The recognition of lectin by poly(Man-r-TMS) was evaluated by measurement with a quartz crystal microbalance (QCM) and adsorption on an affinity membrane, QCM results showed that the mannose-binding protein, concanavalin A, was specifically bound on a poly(Man-r-TMS)-immobilized cell with a higher binding constant than bovine serum albumin. The amount of concanavalin A adsorbed during permeation through a poly(Man-r-TMS)-immobilized membrane was higher than that through an unmodified membrane. Moreover, the concanavalin A adsorbed onto the poly(Man-r-TMS)-immobilized membrane was recoverable by permeation of a mannose derivative at high concentration.     

A measurement system for odor classification based on the dynamic response of QCM sensors

In this paper, an innovative measurement system for odor classification, based on quartz crystal microbalances (QCMs), is presented. The application proposed in this paper is the detection of typical wine aroma compounds in mixtures containing ethanol. In QCM sensors, the sensitive layer is, e.g., a polymeric layer deposited on a quartz surface. Chemical mixtures are sorbed in the sensitive layer, inducing a change in the polymer mass and, therefore, in the quartz resonance frequency. In this paper, the frequency shift is measured by a dedicated, fully digital front-end hardware implementing a technique that allows reducing the measurement time while maintaining a high-frequency resolution . The developed system allows, therefore, measuring variations of the QCM resonance frequency shifts during chemical transients obtained with abrupt changes in odor concentration. Hence, the reaction kinetics can be exploited to enhance the sensor selectivity. In this paper, some measurements obtained with an array of four sensors with different polymeric sensitive layers are presented. An exponential fitting of the transient responses is used for feature extraction. Finally, to reduce data dimensionality, principal component analysis is used.     

A quartz crystal microbalance-based sensor system coated with functional polymers for SO2 and NO2 detection

In this work, a quartz crystal microbalance (QCM)-based adsorption sensor system with high sensitivity, selectivity, and reproducibility is designed and fabricated. The functional polymers such as polypyrrole, poly(3,4-ethylenedioxythiophene) (PEDOT), and polystyrene are coated on 8 MHz AT-cut quartz crystal surfaces as sensing materials for SO2 and NO2. All sensing materials on the QCM surface are characterized experimentally by SEM and AFM. The frequency shifts of the QCM by adsorption and desorption of gases are measured and analyzed to assess the practical applicability of the sensor system. The overall results show that the QCM coated with polypyrrole is highly selective for SO2 gas and that coated with PEDOT is for NO2. It is proven that the QCM-based adsorption sensor system is possible for monitoring SO2 and NO2 gases in the mixture of ppm level.     

Quartz crystal microbalance with integrated surface plasmon grating coupler

We have integrated a surface plasmon grating coupler into a quartz crystal microbalance (QCM) for studying surface association/dissociation reactions. In the integrated system only QCM measurement is needed to record both the optical and the acoustic signals in the same association/dissociation reaction. This integration considerably simplifies a conventional combination instrument of a grating-coupled surface plasmon resonance (SPR) spectrometer and a quartz crystal microbalance by eliminating a number of SPR components. Moreover, in the integrated system detection of the light reflections is not needed by which one bypasses the interference problem caused by two coherent light reflections off the glass window used to seal the fluid sample and off the sensor surface. The utility of the integrated system is demonstrated using a layer-by-layer polyelectrolyte multilayer deposition protocol, in which the complete features of a conventional grating-coupled SPR/QCM combination instrument are retained, including detection of optical and acoustic changes, as well as monitoring of adsorption kinetics.