A study of glycoprotein-lectin interactions using quartz crystal microbalance

A study of biospecific interactions between lectins and glycoproteins using a quartz crystal microbalance biosensor with dissipation monitoring (QCM-D) was reported. Four lectins were covalently immobilised on the thiol-modified gold electrode of the QCM chips in order to obtain sensing surfaces. The frequency shift served as analytical signal and the dissipation shift provided additional information about the viscoelastic properties of the glycoprotein-lectin complex formed on the surface of the QCM chip. The working conditions of the assay were optimised. The interaction between different lectins and glycoproteins was characterised by specific frequency shifts and each glycoprotein displayed its own unique lectin-binding pattern. This lectin pattern can serve as a finger print for the discrimination between various glycoproteins. The biosensor enabled quantitative determination of glycoproteins in the concentration range of 50 μg mL⁻¹ to 1 mg mL⁻¹ with good linearity and R.S.D. of less than 6.0%. An additional advantage of the proposed biosensor was the possibility to re-use the same lectin surfaces during a long period of time (2 month) without changes in analytical response. This was experimentally achieved by the application of a proper regeneration solution (10 mM glycine-HCl, pH 2.5). The lectin-based quartz crystal microbalance technique is suitable both for rapid screening and for quantitative assay of serum glycoproteins.     

A quartz crystal microbalance study on a metallic nickel electrode

Electrochemical processes taking place on a Ni electrode have been investigated with the electrochemical quartz crystal microbalance. At potentials negative of ca. –500 mV vs. SCE, a closed frequency loop is observed without irreversible changes in the mass of the electrode. The phase transition - -Ni(OH)₂, taking place at potentials positive to –500 mV vs. SCE, is accompanied by an irreversible increase in the mass of the electrode. When Ni(OH)₂ is further oxidized, the frequency increase is followed by a decrease, indicating the transport of various species in both directions, i.e. from and into the electrode. During the Ni(OH)₂ oxidation reaction the transport of species responsible for the mass increase is slower than the charge transfer process.Contribution to the 3rd Baltic Conference on Electrochemistry, GDASK-SOBIESZEWO, 23–26 April 2003. Dedicated to the memory of Harry B. Mark, Jr. (February 28, 1934–March 3rd, 2003)     

The quantification of sodium in mineral waters using a quartz crystal microbalance

A sensor for sodium based on a piezoelectric quartz crystal is proposed. The quartz crystal was coated with 5% of bis[(12-crown-4)methyl] dodecylmethylmalonate, 33% of PVC and 62% of NPOE to which KTpClPB in a 22% molar proportion to the ionophore was added. Coating amounts producing a frequency decrease around 18 kHz show optimum linear calibration ranges for the analysis of sodium in commercial mineral waters. Besides sensitivity, coating stability and selectivity of the sensor over other cations were adequate for those analyses. The results obtained analysing commercial mineral waters by the proposed method are not significantly different (α=0.05) from the ones obtained by atomic spectrometry.     

A new electrochemical cell adapted to quartz crystal microbalance measurements

As the resonance frequency of an oscillating quartz changes upon the deposition of a given mass to the crystal surface, it can be used as a very sensitive mass measuring device. Despite a growing interest in the use of electrochemical quartz crystal microbalance (EQCM), there is still no commercial available measurement cell which can satisfy all the conditions needed in electrochemical investigations. The design and characteristics of a new electrochemical cell adapted to EQCM measurements are presented. The sensitivity of the microbalance, which is determined by several calibration runs of silver electrodeposition is (183.2±2.9) Hz μg⁻¹ cm². This value, which is close to the theoretical one, confirms the validity of the system we have developed. The calibration procedure and the EQCM using range are then discussed.     

New approach for natural products screening by real-time monitoring of hemoglobin hydrolysis using quartz crystal microbalance

The hemoglobin (Hb) released from erythrocytes is a primary nutritive component for many blood-feeding parasites. The aspartic protease cathepsin D is a hemoglobinase that is involved in the Hb degradation process and is considered an interesting target for chemotherapy intervention. However, traditional enzymatic assays for studying Hb degradation utilize spectrophotometric techniques, which do not allow real-time monitoring and can present serious interference problems. Herein, we describe a biosensor using simple approach for the real-time monitoring of Hb hydrolysis as well as an efficient screening method for natural products as enzymatic inhibitors using a quartz crystal microbalance (QCM) technique. Hemoglobin was anchored on the quartz crystal surface using mixed self-assembled monolayers. The addition of the enzyme caused a mass change (frequency shift) due to Hb hydrolysis, which was monitored in real time. From the frequency change patterns of the Hb-functionalized QCM, we evaluated the enzymatic reaction by determining the kinetic parameters of product formation (k_cat). The QCM enzymatic assay using immobilized human Hb was shown to be an excellent approach for screening possible inhibitors in complex mixtures, opening up a new avenue for the discovery of novel inhibitors.     

Batch Quartz Crystal Microbalance Immunosensor using a Protein-Immobilized Hydrophilic Polymer

This paper describes a novel batch-type quartz crystal microbalance immunosensor that incorporates a commercially available stainless-steel holder. The sensor was coated with a hydrophilic polymer interface containing phosphatidylcholine analogs that attract water and amino groups for the covalent attachment of receptors. The hydrophilic polymer on a gold substrate captured antibodies and reduced nonspecific adsorption. Operation of the immunosensor required only one sample injection. A clear response triggered by antigen-antibody binding was observed and allowed measurement of the IgG antibody concentration with a superior signal-to-noise ratio using only commercially available components. We thus demonstrate an inexpensive and readily available sensor.     

基于分子印迹技术的丙溴磷压电石英晶体微天平研制

介绍了一种用于检测丙溴磷农药的分子印迹压电生物传感器的构建方法。采用沉淀聚合法合成了农药丙溴磷的分子印迹聚合物,将其固定于石英晶体微天平电极表面构建传感器;采用环境扫描电镜以及原子力显微镜对聚合物形貌、传感器电极表面形貌特征进行分析,并利用传感器对丙溴磷农药进行检测分析,其质量浓度在10~1000 ng/mL范围内,传感器频率改变与丙溴磷浓度之间的响应呈线性关系,线性方程为y=0.139ρ+2.26(r=0.9984)。结果表明,构建的分子印迹压电生物传感器能够对农药进行初步检测,具有较高的灵敏性和较好的特异识别能力。     

Electrochemical quartz crystal microbalance study of copper ad-atoms on gold and platinum electrodes Part I. Adsorption of anions in sulfuric acid

The deposition and dissolution processes of copper ad-atoms on a gold or a platinum electrode in sulfuric acid electrolyte solution were investigated by using the electrochemical quartz crystal microbalance. It was found that the weight loss in the removal of the Cu-adlayer from the Au substrate was considerably larger than that expected from Faraday's law whereas the deviation for the Pt substrate was very small. The adsorption of bisulfate or sulfate anions both on Cu ad-atoms and on the electrode substrates was discussed quantitatively. It was demonstrated that higher coverage with Cu ad-atoms and lower adsorbability with bisulfate or sulfate anions were obtained on the Pt electrode than on the Au, and these effects could be ascribed to the difference in electronegativity between Pt and Au substrates.     

Antibody immobilization to phospholipid polymer layer on gold substrate of quartz crystal microbalance immunosensor

To modify gold electrode for immunosensor to construct an artificial cell membrane structure, water-soluble amphiphilic phospholipid polymer, poly[2-methacryloyloxyehtyl phosphorylcholine-co-n-butyl methacrylate-co-p-nitrophenyloxycarbonyl poly(ethylene glycol) methacrylate (PMBN)] was applied. The polymer had active ester groups for immobilization of biomolecules and it was converted partially to thiol groups for binding to gold substrates. The partially thiolated PMBN was adsorbed on a gold electrode of quartz crystal microbalance (QCM). Surface characterization of adsorbed PMBN layers was thoroughly investigated with reflectance anisotropy spectroscopy, ellipsometry spectroscopy, dynamic contact angle and X-ray photoelectron spectroscopy measurements. Among several PMBN, having different degree of thiolation, it was concluded that 21.5% thiolated PMBN layer had the most well-ordered phosphorylcholine groups in its outer surface. The proteins adsorption test revealed that the phosphorylcholine group on the outer side of PMBN layers, which was substituted their active ester groups by glycine, showed suppress the non-specific adsorption of proteins, such as bovine serum albumin and γ-globulin. Also, through antigen–antibody binding evaluation, the anti-C-reactive protein antibody immobilized on the PMBN surface worked well and it was confirmed that denaturation of the antibody on the PMBN layers was hardly occurred in spite of 60 days storage at 4 °C. The antibody conjugated phospholipid polymer layer with well-ordered phosphorylcholine group could be outstanding functional membrane for biomedical diagnostic devices without non-specific binding and reduction of immunologic activity of immobilized antibody.     

Application of the quartz crystal microbalance for the investigation of nanotribological processes

The electrochemical preparation of copper layers was investigated using a quartz crystal microbalance (QCM) with damping monitoring. The damping increase during the deposition could be separated into two contributions arising from an internal and an external friction process. External friction comes from the coupling of the shear motion of the rough metal surface to the viscous liquid. Internal friction occurred only in the coarse grained layers and can be explained by phonon excitations at the grain boundaries.Presented at the 3rd International Symposium on Electrochemical Processing of Tailored Materials held at the 53rd Annual Meeting of the International Society of Electrochemistry, 15–20 September 2002, Düsseldorf, Germany     

A quartz crystal microbalance sensor for the determination of nitroaromatics in landfill gas

A new sensor based on oxidative combustion of nitroaromatics to NO₂ and its detection with a quartz crystal microbalance (QCM) coated with copper phthalocyanine (CuPc) was developed for determination of nitroaromatics in landfill gas. An alternative method based on gas chromatography–mass spectrometry (GC–MS) was also used in order to assess the performance of the proposed method. The results show that the analytical apparatus based on the QCM is less expensive than the GC–MS, and that the analytical error is 0.8% for both methods.     

Synthesis of isoprenoid chain-contained chemical probes for an investigation of molecular interactions by using quartz crystal microbalance

Five probes including four that contained isoprenoid chain were synthesized. These probes were assembled onto the gold-coated quartz crystal chips for analysis of their interactions with four yeast proteins by using the quartz crystal microbalance technology. Results showed that 3-phosphoglycerate phosphokinase and triosephosphate isomerase had clear interactions with certain probes, while glutathione reductase and phosphoglucose isomerase gave much lower interaction signals. It also suggested that 3-phosphoglycerate phosphokinase had two sites interacting with the probe attached with a geranyl moiety. Further molecule simulation experiments provided supportive information on these intermolecular interactions. Together, our data suggested that there are hydrophobic interactions, with relatively good selectivity, between isoprenoid chain and proteins.     

Improve the signal-to-noise ratio of a quartz crystal microbalance in an impedance analysis method

A new data treatment method for the improvement of the signal-to-noise ratio of a quartz crystal microbalance (QCM) was proposed, where an averaged resonant frequency was calculated according to its conductance peak in an impedance analysis method. The relationship between the averaged resonant frequency and the medians of the conductance peak at different sampling heights was derived. It was shown that the signal-to-noise ratio of the averaged resonant frequency was about eight times of that of the resonant frequency calculated directly from its equivalent circuit parameters. The averaged resonant frequency of the QCM was applied to monitor the self-assembled process of a 6-O-(2′-(α-thiohydroxyacetamide)-ethyl)-diethoxylsilyl-β-cyclodextrin (OTED-β-CD), on gold surface as well as the adsorption of nitrophenol isomers onto the OTED-β-CD self-assembled monolayer film.     

Physical adsorption of protamine for heparin assay using a quartz crystal microbalance and electrochemical impedance spectroscopy

This study attempted to determine absolute heparin concentration in phosphate buffer solution (PBS, pH 7.4) by using quartz crystal microbalance (QCM) as an affinity biosensor. Electrochemical impedance spectroscopy (EIS) was also used to investigate immobilization of protamine and heparin assay. In addition, the effectiveness of physical adsorption in immobilizing protamine was confirmed by examining the preparation conditions, including the incubation time and protamine concentration. It induced maximum decrease (ca. −100 Hz) in oscillating frequency of QCM by applying 20 mg/ml protamine and 20 min for incubation in PBS. Heparin adsorption onto protamine-modified electrode in PBS revealed an exponential-like binding curve and long duration for reaching the steady state in frequency response of QCM. Moreover, two linear calibration curves were obtained judging from the initial slope (df/dt) and the frequency change (Δf) of QCM obtained after a binding interval (600 s) for heparin concentrations from 0 to 3.0 and 7.0 U/ml, respectively. In EIS analysis, calibration curves with linear concentration range of 0-3.0 U/ml were obtained for heparin in PBS when ferrocyanide was used as an electroactive marker.     

Study of electrochemical palladium behavior by the quartz crystal microbalance. I. Acidic Solutions

Sorption of hydrogen in a palladium electrode with a limited volume has been studied using the quartz crystal microbalance (QCM). During the hydrogen sorption process in the palladium electrode, strains are generated inside the metal which result in changes in the frequency of the crystal. These stresses change in a non-linear manner during electrode saturation, with α- and β-phases. This effect creates significant problems with the objective estimation of the amount of sorbed hydrogen inside the palladium electrode using the QCM method. This method is more accurate for the study of electrode surface processes, i.e. specific anion adsorption on the electrode surface or electrode dissolution. Received: 6 August 1998 / Accepted: 1 December 1998     

Development of Piezoelectric Immunosensor for the Detection of Probiotic Bacteria

A Quartz Crystal Microbalance (QCM) based direct immunosensor was developed for real-time detection of probiotic bacteria. To optimize the immunosensor system, model measurements were carried out with bovine serum albumin (BSA) and anti-BSA IgG (a-BSA) antibody. During the model experiments, two kinds of self-assembled monolayers were created to compare their efficiency on antigen binding. Sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido] hexanoate (sulfo-LC-SPDP) and 16-mercapto-hexadecanoic acid (MHDA) cross-linking agents were used for immobilizing anti-BSA antibody onto the gold surface of the AT-cut quartz wafer. Two different measuring procedures, flow-through and stopped-flow methods, were applied, and the frequency responses obtained by both analytical methods were compared.

After the model experiments probiotic bacteria, Bifidobacterium bifidum O1356 and Lactobacillus acidophilus O1132 serotypes were detected from buffer solution and from real samples (spiked milk samples, acidophilus, and bifidus milk samples).

Using the novel immunosensor, the target bacteria could be detected in the range of 10⁴–10⁷ colony-forming units (CFU)/ml within 60 minutes. The selectivity of a-Bifidobacterium bifidum and a-Lactobacillus acidophilus antibody coated sensors were also tested.     

Biofouling of dextran-derivative layers investigated by quartz crystal microbalance

This study reports the fouling of carboxymethyl dextran (CMD) layers in cell culture medium, fibronectin, and serum solutions. CMD layers were covalently immobilized onto amine groups available either on an n-heptylamine plasma polymer (HApp) layer or onto a polyethylenimine (PEI) coating grafted to an acetaldehyde plasma polymer (AApp) layer. The successful immobilization of the graft layers was demonstrated by X-ray photoelectron spectroscopy (XPS). Primary amines on HApp and AApp + PEI surfaces were quantified using a colorimetric assay. Quartz crystal microbalance (QCM) was used to investigate in real-time the fouling of the graft layers upon incubation in cell culture medium (RPMI), fibronectin, and foetal bovine serum (FBS) solutions. HApp, AApp and AApp + PEI layers exhibited large fouling in fibronectin and FBS solutions, while fouling in RPMI cell culture medium was not significant. Protein repellent properties of CMD layers in FBS and fibronectin have been demonstrated compared to the other tested surfaces. QCM has shown that both CMD layers were fouled to a small extent in RPMI medium.     

The use of trichloroacetic acid imprinted polymer coated quartz crystal microbalance as a screening method for determination of haloacetic acids in drinking water

An alternative screening method for haloacetic acids (HAAs) disinfection by-products in drinking water is described. The method is based on the use of piezoelectric quartz crystal microbalance (QCM) transducing system, where the electrode is coated with a trichloacetic acid-molecularly imprinted polymer (TCAA-MIP). This MIP comprises a crosslinked poly(ethyleneglycoldimethacrylate-co-4-vinylpyridine). The coated QCM is able to specifically detect the analytes in water samples in terms of the mass change in relation to acid-base interactions of the analytes with the MIP. The TCAA-MIP coated QCM showed high specificity for the determination of TCAA in aqueous solutions containing inorganic anions, but its sensitivity reduced in water samples containing hydrochloric acid due to a mass loss at the sensor surface. Cross-reactivity studies with HAA analogs (dichloro-, monochloro-, tribromo-, dibromo-, and monobromo-acetic acids) and non-structurally related TCAA molecules (acetic acid and malonic acid) indicated that recognition of the structurally related TCAA compounds by the TCAA-MIP-based QCM is due to a carboxylic acid functional group, and probably involves a combination of both size and shape selectivity. The total response time of sensor is in the order of 10 min. The achieved limits of detection for HAAs (20-50 μg l⁻¹) are at present higher than the actual concentrations found in real-life samples, but below the guidelines for the maximum permissible levels (60 μg l⁻¹ for mixed HAAs). Recovery studies with drinking water samples spiked with TCAA or spiked with mixtures of HAAs revealed the reproducibility and precision of the method. The present work has demonstrated that the proposed assay can be a fast, reliable and inexpensive screening method for HAA contaminants in water samples, but further refinement is required to improve the limits of detection.     

Immersion angle dependence of the resonant-frequency shift of the quartz crystal microbalance in a liquid: effects of longitudinal wave

We investigated the effects of the longitudinal wave on the immersion angle dependence of the resonant-frequency shift, ΔF, of the quartz crystal microbalance, QCM. In order to study exactly the effects, we employed the three types of cells: normal cell, cell with the glass beads and cell with sponge. The longitudinal wave exists in the normal cell. On the other hand, both the cell with the glass beads and the cell with sponge eliminate the longitudinal wave. As results, we have found that the tendencies of ΔF are the same in the three types of cells. That is, we conclude that the longitudinal wave does not have effects on the immersion angle dependence of ΔF.     

Development of an immunosensor for human ferritin, a nonspecific tumor marker, based on a quartz crystal microbalance

A new human ferritin immunosensor was developed using anti-human ferritin antibodies (Abs) immobilized on the gold disc of a quartz crystal microbalance (QCM). Two kinds of self-assembled monolayers (SAMs) prepared by cystamine-glutaraldehyde and cystamine method were applied to immobilize anti-ferritin monoclonal antibodies (MoAbs) and polyclonal antibodies (PoAbs) on the quartz, respectively. The reusabilities of quartz crystal adopting the SAMs were found to be better than those of the other immobilization methods used. The 10 cycles of measurements could be performed on the gold surface of the same crystal regenerated with a solution of glycine·HC1. This sensor system could be continuously performed for 15 days, the relative frequency shifts (the frequency shifts measured are relative to the response at the first day) were all found to be above 95%. A linear relationship existed between the frequency shifts (Hz) and the log values of human ferritin concentrations in the range from 0.1 to 100 ng/ml in buffer and mouse serum. This human ferritin immunosensor had some advantages: high sensitivity, high specificity, low sample requirement, high reusability, no label and no pretreatment etc.