Quartz crystal microbalance as a sensing active element for rupture scanning within frequency band

A new method based on the use of quartz crystal microbalance (QCM) as an active sensing element is developed, optimized and tested in a model system to measure the rupture force and deduce size distribution of nanoparticles. As suggested by model predictions, the QCM is shaped as a strip. The ratio of rupture signals at the second and the third harmonics versus the geometric position of a body on QCM surface is investigated theoretically. Recommendations concerning the use of the method for measuring the nanoparticle size distribution are presented. It is shown experimentally for an ensemble of test particles with a characteristic size within 20–30 nm that the proposed method allows one to determine particle size distribution. On the basis of the position and value of the measured rupture signal, a histogram of particle size distribution and percentage of each size fraction were determined. The main merits of the bond-rupture method are its rapid response, simplicity and the ability to discriminate between specific and non-specific interactions. The method is highly sensitive with respect to mass (the sensitivity is generally dependent on the chemical nature of receptor and analyte and may reach 8 × 10⁻¹⁴ g mm⁻²) and applicable to measuring rupture forces either for weak bonds, for example hydrogen bonds, or for strong covalent bonds (10⁻¹¹–10⁻⁹ N). This procedure may become a good alternative for the existing methods, such as AFM or optical methods of determining biological objects, and win a broad range of applications both in laboratory research and in biosensing for various purposes. Possible applications include medicine, diagnostics, environmental or agricultural monitoring.     

Quartz Crystal Microbalance monitoring the real-time binding of lectin with carbohydrate with high and low molecular mass

A Quartz Crystal Microbalance (QCM) was used to monitor the mass changes on a quartz crystal surface containing immobilized lectins that interacted with carbohydrates. The strategy for lectin immobilization was developed on the basis of a multilayer system composed of Au–cystamine–glutaraldehyde–lectin. Each step of the immobilization procedure was confirmed by FTIR analysis. The system was used to study the interactions of Concanavalin A (ConA) with maltose and Jacalin with Fetuin. The real-time binding of different concentrations of carbohydrate to the immobilized lectin was monitored by means of QCM measurements and the data obtained allowed for the construction of Langmuir isotherm curves. The association constants determined for the specific interactions analyzed here were (6.4 ± 0.2) × 10⁴ M^− 1 for Jacalin–Fetuin and (4.5 ± 0.1) × 10² M^− 1 for ConA–maltose. These results indicate that the QCM constitutes a suitable method for the analysis of lectin–carbohydrate interactions, even when assaying low molecular mass ligands such as disaccharides.     

石英晶体微天平传感器及其在生物检测中应用的研究进展

石英晶体微天平(Quartz crystal microbalance,QCM)是一种具有灵敏度高、免标记、可实时在线检测等优点的重要分析工具。在生物检测领域,QCM与多种信号放大方法相结合,广泛应用于对生物分子的高灵敏检测。新型耗散型QCM(QCM-D)通过对薄膜厚度、粘弹性等的研究,主要用于考察生物分子的吸附分离、构型变化等微观过程。本文主要阐述了QCM及QCM-D生物传感器的构建及其在DNA、蛋白质、细胞和微生物检测中的研究进展。     

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.     

Lectin-modified piezoelectric biosensors for bacteria recognition and quantification

The use of lectins for microorganism biosensors fabrication is proposed. Lectins are immobilised onto a gold-plated quartz crystal for direct piezoelectric label-free transduction of the bacteria–lectin binding event using an electrochemical quartz crystal microbalance (EQCM). Concanavalin A (Con A) and Escherichia coli were used for the evaluation of the lectin immobilisation method and the biosensor performance. Adsorption on nonpolarised and polarised (−0.200 V) gold-coated quartz crystals and immobilisation through avidin–biotin binding were checked for Con A surface attachment. Lectin–bacteria binding was evaluated in all cases. With a crystal modified with Con A via avidin–biotin immobilisation we obtained a linear calibration plot between 5.0 × 10⁶ and 2.0 × 10⁷ cfu mL⁻¹ by measuring frequency changes with E. coli concentration 1 h after bacteria addition. A remarkable increase in sensitivity was achieved when the analytical solution contained free biotinylated Con A, as a consequence of multiple lectin adhesion to Escherichia coli cell wall, which produced an accumulation of Con A–E. coli conjugates in the form of multilayers at the electrode surface. A detection limit of approximately 1.0 × 10⁴ cfu mL⁻¹ was achieved. Moreover nonspecific adsorptions were minimised. Using Con A and lectin from Arachis hypogaea, different response profiles were achieved for Escherichia coli, Staphylococcus aureus and Mycobacterium phlei, thus demonstrating the feasibility of bacteria discrimination. An approach involving filtering of free and lectin-bound bacteria and introduction of a filter in the measuring cell allowed a significant frequency change to be obtained for an E. coli concentration of 1.0 × 10³ cfu mL⁻¹ in order to further increase the sensitivity and discriminate between viable and nonviable cells; an approach using electrochemical measurements of bacterial catalase activity was also checked.     

预富集装置在QCM传感器检测芥子气中的运用

在化学战剂的诸种检测方法中, 质量型微传感器以其响应快速、使用简便等优点成为一种理想的检测手段. 但是这种传感器的使用往往受到检出限的限制, 对于低浓度的毒剂不能及时报警. 预富集技术的运用可以提高微传感器的检测限. 本文研制了一种预富集装置并对其进行了初步测试. 芥子气通过此预富集装置之后在QCM(石英晶体微天平)传感器上的检出限可以达到0.1 mg/m3.     

Quartz Crystal Microbalance Biosensor for Saxitoxin Based on Immobilized Sodium Channel Receptors

This work explored the possibility of coupling the toxin receptor-binding principle with the piezoelectric transduction principle. The sensing component of the saxitoxin biosensor involves a piezoelectric quartz crystal that was coated with sodium channel receptors. The sodium channel receptors were isolated from the electroplax organ of Electrophorus electricus. Binding of the sodium channel extracts to the quartz crystal was facilitated by pre-coating the gold electrode with a hydrophobic self-assembled monolayer of dodecanethiol. The instrumentation system consisted of a flow cell that held the quartz crystal, an oscillator circuit, an injection port, and a frequency counter that was connected to a personal computer. The various immobilization and measurement parameters were optimized. Binding of saxitoxin standards with the immobilized sodium channels was monitored through the decrease in the crystal oscillation frequency readings (ΔF) upon the introduction of saxitoxin into the flow cell. A calibration curve for saxitoxin was constructed by plotting the ΔF values vs. saxitoxin concentrations in the range from 0.1 to 2.0 μg/mL. A correlation coefficient of 0.9653 was obtained. The saxitoxin biosensor developed has the potential to be applied to the rapid screening of total paralytic shellfish poisoning toxins.     

Applying the Quartz Crystal Microbalance Technique to Detect the Epithelial Cell Tight Junction Integrality of Caco-2 Cells

This paper describes a method that utilizes the quartz crystal microbalance (QCM) technique to measure the tight junction of the Caco-2 cell. The Caco-2 cells are placed on the QCM surface for cell growth and tight junction measurements. We found that the QCM resonance frequency changes less when a better tight cell junction is formed. The quantity of QCM frequency change is less than 100 Hz as the tight junction cell integrality is completed up to 15 days. The QCM and traditional detection system transepithelial electrical resistance were similar as the cell growth affects the two systems over time.     

聚甲基丙烯酸甲酯-牛血清白蛋白核壳纳米粒子在金表面的吸附过程及在传感器中的应用

利用铜离子引发体系, 制备出核层为聚甲基丙烯酸甲酯(PMMA)壳层为牛血清白蛋白(BSA)的PMMA-BSA核壳纳米粒子. 通过透射电子显微镜(TEM)表征, 直接观察到PMMA-BSA纳米粒子的核壳结构.结合X射线光电子能谱(XPS)测试, 分析PMMA-BSA纳米粒子的表面成分, 证明PMMA-BSA纳米粒子的壳层是BSA. 利用带耗散的石英晶体微天平(QCM-D)研究了PMMA-BSA纳米粒子在金片表面的吸附行为. 频率的迅速下降, 耗散因子的快速上升, 说明PMMA-BSA粒子快速地吸附到金片表面. 利用磷酸盐缓冲液反复冲洗时, 频率和耗散没有变化, 表明PMMA-BSA 纳米粒子在金片上吸附较牢固. 以金电极为基底电极, 吸附PMMA-BSA纳米粒子后, 利用戊二醛修饰粒子壳层, 再通过氨基与醛基的反应来固定葡萄糖氧化酶, 制备出电流型葡萄糖传感器. 电化学测试表明该传感器对葡萄糖具有良好的电流响应, 在0.3 V的工作电位下, 响应电流与葡萄糖浓度在0.20-5.85 mmol·L^-1范围内呈现出较好的线性关系, 相关系数为0.989. 传感器的灵敏度高达28.6 μA·L·mmol^-1·cm^-2, 响应时间仅为11 s. 传感器还具有良好的稳定性, 在25℃下储存30 d, 响应电流仅下降了16%.     

Application of a flow type quartz crystal microbalance immunosensor for real time determination of cattle bovine ephemeral fever virus in liquid

Bovine ephemeral fever (BEF) is a viral disease of cattle. A flow type quartz crystal microbalance (QCM) immunosensor was developed for the real time determination BEF virus (BEFV) that is suitable for clinical point-case diagnosis. Self-assembled monolayer (SAM) of thiols and sulphides by the cystamine–glutaraldehyde method was used for the immobilization of BEFV monoclonal antibody on the gold surface of a quartz crystal microbalance (QCM). A positive correlation was found between the virus concentration and frequency changes (R² = 0.9962) on this QCM system. The reproducible rates for the 50 and 10 μg/mL samples were 4 and 13.9%, respectively. There was no interference from non-specifically adsorbed phage. Using this flow type QCM immunosensor, BEFV could specifically be detected with sensitivity comparable to a conventional enzyme-linked immunosorbent assay (ELISA). The measurement could be obtained directly, within several minutes, rather than hours as required visualizing the results of ELISA. In addition, the observation of reproducible and constant changes after successive additions of BEFV suggests that a QCM immunosensor in a flow cell could be developed for automated or continuous real time operation.     

Polyaniline film deposition from the oxidative polymerization of aniline using K2Cr2O7

The deposition of the polyaniline (PANI) films was monitored using the quartz crystal microbalance (QCM) technique. The films were grown from an aqueous dilute hydrochloric acid solution by the chemical oxidation of aniline using potassium dichromate (KDC). The effect of the initial molar ratio of the KDC/aniline on the yield and the growth rate of the PANI films were studied. There is no optimum initial molar ratio of KDC/aniline of PANI film deposition. Also there was a small depletion period and no degradation to the deposited PANI films. The order of the polymerization kinetics was studied with respect to KDC. The UV-visible spectra of the PANI films grown onto a glass support immersed into the bulk solution were measured. The absorption of the PANI film with the time of polymerization was compared to the growth of the PANI film thickness with time determined from the QCM technique. The characteristics of the PANI film deposition were compared to the corresponding ones that were observed during the oxidative polymerization of aniline with ammonium persulphate (APS).     

Direct introduction of a hydrazide group into a quartz crystal microbalance surface with dodecanoic hydrazide embedded in a hybrid bilayer membrane

Hydrazide group has a potential of immobilizing an antibody on a sensor surface in a way that ensures an optimal orientation and efficiency of the antibody. However, a multi-step chemical process, required for the preparation of a hydrazide group, is a barrier to its extensive application. This paper describes a new method to introduce a hydrazide group to a sensor surface by a one-step process using dodecanoic hydrazide. The method is based on an ability of the dodecanoic hydrazide to be incorporated into a hybrid bilayer membrane (HBM) layer, thereby presenting its hydrazide group to the surface. Liposome containing dodecanoic hydrazide was added to a hydrophobic self-assembled monolayer surface of a quartz crystal microbalance for the formation of a HBM. Then, the hydrazide group, presented in the surface of the HBM layer, was utilized for the oriented immobilization of an antibody via its carbohydrate moiety which was partially oxidized prior to the conjugation reaction. Activity and stable status of the incorporated dodecanoic hydrazide was revealed by the efficiency and reproducibility of the resulting immunosensor chip.     

Development of a rubber elongation factor, surface-imprinted polymer-quartz crystal microbalance sensor, for quantitative determination of Hev b1 rubber latex allergens present in natural rubber latex products

Molecularly imprinted polymers (MIPs) for screening to detect rubber latex allergens (Hev b1) in natural rubber based products were designed as artificial recognition polymeric materials coated onto a quartz crystal microbalance (QCM). The polymers were prepared using a stamp imprinting procedure after mixing optimum amounts of methacrylic acid–vinylpyrrolidone–dihydroxyethylene bisacrylamide and Hev b1 latex allergen proteins, obtained from rubber gloves. QCM measurements showed that the resulting polymer layers after removal of the proteins used in their preparation could incorporate structures and features down to nanometer scale of protein templates into the imprinted polymer much better than a non-specific control polymer under controlled sensor conditions and an optimized polymerization process. This selective polymer but not the non-selective polymer clearly distinguished between the latex allergen Hev b1 and proteins such as lysozyme, ovalbumin and bovine serum albumin, with a selectivity factor of from 2 to 4, and the response of the rubber elongation factors by an astonishing factor of 12. The imprinted cavities recognized specific binding sites and could distinguish among related hevein latex allergenic proteins isolated from fresh natural rubber latex; Hev b1, Hev b2, and Hev b3 with a selectivity factor of from 4 to 6. The different QCM measurements obtained presumably reflected slightly different conformations and affinities to the MIP binding sites. The sensor layers selectively adsorbed Hev b1 within minutes in amounts ranging from 10 to 1500 μg L⁻¹ and with a detection limit of 1 μg L⁻¹. This work has demonstrated that this new sensor provides a fast and reliable response to natural rubber latex protein, even after being extracted from the matrix of rubber gloves.     

石英晶体微天平的纳米微球质量放大技术及短序列DNA测定

提出了一种新型质量放大系统用于提高石英晶体微天平检测的灵敏度.将DNA嵌入剂放线菌素D作为导向分子修饰在磁性微球上,通过石英晶体微天平技术识别测定短序列DNA,使最低检测限从6.2×10^-8mol/L下降到2.0×10^-12mol/L.     

Development of a sensitive detection method of cancer biomarkers in human serum (75%) using a quartz crystal microbalance sensor and nanoparticles amplification system

A simple and sensitive sensor method for cancer biomarkers [prostate specific antigen (PSA) and PSA-alpha 1-antichymotrypsin (ACT) complex] analysis was developed, to be applied directly with human serum (75%) by using antibody modified quartz crystal microbalance sensor and nanoparticles amplification system. A QCM sensor chip consisting of two sensing array enabling the measurement of an active and control binding events simultaneously on the sensor surface was used in this work. The performance of the assay and the sensor was first optimised and characterised in pure buffer conditions before applying to serum samples. Extensive interference to the QCM signal was observed upon the analysis of serum. Different buffer systems were then formulated and tested for the reduction of the non-specific binding of sera proteins on the sensor surface. A PBS buffer containing 200 μg mL⁻¹ BSA, 0.5 M NaCl, 500 μg mL⁻¹ dextran and 0.5% Tween 20, was then selected which eliminated the interfering signal by 98% and enabled the biomarker detection assay to be performed in 75% human serum. By using Au nanoparticles to enhance the QCM sensor signal, a limit of detection of 0.29 ng mL⁻¹ PSA and PSA-ACT complex (in 75% serum) with a linear dynamic detection range up to 150 ng mL⁻¹ was obtained. With the achieved detection limit in serum samples, the developed QCM assay shows a promising technology for cancer biomarker analysis in patient samples.     

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.     

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.     

Development of immunochips for the detection of dengue viral antigens

Recently, dengue virus has become a new emerging disease in the world. However, the procedures currently used for the detection of dengue virus are cumbersome and time-consuming. This is unfavorable for early stage epidemiological control and effective medical treatment. A new detection system was developed based on the quartz crystal microbalance (QCM) coating using two monoclonal antibodies that act specifically against the dengue virus envelope protein (E-protein) and non-structural 1 protein (NS-1 protein), respectively. Three different immobilizing methods, the glutaraldehyde (GA) method, protein A method and carbodiimide method (1-ethyl-3-(3-dimethyl- aminopropyl) carbodiimide, EDC) were used to prepare the immunochips. The “cocktail” immunochip, which has both antibodies attached, was also fabricated and used in comparison. The results showed that the protein A method was the best among the three. The sensitivity of the immunochip was 100-fold greater than the conventional sandwich ELISA method. The cocktail immunochip had a higher signal level than the normal immunochip. The time required for detection was shorter (about 1 h) and a blood specimen could be used to detect the virus in the viremia phase.     

Quartz crystal microbalance sensor array for the detection of volatile organic compounds

A sensor array system consisting of five quartz crystal microbalance (QCM) sensors (four for measuring and one for reference) and an artificial neural network (ANN) method is presented for on-line detection of volatile organic compounds. Three ionic liquids, 1-butyl-3-methylimidazolium chloride (C₄mimCl), 1-butyl-3-methylimidazolium hexafluorophosphate (C₄mimPF₆), 1-dedocyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (C₄mimNTf₂), and silicone oil II, which is widely used as gas chromatographic stationary phase, have been selected as sensitive coatings on the quartz surface allowing the sensor array effective to identify chemical vapors, such as toluene, ethanol, acetone and dichloromethane. The success rate for the qualitative recognition reached 100%. Quantitative analysis has also been investigated, within the concentration range of 0.6-6.1 mg/L for toluene, 0.9-7.5 mg/L for ethanol, 2.8-117 mg/L for dichloromethane, and 0.7-38 mg/L for acetone, with a prediction error lower than 8%.     

Comparison of two methods for coating piezoelectric crystals

Quartz piezoelectric crystals were coated with triethanolamine using two procedures: spray and syringe methods. The reproducibility of both coating methods was evaluated comparing the relative standard deviation of batches of four crystals coated by each of the procedures. The relative standard deviation obtained with the spray method is a hyperbolic function of the coating frequency and is, at least, three times lower than the one calculated from results of the syringe method, which is not frequency dependent. An explanation for the poorer reproducibility of the syringe method is given, based on the radial sensitivity theory and the non-uniformity of the coating distribution over the crystal, as evidenced by microscopic photographs.