Quartz crystal microbalance genosensor for sequence specific detection of attomolar DNA targets

A mass sensitive quartz crystal microbalance (QCM) based genosensor has been developed using breast cancer 1 (BRCA1) gene as a model gene. We modified the traditional sandwich assay by conjugating reporter probe DNA (DNA-r) with an assembly of gold nanoparticles leading to an increased mass on the surface, which enhanced the sensitivity to few orders of magnitude. The unique cleavage function of endonuclease is used for achieving the selectivity to complementary DNA over mismatched DNA. With this combination, the sensor exhibited excellent sensitivity with a detection limit of 10 aM BRCA1 gene and it showed good selectivity for even single base mismatch DNA targets. This ultrasensitive and cost-effective DNA detection protocol can be extended to the direct analysis of any non-amplified genomic DNA.     

Quartz crystal microbalance immunosensor for the detection of antibodies to double-stranded DNA

We report the development of a novel quartz crystal microbalance immunosensor with the simultaneous measurement of resonance frequency and motional resistance for the detection of antibodies to double-stranded DNA (dsDNA). The immobilization of poly(l-lysine) and subsequent complexation with DNA resulted in formation of a sensitive dsDNA-containing nanofilm on the surface of a gold electrode. Atomic force microscopy has been applied for the characterization of a poly(l-lysine)–DNA film. After the blocking with bovine serum albumin, the immunosensor in flow-injection mode was used to detect the antibodies to dsDNA in purified protein solutions of antibodies to dsDNA and to single-stranded DNA, monoclonal human immunoglobulin G, DNase I and in blood serum of patients with bronchial asthma and systemic lupus erythematosus. Experimental results indicate high sensitivity and selectivity of the immunosensor. In memoriam Prof. Victor G. Vinter     

Quartz crystal microbalance biosensor for recombinant human interferon-beta detection based on antisense peptide approach

Quartz crystal microbalance (QCM) biosensors for recombinant human interferon-β (rhIFN-β) were constructed by utilizing antisense peptides adhering to the QCM gold surfaces. Two antisense peptides, both corresponding to the N-terminal fragment 1-14 of rhIFN-β, were used in this study. Antisense peptide AS-1 was the original antisense peptide and AS-2 was the modified antisense peptide based on the antisense peptide degeneracy. Both antisense peptides were immobilized on the gold electrodes of piezoelectric crystals, respectively, via a self-assembling monolayer of 1,2-ethanedithiol. The binding affinity between rhIFN-β and each immobilized antisense peptide in solution was evaluated using a quartz crystal microbalance-flow injection analysis (QCM-FIA) system. The dissociation constant of rhIFN-β on the antisense peptide AS-1 and AS-2 biosensor was (1.89 ± 0.101) × 10⁻⁴ and (1.22 ± 0.0479) ×10⁻⁵ mol L⁻¹, respectively. The results suggested that AS-2 had a higher binding affinity to rhIFN-β than AS-1. The detection for rhIFN-β using each biosensor was precise and reproducible. The linear response ranges of rhIFN-β binding to both biosensors were same with a concentration range of 0.12-0.96 mg mL⁻¹. The results demonstrated the successful construction of highly selective QCM biosensors using antisense peptide approach, and also confirmed the feasibility of increasing antisense peptide binding affinity by appropriate sequence modification.     

Quartz crystal microbalance for immunosensing

An immunosensing system is described for the detection of specific antibodies in human sera using a piezoelectric immunosensor and a flow through system. A quartz crystal microbalance theory, regarding mass and viscoelastic effects in multilayer systems, which is derived from the Mason circuit, gives the theoretical background for the interpretation of measurement data obtained from an oscillator circuit. An acoustic model for the antigen/antibody reaction is described (Part 1). An immunospecific detector was developed by immobilizing synthetic peptides or recombinant proteins, comprising major diagnostic epitops of HIV, on the surface of the transducer. Attempts have been made to use the sensor for the initial screening for antibodies, specific against the Human Immunodeficiency Virus (HIV), in human serum samples. Results are obtained within a few minutes and with a selectivity comparable to a licensed HIV ELISA (Part II).     

RNA-templated single-base mutation detection based on T4 DNA ligase and reverse molecular beacon

A novel RNA-templated single-base mutation detection method based on T4 DNA ligase and reverse molecular beacon (rMB) has been developed and successfully applied to identification of single-base mutation in codon 273 of the p53 gene. The discrimination was carried out using allele-specific primers, which flanked the variable position in the target RNA and was ligated using T4 DNA ligase only when the primers perfectly matched the RNA template. The allele-specific primers also carried complementary stem structures with end-labels (fluorophore TAMRA, quencher DABCYL), which formed a molecular beacon after RNase H digestion. One-base mismatch can be discriminated by analyzing the change of fluorescence intensity before and after RNase H digestion. This method has several advantages for practical applications, such as direct discrimination of single-base mismatch of the RNA extracted from cell; no requirement of PCR amplification; performance of homogeneous detection; and easily design of detection probes.     

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%.     

Quartz crystal microbalance immunoassay for carcinoma antigen 125 based on gold nanowire-functionalized biomimetic interface

Gold nanowires with of designed length on a solid substrate have been proven as an efficiently immobilized affinity support for the detection of carcinoma antigen 125 (CA 125) in this study. The presence of gold nanowires provides a well-defined three-dimensional structure, and greatly amplifies the coverage of the anti-CA 125 protein on the probe surface. Moreover, the amount of anti-CA 125 varied with the change of the morphology of the probe, and achieved an optimal quartz crystal microbalance (QCM) response towards anti-CA 125 adsorption at the number of gold nanolayers of 5. The formed immune-probe exhibits good QCM responses for the detection of CA 125, and allows the detection of CA 125 at concentrations as low as 0.5 U ml(-1). The QCM immunosensor exhibited good precision, high sensitivity, acceptable stability, accuracy and reproducibility. The as-prepared immunosensors were used to analyze CA 125 in human serum specimens. Analytical results suggest that the developed immunoassay method is a promising alternative approach for detecting CA 125 in the clinical diagnosis. Compared with conventional ELISA, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the route provides an alternative approach to incorporate multiple gold nanolayers onto the solid matrix for biosensing applications.     

Quartz crystal microbalance determination of organophosphorus and carbamate pesticides

We have developed a quartz crystal microbalance (QCM) biosensor for the determination of organophosphorus and carbamate pesticides. A change in resonant frequency is observed as a result of mass adsorption, and we have used this as the basis for sensor development. Specifically, we have used a two-enzyme system (acetylcholine-esterase and choline oxidase) which converts acetylcholine to betaine producing hydrogen peroxide as a by-product. In a third enzyme reaction (peroxidase), the peroxide is able to oxidise benzidines (3,3′-diaminobenzidine) into an insoluble product that precipitates out and can adsorb to surfaces. Non-ionic surfactants have been used for the first time to enhance the surface deposition of suspended precipitate, thereby improving sensor sensitivity. Pesticides are known to inhibit esterase activity (thereby reducing the amount of QCM-detectable precipitate produced). We have shown that the QCM-enzyme sensor system can be used to determine carbaryl and dichlorvos down to 1 ppm.     

Quartz crystal microbalance studies on bilirubin adsorption on self-assembled phospholipid bilayers

Bilirubin adsorption on self-assembled phospholipid bilayers was studied using quartz crystal microbalance, and factors influencing its adsorption such as pH, temperature, and solution ionic strength were discussed in detail. The results show the amount of adsorbed bilirubin on self-assembled phospholipid bilayers is small at higher temperature and large at higher pH and solution ionic strength, and the adsorption kinetic parameter estimated from the in situ frequency measurement is (1.8+/-0.27)x10(6) M(-1) (mean +/- S.D.). With the present method, the desorption of adsorbed bilirubin caused by human serum albumin and the photoinduced decomposition of adsorbed bilirubin under light illumination were also examined. QCM measurement provides a useful method for monitoring the adsorption/desorption process of bilirubin on self-assembled phospholipid bilayers.     

Solubility of water in polyimides: Quartz crystal microbalance measurements

The solubility of water vapor in four different polyimides at 100% relative humidity and 22°C was measured by means of a quartz crystal microbalance system. The results confirm earlier conclusions that diffusion in these polymers is Fickian, and are sufficiently precise that the order of solubilities is unambiguous. The differences between the values are interpreted in terms of morphological variations rather than variations in chemical affinity for water. Diffusion coefficients are also reported.     

Quartz crystal microbalance study of the kinetics of surface initiated polymerization

Surface initiated polymerization (SIP) is a valuable tool in synthesizing functional polymer brushes, yet the kinetic understanding of SIP lags behind the development of its application. We apply quartz crystal microbalance (QCM) to address two issues that are not fully addressed yet play a central role in the rational design of functional polymer brushes, namely quantitative determination of the kinetics and the initiator efficiency (IE) of SIP. SIP are monitored online using QCM. Two quantitative frequency-thickness (f-T) relations make the direct determination and comparison of the rate of polymerization possible even for different monomers. Based on the bi-termination model, the kinetics of SIP is simply described by two variables, which are related to two polymerization constants, namely a = 1/(k _p,s,app−[M][R·]₀) and b = k _t,s,app/(k _p,s,app[M]). Factors that could alter the kinetics of SIP are studied, including (i) the molecular weight of monomers, (ii) the solvent used, (iii) the initial density of the initiator, (iv) the concentration of monomer, [M], and (v) the catalyst system (ratio among the ingredients, metal, ligands, and additives). The dynamic nature of IE is also described by these two variables, IE = a/(a + bt). Instead of the molecular weight and the polydispersity, we suggest that film thickness, the two kinetic parameters (a and b), and the initial density of the initiator and IE be the parameters that characterize ultra-thin polymer brushes. Besides the kinetics study of SIP, the reported method has many other applications, for example, in the fast screening of catalyst system for SIP and other polymerization systems.     

Quartz crystal microbalance studies of the contact between soft, viscoelastic solids

Mechanical contact between a viscoelastic lens and a viscoelastic film has been probed by means of a quartz crystal microbalance operated in the impedance analysis mode. The frequency shift induced by the formation of the contact decreases with increasing film thickness because of the finite penetration depth of the acoustic shear wave. The dependence of frequency and bandwidth on film thickness and contact area is described within a sheet-contact model, which can be employed to quantitatively analyze mechanical contact in a wide range of materials problems. The model was tested by bringing a quartz crystal coated with an elastomeric gel into contact with a hemispherical cap of a similar gel. Both gels consisted of the thermoreversible gel Kraton G swollen in mineral oil. The experiments support the model well.     

Quartz crystal microbalance immunosensor for highly sensitive 2,3,7,8-tetrachlorodibenzo-p-dioxin detection in fly ash from municipal solid waste incinerators

A quartz crystal microbalance (QCM) immunosensor was developed for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxins (TCDD) in environmental pollutants. An anti-TCDD antibody was immobilized on the gold surface of the QCM via chemical coupling, and its immunologic activity was then maintained by treatment with an artificial stabilizing reagent such as poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate). A competitive immunoreaction with TCDD conjugated ovalbumin (TCDD-ovalbumin) was used to detect TCDD. A calibration curve was obtained through the competitive immunoreaction, and linearity was shown from 100 ng mL(-1) to 0.1 ng mL(-1). Also, the cross-reactivities of the anti-TCDD monoclonal antibody were thoroughly evaluated with several TCDD derivatives. The relationships between GC-MS, ELISA, and QCM were compared using fly ash samples from a municipal solid waste, which were prepared using an accelerated solvent extractor. For 23 samples, the experimental relationship between the TCDD concentration by QCM vs. the TCDD concentration by ELISA was y= 1.07x + 2.70, r= 0.99, and the TCDD concentration by QCM vs. the toxic equivalent quantity (TEQ) value by GC-MS was y= 2.46x - 14.98, r= 0.89.     

Quartz crystal microbalance study of protein adsorption kinetics on poly(2-hydroxyethyl methacrylate)

The interaction of macromolecules with artificial biomaterials may lead to potentially serious complications upon implantation into a biological environment. The interaction of one of the most widely used biomaterials, polyHEMA, with lysozyme, bovine serum albumin (BSA), and lactoferrin was investigated using quartz crystal microbalance (QCM). The concentration dependence of adsorption was measured for the aforementioned proteins individually as well as for lysozyme-BSA, and lysozyme-lactoferrin combinations. An extension of Voinova's viscoelastic model to n layers was used to create thickness-time graphs for adsorption. For each of lactoferrin and lysozyme, two distinctly different timescales of adsorption could be differentiated. However, the mechanisms of adsorption appeared to differ between the two. Negative dissipation shifts were measured for low concentrations of lysozyme, trending to positive dissipation at higher concentrations. This suggested that lysozyme was adsorbed initially into the matrix, stiffening the hydrogel, and later onto the surface of polyHEMA. Additionally, trials with commercial no-rub cleaning solutions indicated little added effectiveness over buffer solutions. Mixtures of proteins showed behaviour which differed in some cases from the simple combination of single protein adsorption experiments.     

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.     

Quartz crystal microbalance determination of vapors of volatile organic compounds on carbon nanotubes under batch conditions

Specific features of the sorption of vapors of monoatomic aliphatic alcohols and aromatic compounds on coatings formed from carbon nanotubes of various genesis studied by the method of piezoelectric micro weighing are considered. The morphology of the coatings is considered in dependence on the conditions of synthesis and aftertreatment of carbon nanotubes. A new method of the processing of signals from an array piezoelectric sensors is proposed; it allows an increase in the selectivity of the determination of volatile organic compounds in an equilibrium gas phase by 2–2.5 times. The advantages of the application of carbon nanotubes as sorption coatings in comparison to the standard polymeric sorbents for piezoelectric micro weighing are demonstrated: the detection limit decreases by 4–15 times and the duration of measurement by 5–55 times.     

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 simulation of the directionality of Si etching in CF4 glow discharges

Two quartz crystal microbalances have been mounted in a planar rf discharge system in such a way that the potential of the microbalances with respect to the glow discharge can be varied. This apparatus allows a rapid simulation of the etching directionality that can be expected in real pattern transfer situations in that operating one microbalance at ground and one at a negative potential gives a measure of the sidewall and vertical etch rates, respectively. The voltage threshold for ion-assisted etching has been determined to be 20 V which is the approximate value of the plasma potential in this asymmetric system.     

Development of a sensor for calcium based on quartz crystal microbalance

A new sensor for calcium based on a piezoelectric quartz crystal is presented. The selectivity depends on the ratio lipophilic salt/ionophore of the composition of the coating of the quartz crystal. A crystal coated with a THF solution of PVC (34.5% w/w), DOS (62.1% w/w) and 10,19-bis[(octadecylcarbamoyl) methoxyacetyl]-1,4,7,13,16-pentaoxa-10,19-diaza cycloheneicosane (3.4% w/w) and a salt/ionophore molar proportion of 60%, corresponding to a frequency decrease of the dry crystal of 6.0 kHz, showed a detection limit to calcium of 2.2 mg/L. Both a standard calcium chloride solution and a commercially balanced salt solution for tissue culture were analyzed subsequent to ion chromatographic separation. The results obtained with the sensor developed were compared with those obtained with a conductivity detector. Although the results from both detectors agreed for the standard solution, only the quartz crystal sensor was able to give reliable results for the tissue culture solution.