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PubMed2003

Evaluation and application of conducting polymer entrapment on quartz crystal microbalance in flow injection immunoassay.

An immunosensor employing conducting polymer entrapment (CPE) method to immobilize immuno-protein on the quartz crystal microbalance (QCM) for clinical flow-injection-analysis (FIA) purpose was exploited. By comparing the CPE approach and the conventional physical immobilization (PI) method, a frequency shift of the former was 47% higher than that of the latter when measuring at 0.5 mg/ml human serum albumin antibody concentration. This implied that CPE was a feasible approach for developing QCM-FIA process. An immunoassay of anti-pseudorabies virus antibody in mouse sera further exemplified its practical potential in diagnostic implication.

Animals↗
PubMed2003

A new approach for immobilization of oligonucleotides onto piezoelectric quartz crystal for preparation of a nucleic acid sensor for following hybridization.

The aim of this study is to develop a nucleic acid sensor based on piezoelectric crystal microbalance system (QCM) for following hybridization. Piezoelectric quartz crystal surfaces were first treated in a glow-discharge apparatus with ethylene diamine (EDA) plasma at 15 W (discharge power), 2.5 min (incubation time) and 35 ml/min (monomer flow rate) to create amino groups on the crystal surfaces. The thickness of the EDA-plasma film formed was about 43+/-24 A. Then, the amino groups on the crystal surfaces were converted to aldehyde groups by reacting the amino groups with glutaraldehyde (GA) at different conditions. A GA concentration of 2.5% and an incubation time of 2 h were selected as optimal values at this step, corresponding to a GA surface concentration of about 270 ng/cm2. A double strand Oligonucleotides, having one extra base on 5'-end of one of the complementary strands, were immobilized through the amino groups of this base onto the GA-modified crystals. Optimal immobilization conditions were as follows: oligonucleotide concentration: 1 microg/ml; time: 3 h; pH: 9.2 carbonate buffer; ionic strength: 0.1; and temperature: 20 degrees C. The QCM sensor carrying the covalently bound strand was used in the hybridization experiments, which showed that equilibrium is achieved in about 5 min, and the frequency shift measured is related to the concentration of the target strand to be measured within the medium.

Adsorption↗
PubMed2003

Real time kinetic analysis of the interaction between immunoglobulin G and histidine using quartz crystal microbalance biosensor in solution.

Quartz crystal microbalance (QCM) biosensor integrated in a flow injection analysis (FIA) system was used for the investigation of the specific interaction between immunoglobin G (IgG) and histidine. The histidine was immobilized on the gold electrodes of the piezoelectric crystal using appropriate procedures based on self-assembling of the dithiothreitol (DTT). The specific interaction of the immobilized ligand with IgG in solution was followed as a change in the resonant frequency of the modified crystal and studied in real time without any additional labels. With the mass sensitive biosensor system, the differences in affinity of three different species of IgG: human IgG, goat IgG and mouse IgG were easily distinguished and their respective kinetic rate constants (kass and kdiss) and equilibrium association constants (KA) were determined from the curves of frequency versus time. For the interactions, KA were 2.92 x 10(4), 3.23 x 10(4) and 4.08 x 10(4) M(-1) for human IgG, goat IgG and mouse IgG, respectively.

Adsorption↗
PubMed2003

Quartz crystal biosensor for real-time monitoring of molecular recognition between protein and small molecular medicinal agents.

A quartz crystal microbalance (QCM) biosensor integrated into a flow injection analysis (FIA) system was used for the real-time investigation of molecular recognition between a protein and small molecular medicinal agents. Two sulfa-drugs, sulfamethazine (SMZ) and sulfamethoxazole (SMO), were, respectively, immobilized on the gold electrodes of the piezoelectric crystals using appropriate procedures based on self-assembly of the dithiothreitol (DTT). The binding interactions of the two immobilized drug ligands, with various proteins in solution, were followed as changes in the resonant frequency of the modified crystals. Results obtained from this rapid screen analysis clearly indicated that the two drug ligands appeared quite different in this molecular recognition procedure although their structures were similar. SMZ-immobilized sensor showed specific interaction only with IgG, while SMO-immobilized sensor showed negligible specific binding with IgG, but binding with trypsin and chymotrypsin. Further studies on the specific interaction between immobilized SMZ and three different species of IgG--human IgG, goat IgG and mouse IgG were carried out and the marked species-dependent difference was observed. The resultant sensorgrams were rapidly analyzed by using an in-house kinetic analysis software based on genetic algorithm (GA) to derive both the kinetic rate constants (kass and kdiss) and equilibrium association constants (KA) for IgG-SMZ interactions. For the interactions, KA were 5.48 x 10(5), 2.75 x 10(5) and 1.86 x 10(5) M(-1) for human IgG, goat IgG and mouse IgG, respectively. The kinetic data provided further insight into the structural/functional relationships of different IgG on a molecular level.

Adsorption↗
PubMed1997

Quartz crystal microbalances for quantitative biosensing and characterizing protein multilayers.

The use of quartz crystal microbalances (QCMs) for quantitative biosensing and characterization of protein multilayers is demonstrated in three case studies. Monolayers of QCM-based affinity biosensors were investigated first. Layers of a thiol-containing synthetic peptide constituting an epitope of the foot-and-mouse-disease virus were formed on gold electrodes via self-assembly. The binding of specific antibodies to epitope-modified gold electrodes was detected for different concentrations of antibody solutions. Oligolayers were studied in a second set of experiments. Dextran hydrogels were modified by thrombin inhibitors. The QCM response was used in a competitive binding assay to identify inhibitors for thrombin at different concentrations. Multilayers of proteins formed by self-assembly of a biotin-conjugate and streptavidin were investigated next. The QCM frequency response was monitored as a function of layer thickness up to 20 protein layers. A linear frequency decay was observed with increasing thickness. The decay per layer remained constant, thus indicating perfect mass coupling to the substrate. Frequency changes a factor of four higher were obtained in buffer solution as compared to measurements in dry air. This indicates a significant incorporation of water (75% weight) in the protein layers. This water behaves like a solid concerning the shear mode coupling to the substrate. The outlook discusses briefly the need for controlled molecular engineering of overlayers for subsequent QCM analysis, and the importance of an additional multiparameter analysis with other transducer principles and with additional techniques of interface analysis to characterize the mechanical coupling of overlayers as biosensor coatings. A promising trend concerns the use of QCM-arrays for screening experiments.

Biosensing Techniques↗
PubMed1998

A piezoelectric quartz crystal sensor for the determination of coagulation time in plasma and whole blood.

A Peizoelectric Quartz Crystal (PQC) system possessing an adequate sensitivity to a slight viscosity change is employed to monitor the blood coagulation. In the case of an anticoagulated plasma specimen, prothrombin time (PT) was determined by the PQC sensor on the basis of the introduction of tissue thromboplastin (TF) and calcium ion. Discussing the PQC data statistically, we found that at 90% of the total frequency shift (delta f) the best linear relationship in a double-logarithmic plot of PT versus TF concentration in the range of 5.466-22.311 unit/ml is shown. The PQC result was compared with commercial optical coagulometry and showed a good linear correlation (ca. 0.98). The clotting and recalcification of whole blood were also studied by the PQC system. The testing time for one measurement could be completed within 20 min. and had a good agreement with that obtained conventionally.

Biosensing Techniques↗
PubMed2002

High-level gamma dosimetry using phototransferred thermoluminescence in quartz.

Phototransferred thermoluminescence (PTTL) in natural quartz, separated from sand, has been studied for its application in high-level gamma dosimetry. The paper reports dose-vs.-PTTL response of 110 degrees C and 180 degrees C peaks in the dose range of 10 Gy to 6 kGy. Using this method, the dose rate delivered to the sewage sludge during irradiation at SHRI facility, Vadodara, India, was estimated to be 0.35 +/- 0.02 kGy/h.

Gamma Rays↗
PubMed1993

Quartz crystal microbalance for the detection of microgram quantities of human serum albumin: relationship between the frequency change and the mass of protein adsorbed.

We have developed a piezoelectric immunosensor for the detection of microalbumin. Human serum albumin (HSA) in the range 0.1-100 micrograms mL-1 could be detected using a flow cell; the immunosensor is sensitive enough to monitor levels of albuminuria. The immunosensor did not respond to bovine serum albumin, only to HSA, implying that the specificity for HSA was high. We investigated the relationship between the frequency change (delta F) and adsorption per unit area of piezoelectrically active quartz crystal (delta M). delta M was estimated with radioisotope-labeled anti-HSA or HSA. When anti-HSA was adsorbed onto the surface of the crystal or HSA was bound to anti-HSA supported by the crystal, values of magnitude of delta F/delta M were larger than the value predicted from theory (Sauerbrey's equation). Furthermore, magnitude of delta F/delta M for HSA was larger than that for anti-HSA.

Adsorption↗
PubMed1993

Osteoblast attachment monitored with a quartz crystal microbalance.

A quartz crystal microbalance is used in aqueous solutions to monitor the rate of attachment of osteoblasts, bone-forming cells, to the surface of the crystal. Changes in resonant frequency of the crystal are measured for various surface coverages by osteoblasts. Crystal surface coverages are determined by digital image processing of scanning electron micrographs. A linear relationship is established between the surface coverages and the changes in resonant frequency of the crystal. The osteoblasts are observed to behave viscoelastically. Hence, the Sauerbrey equation can not be used to describe the relationship between the change in mass of osteoblasts on the surface and the change in resonant frequency of the crystal. Apparent viscosities at 5.0 MHz are also determined for osteoblasts.

Animals↗
PubMed2000

Graphite microparticles as coatings for quartz crystal microbalance-based gas sensors.

The use of graphite particles (1-2 microm) as coatings on quartz crystal microbalances (QCMs) for detection and monitoring of toluene and other volatile organic compounds (VOCs) is described. Unlike the more commonly used polymeric coatings with low glass transition temperatures (Tg), particulate graphitic coatings are not as susceptible to loss of acoustic energy when coating thickness or operational temperature increases. This situation enables the use of relatively thick coatings, which increases the absolute amount of vapor sorbed in the coating and, consequently, lowers the level of detection and enhances operation over a wide temperature range. The use of small size particles also results in a coating with a more porous structure, which facilitates uptake and release of VOCs in comparison to coatings made from high Tg polymers, which have a lower porosity. These attributes, coupled with the inherent stability of graphitic materials, make particulate graphite coatings especially suitable for applications at high temperatures. The advantages of using particulate graphite as a coating on QCMs are demonstrated by comparison to the performance of a few low-Tg polymers [i.e., poly(isobutylene) and poly(diphenoxyphosphazene)] and high-Tg polymers (i.e., polystyrene).

Crystallization↗
PubMed1990

Synthetic chemoreceptive membranes. Sensing bitter or odorous substances on a synthetic lipid multibilayer film by using quartz-crystal microbalances and electric responses.

Specific adsorptions of bitter or odorous substances on a synthetic lipid multibilayer matrix (2C18N+2C1/PSS-) were detected by observing frequency changes of a multibilayer-coated quartz-crystal microbalance (QCM). Partition coefficient (P) and diffusion constants (D) of these substances in the lipid matrix could be obtained quantitatively by using the QCM method. There were good correlations between partition coefficients of various bitter or odor substances to the synthetic multibilayer film on the QCM and the intensity of bitter tastes or olfactory receptions in humans: the stronger the intensity of a bitter substance or odorant, the greater the adsorption on the lipid matrix. This indicates that the lipid-coated QCM acts as a sensitive and selective sensor for bitter taste and odor. Electric responses (changes of membrane potential and membrane resistance) of the 2C18N+2C1/PSS-film occurred consecutively by the adsorption of these substances. The bitter or odor substance showing the stronger intensity induced membrane potential change in lower concentrations. It was found that bitter substances having sterically bulky molecular structures adsorb on the surface of the lipid matrix, and the phase-boundary potential of the membrane is thereby changed. On the contrary, odor substances with relatively small or slender structures can penetrate into the lipid matrix and cause reduction of the membrane resistance (the increase of ion permeability). The selective adsorption behavior of bitter and odor substances by molecular shapes was confirmed by adsorption studies of simple C9-10 hydrophobic alcohols having various molecular structures.

Adsorption↗
PubMed2001

Direct and quantitative detection of bacteriophage by "hearing" surface detachment using a quartz crystal microbalance.

We show that it is possible to detect specifically adsorbed bacteriophage directly by breaking the interactions between proteins displayed on the phage coat and ligands immobilized on the surface of a quartz crystal microbalance (QCM). This is achieved through increasing the amplitude of oscillation of the QCM surface and sensitively detecting the acoustic emission produced when the bacteriophage detaches from the surface. There is no interference from nonspecifically adsorbed phage. The detection is quantitative over at least 5 orders of magnitude and is sensitive enough to detect as few as 20 phage. The method has potential as a sensitive and low-cost method for virus detection.

Adsorption↗
PubMed2001

Self-assembled monolayers as the coating in a quartz piezoelectric crystal immunosensor to detect Salmonella in aqueous solution.

A new procedure based on the self-assembled monolayers (SAM) of alkanethiols was developed to immobilize antibodies onto gold electrodes of a quartz crystal microbalance (QCM) for detecting Salmonella paratyphi A. The procedure includes (1) chemisorption of 3-mercaptopropionic acid (MPA) at electrode surfaces to produce a carboxylic acid terminated monolayer, (2) activation by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosuccinimide (NHS) to generate a stable acyl amino ester intermediate, and (3) condensation of antibodies after aminolysis of the NHS adduct. Activation by two coupling agents (EDC and NHS) was shown to enhance the stability of the coating and facilitate the formation of a suitable intermediate to condense antibodies reproducibly and densely at the SAM, leading to high sensitivity and good precision of the developed immunosensor. With 50 min incubation under continuous monitoring, working ranges from 10(2) to 10(5) cells/mL with repeatability < 10% RSD were obtained. On the basis of S/N = 3, the detection limit was 1.7 x 10(2) cells/mL The MO2 piezoimmunosensor developed was specific to differentiate S. paratyphi A against Escherichia coli and other serogroups of Salmonella that are commonly present together.

Antibodies, Bacterial↗
PubMed2002

An aptamer-based quartz crystal protein biosensor.

We developed a quartz crystal biosensor designed to detect concentrations and ligand affinity parameters of free unlabeled proteins in real time. Using a model system with human IgE as the analyte and single-stranded DNA aptamers or an anti-IgE antibody as immobilized ligands, we could demonstrate that aptamers were equivalent to antibodies in terms of specificity and sensitivity. Both receptor types selectively detected 0.5 nmol/L of IgE. In addition, the aptamer receptors tolerated repeated affine layer regeneration after ligand binding and recycling of the biosensor with little loss of sensitivity. Because of the small size and nonprotein nature of the aptamers, they were immobilized in a dense, well-oriented manner, thus extending the linear detection range to 10-fold higher concentrations of IgE. In addition to demonstrating for the first time that an aptamer-based biosensor can specifically and quantitatively detect an analyte in various complex protein mixes, the aptamer-ligand proved to be relatively heat resistant and stable over several weeks. Since aptamers consist of nucleic acids, well-established chemistry can be applied to produce optimized affine layers on biosensors that may be developed to specifically detect proteins in solution for analysis of proteomes.

Antibodies, Anti-Idiotypic↗
PubMed2003

Analysis of NAD(P)+/NAD(P)H cofactors by imprinted polymer membranes associated with ion-sensitive field-effect transistor devices and Au-quartz crystals.

Specific recognition sites for the NAD(P)+ and NAD(P)H cofactors are imprinted in a cross-linked acrylamide-acrylamidophenylboronic acid copolymer membrane. The imprinted membranes, associated with pH-sensitive field-effect transistors (ISFETs) or Au-quartz piezoelectric crystals, enable the potentiometric or microgravimetric analysis of the oxidized NAD(P)+ cofactors and the reduced NAD(P)H cofactors, respectively. The NAD+- and NADP+-imprinted membranes associated with the ISFET allow the analysis of NAD+ and NADP+ with sensitivities that correspond to 15.0 and 18.0 mVdecade(-1) and detection limits of 4 x 10(-7) and 2 x 10(-7) M, respectively. The NADH- and NADPH-imprinted membranes associated with the ISFET device enable the analysis of NADH and NADPH with sensitivities that correspond to 24.2 and 21.8 mV x decade(-1) and lower detection limits that are 1 x 10(-7) and 2 x 10(-7) M, respectively. The ISFET devices functionalized with the NADH and NADPH membranes are employed in the analysis of the biocatalyzed oxidation of lactic acid and ethanol in the presence of lactate dehydrogenase and alcohol dehydrogenase, respectively.

Binding Sites↗
PubMed2003

Detection of polycyclic aromatic hydrocarbons using quartz crystal microbalances.

A chemically coated piezoelectric sensor has been developed for the determination of PAHs in the liquid phase. An organic monolayer attached to the surface of a gold electrode of a quartz crystal microbalance (QCM) via a covalent thiol-gold link complete with an ionically bound recognition element has been produced. This study has employed the PAH derivative 9-anthracene carboxylic acid which, once bound to the alkane thiol, functions as the recognition element. Binding of anthracene via pi-pi interaction has been observed as a frequency shift in the QCM with a detectability of the target analyte of 2 ppb and a response range of 0-50 ppb. The relative response of the sensor altered for different PAHs despite pi-pi interaction being the sole communication between recognition element and analyte. It is envisaged that such a sensor could be employed in the identification of key marker compounds and, as such, give an indication of total PAH flux in the environment.

Anthracenes↗
PubMed2004

Separation of long DNA molecules by quartz nanopillar chips under a direct current electric field.

We have established the nanofabrication technique for constructing nanopillars with high aspect ratio (100-500 nm diameter and 500-5000 nm tall) inside a microchannel on a quartz chip. The size of pillars and the spacing between pillars are designed as a DNA sieving matrix for optimal analysis of large DNA fragments over a few kilobase pairs (kbp). A chip with nanopillar channel and simple cross injector was developed based on the optimal design and applied to the separation of DNA fragments (1-38 kbp) and large DNA fragments (lambda DNA, 48.5 kbp; T4 DNA, 165.6 kbp) that are difficult to separate on conventional gel electrophoresis and capillary electrophoresis without a pulsed-field technique. DNA fragments ranging from 1 to 38 kbp were separated as clear bands, and furthermore, the mixture of lambda DNA and T4 DNA was successfully separated by a 380-microm-long nanopillar channel within only 10 s even under a direct current (dc) electric field. Theoretical plate number N of the channel (380-1450 microm long) was 1000-3000 (0.7 x 10(6)-2.1 x 10(6) plates/m). A single DNA molecule observation during electrophoresis in a nanopillar channel revealed that the optimal nanopillars induced T4 DNA to form a narrow U-shaped conformation during electrophoresis whereas lambda DNA kept a rather spherical conformation. We demonstrated that, even under a dc electric field, the optimal nanopillar dimensions depend on a gyration radius of DNA molecule that made it possible to separate large DNA fragments in a short time.

DNA↗
PubMed2004

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.

Adenosine Triphosphatases↗