Pocketable Biosensor Based on Quartz-Crystal Microbalance and Its Application to DNA Detection.

Quartz-crystal microbalance (QCM) is a technique that can measure nanogram-order masses. When a receptor is immobilized on the sensor surface of a QCM device, the device can detect chemical molecules captured by the mass change. Although QCM devices have been applied to biosensors that detect biomolecules without labels for biomolecular interaction analysis, most highly sensitive QCM devices are benchtop devices. We considered the fabrication of an IC card-sized QCM device that is both portable and battery-powered. Its miniaturization was achieved by repurposing electronic components and film batteries from smartphones and wearable devices. To demonstrate the applicability of the card-sized QCM device as a biosensor, DNA-detection experiments were performed. The card-sized QCM device could detect specific 10-mer DNA chains while discerning single-base differences with a sensitivity similar to that of a conventional benchtop device. The card-sized QCM device can be used in laboratories and in various other fields as a mass sensor.

Kinetics of iterative carbohydrate transfer to polysaccharide catalyzed by chondroitin polymerase on a highly sensitive flow-type 27†MHz quartz-crystal microbalance.

Using a highly sensitive flow-type 27 MHz quartz crystal microbalance, we could detect a small mass change during stepwise and alternating one-sugar transfer of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) to an acceptor, catalyzed by chondroitin polymerase from Escherichia coli strain K4 (K4CP), and analyze the elongation mechanism of K4CP. K4CP was found to bind strongly to a chondroitin acceptor (K(d)=0.97 µM). Although the binding affinity and the catalytic rate constant for each monomer were considerably different, the apparent catalytic efficiency (k(cat)/K(m)) was similar (6.3×10(4) M(-1) s(-1) for GlcA transfer and 3.4×10(4) M(-1) s(-1) for the GalNAc transfer). This is reasonable for the smooth alternating elongation of GlcA and GalNAc on the acceptor. This is the first study to report the determination of kinetic parameters for enzymatic, alternated, sugar elongation.

In situ monitoring of a trace intermediate during DNA phosphorylation by T4 polynucleotide kinase for transient kinetic studies.

Formation and decomposition of the enzyme-substrate (ES) complex during phosphorylation by T4 polynucleotide kinase (T4 PNK) of dsDNAs were monitored using a highly sensitive quartz crystal microbalance (QCM) to determine kinetic parameters, which were characterised in comparison with those of other enzymes such as DNA polymerase and exo- and endo-nucleases.

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.

Simultaneous anomalous reflection and quartz-crystal microbalance measurements of protein bindings on a gold surface.

Protein bindings onto a gold surface were detected simultaneously by QCM (delta F(water)) and anomalous reflection (deltaR) of gold on the same surface in aqueous solutions; the obtained delta F(water)/deltaR values correlated with surface areas and viscosity of proteins.

Hydration and energy dissipation measurements of biomolecules on a piezoelectric quartz oscillator by admittance analyses.

By using a 27-MHz piezoelectric quartz oscillator connected with a vector network analyzer, we obtained resonance frequency decreases (-DeltaFwater) and energy dissipation increases (DeltaDwater) during binding of biotinylated bovine serum albumin, biotinylated ssDNA, biotinylated dsDNA, and biotinylated pullulan to a NeutrAvidin-immobilized 27-MHz quartz crystal microbalance (QCM) plate in aqueous solution, as well as in the wet air phase (98% humidity, -DeltaFwet and DeltaDwet) and in the dry air phase (-DeltaFair and DeltaDair). -DeltaFwater indicates the total mass of the molecule, bound water, and vibrated water in aqueous solutions. -DeltaFwet indicates the total mass of the molecule and bound water. -DeltaFair simply shows the real mass of the molecule on the QCM. In terms of results, (-DeltaFwet)/(-DeltaFair) values indicated the bound water ratios per unit biomolecular mass were on the order of pullulan (2.1-2.2) > DNAs = proteins (1.4-1.6) > polystyrene (1.0). The (-DeltaFwater)/(-DeltaFair) values indicated the hydrodynamic water (bound and vibrated water) ratios per unit biomolecular mass were on the order of dsDNA (6.5) > ssDNA = pullulan (3.5-4.4) > proteins (2.4-2.5) > polystyrene (1.0). Energy dissipation parameters per unit mass in water (DeltaDwater/(-DeltaFair)) were on the order of pullulan > dsDNA > ssDNA > proteins > polystyrene. Energy dissipation in the wet and dry air phases (DeltaDwet and DeltaDair) were negligibly small, which indicates even these biomolecules act as elastic membranes in the air phase (without aqueous solution). We obtained a good linear relationship between [(-DeltaFwater)/(-DeltaFair) - 1], which is indicative of hydration and DeltaDwater/(-DeltaFair) of proteins. The aforementioned values suggest that the energy dissipation of proteins was mainly caused by hydration and that proteins themselves are elastic molecules without energy dissipation in aqueous solutions. On the contrary, plots in cases of denatured proteins, DNAs, and pullulans were relatively deviant toward the large hydration and energy dissipation from the theoretical line as perfect elastic materials, meaning that the large energy dissipation occurs because of viscoelastic properties of denatured proteins, linear DNAs, and pullulans in the water phase, in addition to energy dissipation due to the hydration of molecules. These two parameters could characterize various biomolecules with structural properties in aqueous solutions.

Detection of oligosaccharides labeled with cyanine dyes using matrix-assisted laser desorption/ionization mass spectrometry.

The sensitivity of oligosaccharides in mass spectrometry lags far behind that of peptides. This is a critical factor in realizing the high-throughput analysis of posttranslational modifications in proteomics. We here described that hydrazide derivatives of cyanine dyes (Cy3, Cy5) with a positive charge made excellent labeling reagents for the detection of oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry. Cy3-labeled standard N-glycan could be detected at 200 amol on the MALDI target plate in reflectron mode without any purification procedures after the labeling reaction, which may meet the level of sensitivity required in proteome research. Despite the general recognition that the production of signals of oligosaccharides under MALDI conditions would be highly dependent on the matrix, most of the known N-glycans from chicken ovalbumin could be detected upon Cye derivatization nearly independent of the kind of matrix tested (e.g., nor-harman, 2,5-dihydroxybenzoic acid and alpha-cyano-4-hydroxycinnamic acid) without spoiling the signal strength. Postsource decay afforded simple spectra mainly consisting of Y-type fragment ions, thus simplifying the sequence analysis. In-source decay afforded a similar fragmentation pattern only when acidic matrixes were used. In addition, this derivatization technique was successfully applied to the profiling of N-glycans of gel-separated glycoproteins.

A versatile planar QCM-based sensor design for nonlabeling biomolecule detection.

Despite high theoretical sensitivity, low-cost manufacture, and compactness potentially amenable to lab-on-a-chip use, practical hurdles have stymied the application of the quartz crystal microbalance (QCM) for aqueous applications such as detection of biomolecular interactions. The chief difficulty lies in achieving a sufficiently stable resonance signal in the presence of even minute fluctuations in hydrostatic pressure. In this work, we present a novel versatile planar sensor chip design (QCM chip) for a microliter-scale on-line biosensor. By sealing the quartz resonator along its edges to a flat, solid support, we provide uniform support for the crystal face not exposed to solvent, greatly decreasing deformation of the crystal resonator under hydrostatic pressure. Furthermore, this cassette design obviates the need for direct handling when exchanging the delicate quartz crystal in the flow cell. A prototype 27-MHz sensor signal exhibited very low noise over a range of flow rates up to 100 microL/min. In contrast, signals obtained from a conventional QCM sensor employing an O-ring-based holder were less stable and deteriorated even further with increasing flow rate. Additional control designs with intermediate amounts of unsupported undersurface yielded intermediate levels of stability, consistent with the interpretation that deformation of the crystal resonator under fluctuating hydraulic pressure is the chief source of noise. As a practical demonstration of the design's high effective sensitivity, we readily detected interaction between myoglobin and surface-bound antibody.