George Papadakis

Quantitative Determination of Size and Shape of Surface-Bound DNA Using an Acoustic Wave Sensor

DNA bending plays a significant role in many biological processes, such as gene regulation, DNA replication, and chromosomal packing. Understanding how such processes take place and how they can, in turn, be regulated by artificial agents for individual oriented therapies is of importance to both biology and medicine. In this work, we describe the application of an acoustic wave device for characterizing the conformation of DNA molecules tethered to the device surface via a biotin-neutravidin interaction. The acoustic energy dissipation per unit mass observed upon DNA binding is directly related to DNA intrinsic viscosity, providing quantitative information on the size and shape of the tethered molecules. The validity of the above approach was verified by showing that the predesigned geometries of model double-stranded and triple-helix DNA molecules could be quantitatively distinguished: the resolution of the acoustic measurements is sufficient to allow discrimination between same size DNA carrying a bent at different positions along the chain. Furthermore, the significance of this analysis to the study of biologically relevant systems is shown during the evaluation of DNA conformational change upon protein (histone) binding.

Construction of three-dimensional biomolecule structures employing femtosecond lasers

The authors demonstrate here a method for three-dimensional patterning of proteins and other biological molecules. The method employs femtosecond-laser-induced three-photon polymerization, a technique which enables the construction of arbitrary two- and three-dimensional structures of submicron resolution. Biotin is subsequently attached to the three-dimensional (3D) structures via UV-activated cross-linking. The integrity of the photolytically immobilized biotin is confirmed by detecting the binding of fluorescently labeled avidin via fluorescence microscopy and via a surface acoustic sensor technique. In all, the technique opens the way for the fabrication of structures with a wide range of biomaterials as well as studying their dynamics within complex 3D structures.

Triple-helix DNA structural studies using a Love wave acoustic biosensor

The development of sensors for detecting the conformation of surface-attached molecules is an emerging field with significance in the pharmaceutical industry and in drug design. In this work, triplex-forming oligos (TFOs), a separate class of non-natural DNA bending agents that can affect the mechanical properties of DNA through the formation of triple-helical structures of specific conformation and/or flexibility, are used as a model system in combination with an acoustic biosensor to determine molecular geometrical features. In practice, the degree of bending of a specific DNA target caused by a particular TFO was evaluated by measuring the ratio of acoustic energy change over phase change observed during the binding of pre-formed triplex DNA molecules to the device surface. The DNA bending angle derived via acoustic measurements is in excellent agreement with previously reported values using molecular biology techniques. The reported acoustic technique appears quite appealing for the biophysical study of DNA molecules providing rapid qualitative and quantitative information, at the same time holding promise to be developed as a high-throughput method for the evaluation of DNA conformational changes.

Development of a combined surface plasmon resonance/surface acoustic wave device for the characterization of biomolecules

It is known that acoustic sensor devices, if operated in liquid phase, are sensitive not just to the mass of the analyte but also to various other parameters, such as size, shape, charge and elastic constants of the analyte as well as bound and viscously entrained water. This can be used to extract valuable information about a biomolecule, particularly if the acoustic device is combined with another sensor element which is sensitive to the mass or amount of analyte only. The latter is true in good approximation for various optical sensor techniques. This work reports on the development of a combined surface plasmon resonance/surface acoustic wave sensor system which is designed for the investigation of biomolecules such as proteins or DNA. Results for the deposition of neutravidin and DNA are reported.

The intrinsic viscosity of linear DNA

We measured the intrinsic viscosity of very small synthetic DNA molecules, of 20-395 base pairs, and incorporated them in a nearly complete picture for the whole span of molecular weights reported in the literature to date. A major transition is observed at M approximately 2 × 10(6) . It is found that in the range of approximately 7 × 10(3) ≤ M ≤ 2 × 10(6) , the intrinsic viscosity scales as [η] approximately M(1.05) , suggesting that short DNA chains are not as rigid as generally thought. The corresponding scaling for the range of 2 × 10(6) ≤ M ≤ 8 × 10(10) is [η] approximately M(0.69) . A comparison of our results with existing equations, for much narrower data distributions, is made, and the agreement is very satisfactory considering the huge range of data analyzed here. Experimental concerns such as the effect of ionic strength, polydispersity, temperature, and shear rate are discussed in detail. Some issues concerning the Huggins coefficient, polymer chain stiffness, and the relationship between the Mark-Houwink constants K, α are also presented; it is found that log K = 1.156 - 6.19α.

Bacteria Murmur: Application of an Acoustic Biosensor for Plant Pathogen Detection.

A multi-targeting protocol for the detection of three of the most important bacterial phytopathogens, based on their scientific and economic importance, was developed using an acoustic biosensor (the Quartz Crystal Microbalance) for DNA detection. Acoustic detection was based on a novel approach where DNA amplicons were monitored and discriminated based on their length rather than mass. Experiments were performed during real time monitoring of analyte binding and in a direct manner, i.e. without the use of labels for enhancing signal transduction. The proposed protocol improves time processing by circumventing gel electrophoresis and can be incorporated as a routine detection method in a diagnostic lab or an automated lab-on-a-chip system for plant pathogen diagnostics.

Characterization of DNA-Hv1 histone interactions; discrimination of DNA size and shape.

We have studied the formation of histone Hv1–DNA complexes using an acoustic biosensor and AFM imaging. Our results show that DNA and histone molecules aggregate into amorphous accumulations which form a compact rigid layer on the sensors surface. By measuring changes in the acoustic wave amplitude, it was possible to titrate surface bound DNA with Hv1 and discriminate between DNA molecules of different size and shape. From the kinetic analysis of real time data, K eq was found equal to 3 × 105 M−1.

Effects of tigecycline and daptomycin on murine gut colonization by Candida albicans.

Adult male Crl:CD1 (ICR) BR mice were fed chow containing Candida albicans or regular chow. Both groups were subsequently given tigecycline or daptomycin or normal saline subcutaneously for 10 days. To determine the effect on the stool yeast concentration, stool cultures were performed immediately before, at the end, and 1 week after discontinuation of treatment. Candida‐colonized mice treated with tigecycline or daptomycin had higher counts of the yeast in their stools than control C. albicans‐colonized animals treated with saline. Tigecycline caused a significant increase of 2.1 log10 CFU g−1 of stools in C. albicans concentration, while daptomycin caused a minor increase of 0.4 log10 CFU g−1 of stools. Mice fed regular chow and treated with the study antibiotics or saline did not have any Candida in their stools. Dissemination of Candida was not detected in any animal. These data suggest that tigecycline induces a substantial increase in the intestinal concentration of C. albicans, while daptomycin causes only a minimal increase. However, these increases are not associated with dissemination of the yeast to internal organs. Clinical studies in humans are needed to validate our findings, especially in patients at risk of developing disseminated candidosis.

Screening for mutations in BRCA1 and BRCA2 genes by measuring the acoustic ratio with QCM

Screening for mutations in the tumor-suppressor genes BRCA1 and BRCA2 is of great importance for breast and ovarian cancer prevention. We describe a methodology for mutation screening and detection based on acoustic wave devices. In particular, we detect four mutations located in BRCA1 and BRCA2 genes using the quartz crystal microbalance technique. The detection is based on measurements of the acoustic ratio of dissipation versus frequency change (ΔD/ΔF) of double-stranded DNA molecules bound to the device surface that are produced after PCR amplification and restriction digestion; the acoustic ratio has been shown to be a measure of the intrinsic viscosity of the attached molecules, which, in turn, depends on the size of the dsDNAs. Novel features of this approach are the lack of a hybridization step, the label free sensing of the length, rather than mass, of the DNA molecules and the direct detection of the digested DNA products without prior purification. The method is generic, simple and capable of detecting single base mutations to long genomic rearrangements; it is also suitable and applicable to a Lab-on-a-chip concept.

Acoustic wave biosensor for detecting DNA conformation; A study with QCM-D

This work describes the development of a real-time rapid technique for the quantitative characterization of DNA intrinsic curvature and conformational changes. We present a new approach where a label-free acoustic biosensor (QCM-D) is used for the detection of DNA conformation independently of bound DNA mass. DNA molecules bind to a neutravidin modified device surface by use of a biotin linker. Acoustic results, expressed as the ratio of dissipation over frequency change, DeltaD/Deltaf, provide insight on (intrinsic) viscosity changes [eta] occurring at the sensor/liquid interface as a result of DNA binding. Quantitative results regarding both the size and shape of DNAs were obtained, for the first time, by combining acoustic measurements with a mathematical treatment of solution viscosity theory. More specifically, we show that: DeltaD/Deltaf ~ [eta]. Acoustic measurements can clearly distinguish between ds-DNAs of same shape (rod) but various sizes (lengths of 20 up to 198 bp) and, of same mass and size (90 bp) but in various shapes (ldquostraightrdquo, ldquobentrdquo, ldquotrianglerdquo). Our results agree well with published qualitative observations and suggest that acoustic biosensors can be developed into a powerful tool for studying DNA conformational changes.