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The objective of this work is to present a methodology for the selection of nanoparticles such as liposomes to be used as acoustic probes for the detection of very low concentrations of DNA. Liposomes, applied in the past as mass amplifiers and detected through frequency measurement, are employed in the current work as probes for energy-dissipation enhancement. Because the dissipation signal is related to the structure of the sensed nanoentity, a systematic investigation of the geometrical features of the liposome/DNA complex was carried out. We introduce the parameter of dissipation capacity by which several sizes of liposome and DNA structures were compared with respect to their ability to dissipate acoustic energy at the level of a single molecule/particle. Optimized 200 nm liposomes anchored to a dsDNA chain led to an improvement of the limit of detection (LoD) by 3 orders of magnitude when compared to direct DNA detection, with the new LoD being 1.2 fmol (or 26 fg/μL or 2 pM). Dissipation monitoring was also shown to be 8 times more sensitive than the corresponding frequency response. The high versatility of this new methodology is demonstrated in the detection of genetic biomarkers down to 1-2 target copies in real samples such as blood. This study offers new prospects in acoustic detection with potential use in real-world diagnostics.

[EN] Regular screening of point mutations is of importance to cancer management and treatment selection. Although techniques like next-generation sequencing and digital polymerase chain reaction (PCR) are available, these are lacking in speed, simplicity, and cost-effectiveness. The development of alternative methods that can detect the extremely low concentrations of the target mutation in a fast and cost-effective way presents an analytical and technological challenge. Here, an approach is presented where for the first time an allele-specific PCR (AS-PCR) is combined with a newly developed high fundamental frequency quartz crystal microbalance array as biosensor for the amplification and detection, respectively, of cancer point mutations. Increased sensitivity, compared to fluorescence detection of the AS-PCR amplicons, is achieved through energy dissipation measurement of acoustically ¿lossy¿ liposomes binding to surface-anchored dsDNA targets. The method, applied to the screening of BRAF V600E and KRAS G12D mutations in spiked-in samples, was shown to be able to detect 1 mutant copy of genomic DNA in an excess of 104 wild-type molecules, that is, with a mutant allele frequency (MAF) of 0.01%. Moreover, validation of tissue and plasma samples obtained from melanoma, colorectal, and lung cancer patients showed excellent agreement with Sanger sequencing and ddPCR; remarkably, the efficiency of this AS-PCR/acoustic methodology to detect mutations in real samples was demonstrated to be below 1% MAF. The combined high sensitivity and technology-readiness level of the methodology, together with the ability for multiple sample analysis (24 array biochip), cost-effectiveness, and compatibility with routine workflow, make this approach a promising tool for implementation in clinical oncology labs for tissue and liquid biopsy., This work was supported by the European Union's Horizon H2020-FETOPEN-1-2016-2017 under grant agreement no. 737212 (CATCH-U-DNA).

The ability to derive information on the conformation of surface attached biomolecules by using simple techniques such as biosensors is currently considered of great importance in the fields of surface science and nanotechnology. Here we present a nanoshape sensitive biosensor where a simple mathematical expression is used to relate acoustic measurements to the geometrical features of a surface-attached biomolecule. The underlying scientific principle is that the acoustic ratio (ΔD/ΔF) is a measure of the hydrodynamic volume of the attached entity, mathematically expressed by its intrinsic viscosity [η]. A methodology is presented in order to produce surfaces with discretely bound biomolecules where their native conformation is maintained. Using DNA anchors we attached a spherical protein (streptavidin) and a rod-shaped DNA (47bp) to a quartz crystal microbalance (QCM) device in a suspended way and predicted correctly through acoustic measurements their conformation, i.e., shape and length. The methodology can be widely applied to draw conclusions on the conformation of any biomolecule or nanoentity upon specific binding on the surface of an acoustic wave device.

While relatively successful theoretical models exist to link the signal of an acoustic biosensor to the mass and viscoelasticity of film-forming adsorbates, this is not the case for surfaces discretely covered by analytes such as proteins or DNA. This work extends and examines in detail the underlying physics of a previously published theory that links the response of microbalance sensors to the shapes and sizes of attached molecules, shedding light on the hydrodynamics and energy-dissipation processes. Their analysis offers further physical insight, and could be exploited to improve detection approaches relevant to biology and biomedicine.

The sensitivity of QCM-D to molecular hydrodynamic properties is applied in this work to study conformational changes of the intrinsically disordered protein ZipA. Acoustic measurements can clearly follow ZipA's unstructured domain expansion and contraction with salt content and be correlated with changes in the hydrodynamic radius of 1.8 nm or less.

In this work we provide strong experimental evidence for the hydrodynamic nature of the acoustic wave/biomolecule interaction at a solid/liquid interface. By using a wide range of DNAs of various sizes and by assuming DNA attachment as discrete particles through a neutravidin/biotin link, we prove experimentally that the acoustic ratio (dissipation/frequency) is directly related to the molecules' intrinsic viscosity [η]. The relationship of [η] to the size and shape of biomolecules is described in general and more specifically for linear dsDNA; equations are derived linking the measured acoustic ratio to the number of dsDNA base pairs for two acoustic sensors, the QCM and Love-wave devices operating at a frequency of 35 and 155 MHz, respectively. Single-stranded DNAs were also tested and shown to fit well to the equation derived for the double-stranded molecules while new insight is provided on their conformation on a surface. Other types of DNA are also shown to fit the proposed model. The current work establishes a new way of viewing acoustic sensor data and lays down the groundwork for a surface technique where quantitative information can be obtained at the nanometer scale regarding the shape and size, i.e., conformation of biomolecules at an interface.

DNA hybridization studies at surfaces normally rely on the detection of mass changes as a result of the addition of the complementary strand. In this work we propose a mass-independent sensing principle based on the quantitative monitoring of the conformation of the immobilized single-strand probe and of the final hybridized product. This is demonstrated by using a label-free acoustic technique, the quartz crystal microbalance (QCM-D), and oligonucleotides of specific sequences which, upon hybridization, result in DNAs of various shapes and sizes. Measurements of the acoustic ratio ΔD/ΔF in combination with a "discrete molecule binding" approach are used to confirm the formation of straight hybridized DNA molecules of specific lengths (21, 75, and 110 base pairs); acoustic results are also used to distinguish between single- and double-stranded molecules as well as between same-mass hybridized products with different shapes, i.e., straight or "Y-shaped". Issues such as the effect of mono- and divalent cations to hybridization and the mechanism of the process (nucleation, kinetics) when it happens on a surface are carefully considered. Finally, this new sensing principle is applied to single-nucleotide polymorphism detection: a DNA hairpin probe hybridized to the p53 target gene gave products of distinct geometrical features depending on the presence or absence of the SNP, both readily distinguishable. Our results suggest that DNA conformation probing with acoustic wave sensors is a much more improved detection method over the popular mass-related, on/off techniques offering higher flexibility in the design of solid-phase hybridization assays.

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.

Acoustic devices were employed to characterize variations in the mechanical properties (density and viscoelasticity) of liposomes composed of 1-oleoyl-2-palmitoyl- sn-glycero-3-phosphocholine (POPC) and cholesterol. Liposome properties were modified in three ways. In some experiments, the POPC/cholesterol ratio was varied prior to deposition on the device surface. Alternatively, the ratio was changed in situ via either insertion of cholesterol or removal of cholesterol with beta-cyclodextrin. This was done for liposomes adsorbed directly on the device surface and for liposomes attached via a biotin-terminated poly(ethylene glycol) linker. The acoustic measurements make use of two simultaneous time-resolved signals: one signal is related to the velocity of the acoustic wave, while the second is related to dissipation of acoustic energy. Together, they provide information not only about the mass (or density) of the probed medium but also about its viscoelastic properties. The cholesterol-induced increase in the surface density of the lipid bilayer was indeed observed in the acoustic data, but the resulting change in signal was larger than expected from the change in surface density. In addition, increasing the bilayer resistance to stretching was found to lead to a greater dissipation of the acoustic energy. The acoustic response is assessed in terms of the possible distortions of the liposomes and the known effects of cholesterol on the mechanical properties of the lipid bilayer that encloses the aqueous core of the liposome. To aid the interpretation of the acoustic response, it is discussed how the above changes in the lipid bilayer will affect the effective viscoelastic properties of the entire liposome/solvent film on the scale of the acoustic wavelength. It was found that the acoustic device is very sensitive to the mechanical properties of lipid vesicles; the response of the acoustic device is explained, and the basic underlying mechanisms of interaction are identified.

By using an acoustic wave methodology that allows direct sensing of biomolecular conformations, we achieved the detection of multiple target DNAs using a single probe, exploiting the fact that each bound target results in a hybridized product of a different shape.

High-precision differential scanning calorimetry (DSC) and circular dichroism (CD) have been employed to study the thermal unfolding of chitinase 40 (Chi40) from Streptomyces thermoviolaceus. Chi40 belongs to family 18 of glycosyl hydrolase superfamily bearing a catalytic domain with a "TIM barrel"-like fold, which exhibits deviations from the (beta/alpha)8 fold. The thermal unfolding is reversible at pH = 8.0 and 9.0. The denatured state is characterized by extensive structural changes with respect to the native. The process is characterized by slow relaxation kinetics. Even slower refolding rates are recorded upon cooling. It is shown that the denaturation calorimetric data obtained at slow heating rate (0.17 K/min) are in excellent agreement with equilibrium data obtained by extrapolation of the experimental results to zero scanning rate. Analysis of the DSC results reveals that the experimental data can be successfully fitted using either a non-two-state sequential model involving one equilibrium intermediate, or an independent transitions model involving the unfolding of two Chi40 energetic domains to intermediate states. The stability of the native state with respect to the final denatured state is estimated, deltaG = 24.0 kcal/mol at 25 degrees C. The thermal results are in agreement with previous findings from chemical denaturation studies of a wide variety of (beta/alpha)8 barrel proteins, that their unfolding is a non-two-state process, always involving at least one unfolding intermediate.

The interaction of PC-based unilamellar liposomes incorporating the complementary 5,5 di-n-dodecylbarbituric acid (DBA) or 9-hexadecyladenine (HA) in various molar ratios has been investigated using imaging, spectroscopicand calorimetric techniques. Liposomal interaction was established by turbidimetry, AFM, and optical microscopy and also by isothermal titration calorimetry. For temperatures below the main lipid phase transition, the results provide evidence for hydrogen-bonding between the adenine and the barbituric acid moieties at the lipid-water-lipid interfaces with a stoichiometry ratio of 1:1. The interaction does not lead to disruption of the liposomes but instead it affords larger aggregates. The role of cholesterol in the DBA-HA binding, at high concentrations in the PC:DBA and PC:HA bilayers has also been investigated. Evidence is found that the presence of cholesterol (at molar concentration 2:1 relative to PC) alters the DBA/HA binding mechanism. For all the systems studied, no liposomal binding could be calorimetrically detected above the main lipid phase transition temperature.

The effect of spermine on proton transport across large unilamellar liposomes containing incorporated complexes of the PSII antenna has been studied with the application of a pH-sensitive dye entrapped inside the vesicles. Both monomeric LHCbs and trimeric LHCII increased the permeability of proteoliposomes to protons when in a partly aggregated state within the lipid membrane. We have previously shown that a spermine-induced conformational change in LHCII results in its aggregation and ultimately in the enhancement of excitation energy as heat (qE). In this paper, spermine-induced aggregation of LHCII was found to facilitate proton transport across the proteoliposomes, indicating that a second protective mechanism (other than qE) might exist and might be regulated in vivo by polyamines when photosynthesis is saturated in excess light.

Liposomes based on hydrogenated phosphatidylcholine (PC) incorporating dihexadecyl phosphate (DHP) and 1-(4-(dihexadecylcarbamoyl) butyl)guanidinium p-toluene sulfonate (DBG), respectively, were mixed in aqueous dispersion at room temperature, and their molecular recognition effectiveness was investigated by turbidimetric measurements, microscopy, DSC, and isothermal titration calorimetry. Because of the high binding efficiency of the incorporated recognizable lipids, accurate results could be obtained for low DHP/PC and DBG/PC molar ratios (19:1), especially for enthalpy change measurements. The liposome−liposome interaction was further investigated as a function of the cholesterol content in mixed (PC−cholesterol) liposomes. At appropriate cholesterol concentrations, the organization of the recognizable moieties within the liquid-ordered phase of the bilayer membrane enhances molecular recognition at the liposomal interface.

A series of oxorhenium(V) SNS/S mixed-ligand complexes [ReO(Ln/L)] carrying different types of tridentate ligands (Ln) and the same monodentate coligand (L) [L = C6H5S, L1 = C2H5N(CH2CH2S)2 (1), L2 = (C2H5)2NCH2CH2N(CH2CH2S)2 (2), L3 = C2H5SCH2CH2N(CH2CH2S)2 (3), and L4 = 2,6-(SCH2)2NC5H3 (4)] have been synthesized and characterized by spectroscopic methods and elemental analyses. X-ray structure determination was performed for complexes 3 and 4. Complex 3 adopts the expected distorted trigonal bipyramidal geometry around the metal in a syn configuration, while complex 4, due to the aromatic character of the nitrogen of the SNS donor-atom set, exhibits a distorted square pyramidal geometry. The interaction of complexes 1−4 with glutathione (GSH) was studied by high-performance liquid chromatography, revealing the rapid formation of the respective daughter complexes 5−8, wherein the L coligand has been substituted by GS. The daughter complexes 5−8 have been characterized by ES-MS and 2D NMR spectroscopy. K...

Application of PCR to multiplexing assays is not trivial; it requires multiple fluorescent labels for amplicon detection and sophisticated software for data interpretation. Alternative PCR-free methods exploiting new concepts in nanotechnology exhibit high sensitivities but require multiple labeling and/or amplification steps. Here, we propose to simplify the problem of simultaneous analysis of multiple targets in genetic assays by detecting directly the conformation, rather than mass, of target amplicons produced in the same PCR reaction. The new methodology exploits acoustic wave devices which are shown to be able to characterize in a fully quantitative manner multiple double stranded DNAs of various lengths. The generic nature of the combined acoustic/PCR platform is shown using real samples and, specifically, during the detection of SNP genotyping in Anopheles gambiae and gene expression quantification in treated mice. The method possesses significant advantages to TaqMan assay and real-time PCR regarding multiplexing capability, speed, simplicity and cost.

A novel biophysical approach in combination with an acoustic device is demonstrated as a sensitive, rapid, and label-free technique for characterizing various structures of the DNA Holliday Junction (J1) nanoswitch. We were successful in discriminating the "closed" from the "open" state, as well as confirming that the digestion of the J1 junction resulted in the two, anticipated, rod-shaped, 20 bp long fragments. Furthermore, we propose a possible structure for the ∼10 nm long (DNA58) component participating in the J1 assembly. This work reveals the potential of acoustic devices as a powerful tool for molecular conformation studies.

Acoustic sensors probe the response of a thin layer to the mechanical displacement associated with an acoustic wave. Acoustic measurements provide two simultaneous time-resolved signals; one signal is related to the velocity or frequency of the acoustic wave and is mainly a function of adsorbed mass, while the second signal, related to the oscillation amplitude, is associated with energy dissipation and is a function of the viscoelastic properties of the adsorbed layer. The methods described in this chapter explore the relationship between the acoustic measurements of adsorbed liposomes and the mechanical properties of the lipid bilayer. This is carried out using a well-characterized model system consisting of liposomes prepared from an unsaturated phospholipid and a range of mole fractions of cholesterol. Real-time acoustic measurements are shown to be sensitive to changes in the liposome cholesterol content, regardless of the mode of attachment of the liposome to the device surface. This sensitivity is not due to changes in the density of the bilayer, or to changes in the extent of liposome-surface interactions, thus leaving the mechanical properties of the bilayer as the feature that is probably being measured. Some mechanisms by which the acoustic response could be generated are suggested in this chapter.

Two different types of acoustic sensors, a surface acoustic wave device supporting a Love-wave (Love-SAW) and a quartz crystal microbalance system with dissipation (QCM-D), were used to demonstrate the potential of acoustic devices to probe the binding of a cell membrane receptor to an immobilized ligand. The class I Major Histocompatibility Complex molecule HLA-A2 on the surface of whole cells and anti-HLA monoclonal antibodies immobilized on the sensor were used as an interaction pair. Acoustic measurements consisted of recording the energy and velocity or frequency of the acoustic wave. Results showed that both devices could detect the number of cells in solution as well as the cells bound to the surface. In addition, the Love-wave sensor, which can sense binding events within the relatively short distance of approximately 50 nm from the device surface, was sensitive to the number of bonds formed between the cell membrane and the device surface while the QCM-D, which can sense deeper within the liquid, was found to respond well to stimuli that affected the cell membrane rigidity (cytochalasin D treatment). The above results suggest that acoustic biosensors can be a powerful tool in the study of cell/substrate interactions and acoustic devices of different type can be used in a complementary way.

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.

High-precision differential scanning calorimetry (DSC) and circular dichroism (CD) have been employed to study the thermal unfolding of chitinase 40 (Chi40) from Streptomyces thermoviolaceus. Chi40 belongs to family 18 of glycosyl hydrolase superfamily bearing a catalytic domain with a "TIM barrel"-like fold, which exhibits deviations from the (β/α) 8 fold. The thermal unfolding is reversible at pH = 8.0 and 9.0. The denatured state is characterized by extensive structural changes with respect to the native. The process is characterized by slow relaxation kinetics. Even slower refolding rates are recorded upon cooling. It is shown that the denaturation calorimetric data obtained at slow heating rate (0.17 K/min) are in excellent agreement with equilibrium data obtained by extrapolation of the experimental results to zero scanning rate. Analysis of the DSC results reveals that the experimental data can be successfully fitted using either a nontwo-state sequential model involving one equilibrium intermediate, or an independent transitions model involving the unfolding of two Chi40 energetic domains to intermediate states. The stability of the native state with respect to the final denatured state is estimated, ΔG = 24.0 kcal/mol at 25°C. The thermal results are in agreement with previous findings from chemical denaturation studies of a wide variety of (β/α)8 barrel proteins, that their unfolding is a nontwo-state process, always involving at least one unfolding intermediate. © 2006 Wiley-Liss, Inc.