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Surface plasmon resonance (SPR) and Love wave (LW) surface acoustic wave (SAW) sensors have been established as reliable biosensing technologies for label-free, real-time monitoring of biomolecular interactions. This work reports the development of a combined SPR/LW-SAW platform to facilitate simultaneous optical and acoustic measurements for the investigation of biomolecules binding on a single surface. The system's output provides recordings of two acoustic parameters, phase and amplitude of a Love wave, synchronized with SPR readings. We present the design and manufacturing of a novel experimental set-up employing, in addition to the SPR/LW-SAW device, a 3D-printed plastic holder combined with a PDMS microfluidic cell so that the platform can be used in a flow-through mode. The system was evaluated in a systematic study of the optical and acoustic responses for different surface perturbations, i.e., rigid mass loading (Au deposition), pure viscous loading (glycerol and sucrose solutions) and protein adsorption (BSA). Our results provide the theoretical and experimental basis for future application of the combined system to other biochemical and biophysical studies.

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.

The design and fabrication of a continuous-flow μPCR device with very short amplification time and low power consumption are presented. Commercially available, 4-layer printed circuit board (PCB) substrates are employed, with in-house designed yet industrially manufactured embedded Cu micro-resistive heaters lying at very close distance from the microfluidic network, where DNA amplification takes place. The 1.9-m-long microchannel in combination with desirably high flow velocities (for fast amplification) challenged the robustness of the sealing that was overcome with the development of a novel bonding method rendering the microdevice robust even at extreme pressure drops (12 bars). The proposed fabrication methods are PCB compatible, allowing for mass and reliable production of the μPCR device in the established PCB industry. The μPCR chip was successfully validated during the amplification of two different DNA fragments (and with different target DNA copies) corresponding to the exon 20 of the BRCA1 gene, and to the plasmid pBR322, a commonly used cloning vector in E. coli. Successful DNA amplification was demonstrated at total reaction times down to 2 min, with a power consumption of 2.7 W, rendering the presented μPCR one of the fastest and lowest power-consuming devices, suitable for implementation in low-resource settings. Detailed numerical calculations of the DNA residence time distributions, within an acceptable temperature range for denaturation, annealing, and extension, performed for the first time in the literature, provide useful information regarding the actual on-chip PCR protocol and justify the maximum volumetric flow rate for successful DNA amplification. The calculations indicate that the shortest amplification time is achieved when the device is operated at its enzyme kinetic limit (i.e., extension rate). Graphical abstract.

The objective of this work is to develop a methodology and associated platform for nucleic acid detection at the point-of-care (POC) that is sensitive, user-friendly, affordable, rapid, and robust. The heart of this system is an acoustic wave sensor, based on a Surface Acoustic Wave (SAW) or Quartz Crystal Microbalance (QCM) device, which is employed for the label-free detection of isothermally amplified target DNA. Nucleic acids amplification and detection is demonstrated inside three crude human samples, i.e., whole blood, saliva, and nasal swab, spiked in with 10-100 Salmonella cells. To qualify for POC applications, a portable platform was developed based on 3D printing, integrating inside a single box: (i) simple fluidics based on plastic tubing and a mini peristaltic pump, (ii) a heating plate combined with disposable reaction tubes for isothermal amplification; (iii) a mini antenna analyzer operated through a tablet; and (iv) an acoustic wave device housing unit. The simplicity of the method combined with smartphone operation and detection, rapid sample-to-answer analysis time (30 min), and high performance (detection limit 4 × 10 3 CFU/ml) in three of the most important human samples in diagnostics suggest that the methodology could become a tool of choice for nucleic acid detection at the POC. In addition, the low cost of the platform and assay holds promise for its adoption in resource limited areas. The acoustic detection method is shown to give similar results with a standard colorimetric assay carried out in saliva and nasal swab but can also be used to detect nucleic acids inside whole blood, where a colorimetric assay failed to perform.

The fast and efficient detection of foodborne pathogens is a societal priority, given the large number of food-poisoning outbreaks, and a scientific and technological challenge, given the need to detect as little as 1 viable cell in 25 gr of food. Here, we present the first approach that achieves the above goal, thanks to the use of a micro/nano-technology and the detection capability of acoustic wave sensors. Starting from 1 Salmonella cell in 25 ml of milk, we employ immuno-magnetic beads to capture cells after only 3 h of pre-enrichment and subsequently demonstrate efficient DNA amplification using the Loop Mediated Isothermal Amplification method (LAMP) and acoustic detection in an integrated platform, within an additional ½ h. The demonstrated 4 h sample-to-analysis time comes as a huge improvement to the current need of few days to obtain the same result. In addition, the work presents the first reported Lab-on-Chip platform that comprises an acoustic device as the sensing element, exhibiting impressive analytical features, namely, an acoustic limit of detection of 2 cells/μl or 3 aM of the DNA target and ability to detect in a label-free manner dsDNA amplicons in impure samples. The use of food samples together with the incorporation of the necessary pre-enrichment step and ability for multiple analysis with an internal control, make the proposed methodology highly relevant to real-world applications. Moreover, the work suggests that acoustic wave devices can be used as an attractive alternative to electrochemical sensors in integrated platforms for applications in food safety and the point-of-care diagnostics., (Copyright © 2018. Published by Elsevier B.V.)

The present study demonstrates the sensitive and label-free acoustic detection of dsDNA amplicons produced from whole Salmonella Thyphimurium cells without employing any DNA extraction and/or purification step, in the presence of the lysed bacterial cells and in a hybridization-free assay. A sample-to-answer assay is also shown during DNA detection directly in milk.

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.

We present a polymeric microfluidic chip capable of purifying DNA through solid phase extraction. It is designed to be used as a module of an integrated Lab-on-chip platform for pathogen detection, but it can also be used as a stand-alone device. The microfluidic channels are oxygen plasma micro-nanotextured, i.e. randomly roughened in the micro-nano scale, a process creating high surface area as well as high density of carboxyl groups (COOH). The COOH groups together with a buffer that contains polyethylene glycol (PEG), NaCl and ethanol are able to bind DNA on the microchannel surface. The chip design incorporates a mixer so that sample and buffer can be efficiently mixed on chip under continuous flow. DNA is subsequently eluted in water. The chip is able to isolate DNA with high recovery efficiency (96± 11%) in an extremely large dynamic range of prepurified Salmonella DNA as well as from Salmonella cell lysates that correspond to a range of 5 to 1.9 × 10 8  cells (0.263 fg to 2 × 500 ng). The chip was evaluated via absorbance measurements, polymerase chain reaction (PCR), and gel electrophoresis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

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.

The development of integrated, fast and affordable platforms for pathogen detection is an emerging area where a multidisciplinary approach is necessary for designing microsystems employing miniaturized devices; these new technologies promise a significant advancement of the current state of analytical testing leading to improved healthcare. In this work, the development of a lab-on-chip microsystem platform for the genetic analysis of Salmonella in milk samples is presented. The heart of the platform is an acoustic detection biochip, integrated with a microfluidic module. This detection platform is combined with a micro-processor, which, alongside with magnetic beads technology and a DNA micro-amplification module, are responsible for performing sample pre-treatment, bacteria lysis, nucleic acid purification and amplification. Automated, multiscale manipulation of fluids in complex microchannel networks is combined with novel sensing principles developed by some of the partners. This system is expected to have a significant impact in food-pathogen detection by providing for the first time an integrated detection test for Salmonella screening in a very short time. Finally, thanks to the low cost and compact technologies involved, the proposed set-up is expected to provide a competitive analytical platform for direct application in field settings.

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.

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.

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., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Surface acoustic wave sensors with integrated microfluidics for multi-sample sensing have been implemented in this work towards the quantitative correlation of the acoustic signal with the molecular weight of surface bound proteins investigating different interaction/binding conditions. The results are presented for: (i) four different biotinylated molecules (30 ≤ Mw ≤ 150 kDa) specifically binding to neutravidin; (ii) the same four non-biotinylated molecules, as well as neutravidin, adsorbing onto gold; and (iii) four cardiac marker proteins (86 ≤ Mw ≤ 540 kDa) specifically binding to their homologous antibodies. Surface plasmon resonance was employed as an independent optical mass sensor. A linear relationship was found to exist between the phase change of the acoustic signal and the molecular weight of the proteins in both cases of specific binding. In contrast, non-specific binding of proteins directly onto gold exhibited no such linear relationship. In all three cases phase change was correlated with the bound mass per area. The underlying mechanism behind the different behavior between specific and non-specific binding is discussed by taking into account the geometrical restrictions imposed by the size of the specific biorecognition molecule and the corresponding bound protein. Our results emphasize the quantitative nature of the phase of the acoustic signal in determining the Mw (in the case of specific binding) with a resolution of 15% and the mass of the bound proteins (in all cases), as well as the significance of the biorecognition molecules in deriving the molecular weight from acoustic or optical detectors.

Triplex forming oligonucleotides (TFOs) represent a class of drug candidates for antigene therapy. Based on strict criteria, we investigated the potential of 25 known oncogenes to be regulated by TFOs in the mRNA synthesis level and we report specific target sequences found in seven of these genes.

The present work demonstrates for the first time patterning of a ready-to-use biosensor with several different biomolecules using Dip-Pen Nanolithography (DPN) for the development of a procedure towards more rapid and efficient multi-sample detection. The biosensor platform used is based on a Surface Acoustic Wave (SAW) device integrated with a parallel-channel microfluidic module, termed as "microfluidics-on-SAW" ("μF-on-SAW"), for reproducible multi-sample analysis. Lipids with different functionalized head groups were patterned at distinct, microfluidic-formed rectangular domains with sharp edges all located on the same sensor surface; pattern quality was verified using a fluorescent microscope. The functionality of the head groups, the efficiency of the patterning method, and the suitability of DPN for the surface modification of the acoustic device were subsequently examined through acoustic experiments. The μF-on-SAW configuration was used to detect specific binding between the pre-patterned functionalized lipids with their corresponding biomolecules. The achievement of an improved sensitivity (5-fold compared to previous acoustic configurations) and reduced preparation time by at least 2 h clearly indicates the suitability of DPN as a direct patterning method for ready-to-use acoustic sensor devices like the μF-on-SAW towards integrated, rapid-analysis, multi-sample biosensing microsystem development.

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.

Acoustic biosensors offer the possibility to analyse cell attachment and spreading. This is due to the offered speed of detection, the real-time non-invasive approach and their high sensitivity not only to mass coupling, but also to viscoelastic changes occurring close to the sensor surface. Quartz crystal microbalance (QCM) and surface acoustic wave (Love-wave) systems have been used to monitor the adhesion of animal cells to various surfaces and record the behaviour of cell layers under various conditions. The sensors detect cells mostly via their sensitivity in viscoelasticity and mechanical properties. Particularly, the QCM sensor detects cytoskeletal rearrangements caused by specific drugs affecting either actin microfilaments or microtubules. The Love-wave sensor directly measures cell/substrate bonds via acoustic damping and provides 2D kinetic and affinity parameters. Other studies have applied the QCM sensor as a diagnostic tool for leukaemia and, potentially, for chemotherapeutic agents. Acoustic sensors have also been used in the evaluation of the cytocompatibility of artificial surfaces and, in general, they have the potential to become powerful tools for even more diverse cellular analysis.

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., (2011 Wiley Periodicals, Inc.)

This work describes the application of a newly emerged biosensing configuration incorporating a Surface Acoustic Wave device integrated with a multi-channel microfluidic module for the rapid and efficient analysis of cardiac markers. The examined cardiac markers of creatine kinase MB (CK-MB), cardiac reactive protein (CRP), D-dimer and pregnancy-associated plasma protein A (PAPP-A), comprise a group of both established and emerging heart disease proteins, that has never been probed before with any kind of biochip-related platform. The four markers were successfully detected; kinetics and affinity studies on their interactions with the surface immobilized antibodies are also presented. A concentration detection limit of less than 1 nM was achieved, with a dynamic range of more than two orders of magnitude, covering some of the pathological and healthy areas of interest. Mixtures of biomarkers applied to the device surface were used to prove the specificity of each binding event and investigate the microsystem's performance in the presence of complex fluids, towards future utilization with real samples. The simplicity and multiplexing ability of the integrated platform render the system ideal as a potential diagnostic tool for cardiovascular risk assessment, where simultaneous analysis of various protein markers is required., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The current work describes a novel setup for multi-sample biomolecular analysis. It is based on the assembly of a dual acoustic device chip with a four-channel microfluidic module, forming an array of eight available domains for experiments. Initially, multiple detection was demonstrated via the specific interaction of neutravidin with four different biotinylated proteins, namely protein G, protein A, bovine serum albumin, and immunoglobulin G; results revealed a reproducibility between the microchannel domains better than 90%. Real-time analysis of the binding interactions was used to calculate the affinity and kinetic constants of the four biotinylated molecules binding to surface-immobilized neutravidin; this was the first time that this information was derived using a biosensing device and four biotinylated molecules. Interestingly, all calculated kinetic and affinity constants resemble those typical of antibody-antigen interactions, although the investigated specific binding was of avidin-biotin nature. Finally, under device pre-functionalization conditions, it was possible to probe eight interactions all together, exploiting the full capacity of the microsystem and reducing significantly the analysis time, contrary to the use of the standard acoustic device configuration. The outcome of this full-scale validation opens the way for the integrated acoustic platform to be implemented in even higher throughput detection for future diagnostic/biomedical applications, as well as in fundamental research studies regarding biomolecular interaction investigation and characterization., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The effect of the cell glycocalyx on the binding of a membrane receptor, class I major histocompatibility complex (MHC) human leukocyte antigen (HLA)-A2, to an immobilized anti-HLA antibody was investigated using an acoustic sensor based on a Love wave geometry. The enzyme neuraminidase was used to remove sialic acid residues from the cell glycocalyx. Real-time measurements of the amplitude of the acoustic wave showed that treatment with neuraminidase facilitates HLA/anti-HLA-mediated cell attachment via a 3.6-fold increase of the two-dimensional (2D) binding constant of the interaction. This could be attributed to better approach of binding partners due to favorable condition of the desialylated glycocalyx. The results underline the importance of microtopological factors in membrane receptor binding and reveal the potential of the Love wave sensor and 2D binding parameters for studying cell-substrate binding events.

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.

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., ((c) 2010 Elsevier B.V. All rights reserved.)

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 sensor's 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, Keq was found equal to 3x10(5) M(-1)., (Copyright (c) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

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.

Acoustic devices are sensitive to the mole fraction of cholesterol present in liposomes adsorbed to the device surface as a result of the different mechanical properties of the liposomes. This fact was exploited to develop an acoustic assay to determine the relative affinity of cholesterol for different lipid mixtures. In the assay described here, the initial rate of beta-cyclodextrin-induced removal of cholesterol was measured for liposomes having a range of compositions. The initial rate of cholesterol removal was found to be directly proportional to the concentration of beta-cyclodextrin (betaCD) present over the range of 0-7.5 mg/ml (0-6.6 mM), consistent with other assays measuring the betaCD-accelerated transfer of cholesterol between liposomes. The affinity of cholesterol for 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC) liposomes with a sphingomyelin mole fraction, chi(SPM), of 0.2 was found to be 1.4x higher than that for pure OPPC liposomes. For liposomes composed only of OPPC and cholesterol in varying ratios, the initial rate of cholesterol removal was determined as a function of cholesterol mole fraction (chi(C)). The initial rate of removal showed an increase at chi(C) = 0.13, consistent with phase diagrams showing the start of liquid ordered domain formation, but no such increase at chi(C) = 0.25, in contrast to the predictions of the umbrella model for OPPC/cholesterol interactions.

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.

Direct biosensors are devices operating by monitoring the amount of surface-bound analyte. In this work a new approach is presented where a label-free acoustic biosensor, based on a QCM-D device, and solution viscosity theory, are used to study DNA intrinsic viscosity. The latter is quantitatively related to the DNA conformation and specifically the molecule's shape and size, in a manner that is independent of the amount of bound DNA mass. It is shown that acoustic measurements can clearly distinguish between ds-DNA of same shape (straight rod) but various sizes (from 20 to 198bp (base pairs)) and same mass and size (90bp) but various shapes ("straight", "bent", "triangle"). These results are discussed in the broader context of "coil" and sphere-like molecules detected on surfaces. A mathematical formula is presented relating the length of straight, surface-protruding DNA to the acoustic ratio DeltaD/Deltaf. The development of real-time rapid techniques for the characterization of DNA intrinsic curvature as well as DNA conformational changes upon interaction with proteins is of significance to analytical biotechnology due to the large number of DNA sequences and potential DNA bending proteins involved in genome analysis and drug screening.

Mammalian alpha-defensins all have a conserved triple-stranded beta-sheet structure that is constrained by an invariant tridisulfide array, and the peptides exert bactericidal effects by permeabilizing the target cell envelope. Curiously, the disordered, disulfide-null variant of mouse alpha-defensin cryptdin-4 (Crp4), termed (6C/A)-Crp4, has bactericidal activity equal to or greater than that of the native peptide, providing a rationale for comparing the mechanisms by which the peptides interact with and disrupt phospholipid vesicles of defined composition. For both live Escherichia coli ML35 cells and model membranes, disordered (6C/A)-Crp4 induced leakage in a manner similar to that of Crp4 but had less overall membrane permeabilizing activity. Crp4 induction of the leakage of the fluorophore from electronegative liposomes was strongly dependent on vesicle lipid charge and composition, and the incorporation of cardiolipin into liposomes of low electronegative charge to mimic bacterial membrane composition conferred sensitivity to Crp4- and (6C/A)-Crp4-mediated vesicle lysis. Membrane perturbation studies using biomimetic lipid/polydiacetylene vesicles showed that Crp4 inserts more pronouncedly into membranes containing a high fraction of electronegative lipids or cardiolipin than (6C/A)-Crp4 does, correlating directly with measurements of induced leakage. Fluorescence resonance energy transfer experiments provided evidence that Crp4 translocates across highly charged or cardiolipin-containing membranes, in a process coupled with membrane permeabilization, but (6C/A)-Crp4 did not translocate across lipid bilayers and consistently displayed membrane surface association. Thus, despite the greater in vitro bactericidal activity of (6C/A)-Crp4, native, beta-sheet-containing Crp4 induces membrane permeabilization more effectively than disulfide-null Crp4 by translocating and forming transient membrane defects. (6C/A)-Crp4, on the other hand, appears to induce greater membrane disintegration.

Gaining insights into the dynamic processes of molecular interactions that mediate cell-substrate and cell-cell adhesion is of great significance in the understanding of numerous physiological processes driven by intercellular communication. Here, an acoustic-wave biosensor is used to study and characterize specific interactions between cell-bound membrane proteins and surface-immobilized ligands, using as a model system the binding of major histocompatibility complex class I HLA-A2 proteins to anti-HLA-A2 monoclonal antibodies. The energy of the acoustic signal, measured as amplitude change, was found to depend directly on the number of HLA-A2/antibody complexes formed on the device surface. Real-time acoustic data were used to monitor the surface binding of cell suspensions at a range of 6.0 x 10(4) to 6.0 x 10(5) cells mL(-1). Membrane interactions are governed by two-dimensional chemistry because of the molecules' confinement to the lipid bilayer. The two-dimensional kinetics and affinity constant of the HLA-A2/antibody interaction were calculated (k(a) = 1.15 x 10(-5) mum(2) s(-1) per molecule, k(d) = 2.07 x 10(-5) s(-1), and K(A) = 0.556 mum(2) per molecule, at 25 degrees C), based on a detailed acoustic data analysis. Results indicate that acoustic biosensors can emerge as a significant tool for probing and characterizing cell-membrane interactions in the immune system, and for fast and label-free screening of membrane molecules using whole cells.

The development of smooth hydrophilic surfaces that act as substrates for supported lipid bilayers (SLBs) is important for membrane studies in biology and biotechnology. In this article, it is shown that thin films of poly(dimethylsiloxane) (PDMS) formed on a sensor surface can be used as a substrate for the deposition of reproducible and homogeneous zwitterionic SLBs by the direct fusion of vesicles. Poly(dimethylsiloxane) solution (1% w/v) was spin coated on Love acoustic wave and surface plasmon resonance devices to form a thin PDMS layer. Acoustic, fluorescence, and contact angle measurements were used for the optimization of the PDMS film properties as a function of plasma etching time; parameters of interest involve the thickness and hydrophilicity of the film and the ability to induce the formation of homogeneous SLBs without adsorbed vesicles. The application of PDMS-coated sensor devices to the study membrane of interactions was demonstrated during the acoustic and fluorescence detection of the binding of melittin and defensin Crp4 peptides to model supported lipid bilayers.

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.

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.

The purpose of this work was to develop a simple procedure for the creation of a specific biorecognition layer for histidine-tagged (His-tagged) molecules. Such a layer was prepared by the spontaneous fusion of vesicles containing readily available plain (DOPC) and iminodiacetic acid (DOGS-NTA) phospholipids on a silica surface resulting in the formation of an NTA-containing supported lipid bilayer. The frequency surface acoustic waveguide device which supports Love waves was used to follow the real-time formation of the biorecognition layer. The mole percent of the DOGS-NTA phospholipids in the supported bilayer was optimized by following the kinetics of the fusion for the different NTA-containing lipids. Fluorescently labeled lipids were used with observations of the fluorescence recovery after photobleaching to confirm the presence of lipid bilayers. After saturating all NTA-molecules with Ni(2+), the binding of a His-tagged protein fragment within the concentration range of 0.04 and 0.4 mM to a 5 mol % DOGS-NTA/DOPC was detected; binding curves were used to calculate the apparent association constant k(on) = 2.56 x 10(4) M(-)(1) s(-)(1), dissociation constant k(off) = 1.3 x 10(-)(3) s(-)(1), and equilibrium constant k(eq) = 1.97 x 10(7) M(-)(1). The described method could find significant applications as a generic technique for preparing biorecognition layers for His-tagged proteins. In addition, the acoustic waveguide device, which provides high sensitivity together with flexibility in terms of the substrate material used, is shown to be an attractive alternative to direct optical biosensors.

An acoustic technique was used to monitor the interaction of the pore-forming cytolytic toxin CytB with a positively charged supported lipid bilayer. The acoustic device, which is based on a waveguide geometry, is sensitive to changes in the mass of the supported bilayer. The specificity of the interaction, rate and extent of the association, reversibility and effect of previous depositions of toxin were investigated. The CytB was found to bind irreversibly to the lipids at all fractional coverages even when the protein-to-lipid ratio was high enough to imply that the protein was associating with the external surface of the bilayer. The CytB formed stable structures with the bilayer at high protein surface concentrations and did not appear to disrupt the bilayer in the manner of a detergent. The rate of association with the bilayer was found to be directly proportional to the solution concentration of CytB at higher concentrations but appeared to be low at a CytB solution concentration of 5 microg mL(-1), leading to relatively low amounts of CytB being associated with the bilayer.

In this work, we describe a novel pulse mode shear horizontal-surface acoustic wave (SH-SAW) polymer coated biosensor that monitors rapid changes in both amplitude and phase. The SH-SAW sensors were fabricated on 36 degrees rotated Y-cut X propagating lithium tantalate (36 YX.LT). The sensitivity of the device to both mass loading and visco-elastic effects may be increased by using a thin guiding layer of cross-linked polymer. Two acoustic modes are excited by the electrodes in this crystalline direction. Metallisation of the propagation path of the 36 YX.LT devices allows the two modes to be discriminated. Successive polymer coatings resulted in the observation of resonant conditions in both modes as the layer thickness was increased. Using the 36 YX.LT devices, we have investigated the application of a novel pulse mode system by sensing a sequence of deposition and removal of a biological layer consisting of vesicles of the phospholipid POPC. A continuous wave system was used to verify the accuracy of the pulse mode system by sensing a series of poly(ethylene glycol) (PEG) solutions. The data clearly demonstrates the ability of the 36 YX.LT pulse mode system to provide rapid measurements of both amplitude and phase for biosensing applications.

A simple method for modifying a polymer surface to induce lipid bilayer formation by vesicle fusion is described. A silicate gel was prepared by condensation of tetraethyl orthosilicate (TEOS) in the presence of acid. When applied to a poly(methylmethacrylate) substrate, either a rough or a smooth layer could be produced, depending on the method used for the application. The smooth surface induced formation of a supported lipid bilayer by fusion of lipid vesicles; the rough silicate surface induced adsorption of a vesicle layer. A high-frequency acoustic waveguide device was used to follow the initial adsorption of vesicles, the transition from a vesicle layer to a bilayer, and the formation of a complete bilayer; the time required to form a bilayer was determined as a function of lipid concentration in suspension. The presence of a bilayer on the smooth silicate surface was confirmed by fluorescence recovery after photobleaching. An additional procedure is described to modify a gold surface to induce bilayer formation.

The sensitivity of the acoustic waveguide sensor to mass deposition in the presence of liquid was optimized as a function of the over-layer thickness. The waveguide geometry consisted of a 0.2-2.2-microm poly(methyl)methacrylate (PMMA) over-layer deposited on the surface of a shear acoustic wave device and supported a Love wave. The response of each polymer-coated waveguide was initially assessed by monitoring the frequency and insertion loss of the device in the presence of air. Sensitivity to viscous and mass loading was studied by recording the amplitude and phase of the wave during the application of water and of a supported lipid bilayer, respectively, on the device surface. Supported bilayers are a versatile system for mass calibration in the presence of liquid because they can be formed spontaneously on a hydrophilic surface, resulting in a layer of reproducible mass density. Results clearly showed that the response of both amplitude and phase depends on the over-layer thickness and increases with the thickness of the polymer layer. Phase was generally found to be more sensitive than amplitude to both viscous water and mass loading. The maximum sensitivity to vesicles deposition was measured at 250 cm2 g(-1) and was detected when 1.3 microm of PMMA was used as a waveguide layer. Results showed that the sensitivity of the acoustic wave sensor can be improved by simply increasing the thickness of the PMMA and that supported phospholipid layers can form an ideal system for both mass calibration and interfacial modification.

A direct immunosensor has been developed using an acoustic wave device as a transducer. The device is based on an acoustic waveguide geometry that supports a Love wave. The biorecognition surface, formed on a gold layer, consisted of a biotinylated supported lipid layer which specifically bound streptavidin and, subsequently, biotinylated goat IgG. The modified surface was used as a model immunosensor and successfully detected rabbit anti-goat IgG in the concentration range 3 x 10(-8) - 10(-6) M. Using the anti-goat IgG binding isotherm and the time-resolved measurements of antibody binding, both the binding and rate constants of the reaction were determined. The specificity of each binding step was studied with the acoustic wave device, and it was concluded that the phospholipid bilayer showed a good suppression of nonspecific binding. Comparative measurements using surface plasmon resonance allowed the response of the immunosensor to be quantitatively correlated with mass binding to the surface.