A simple and efficient method to fabricate a glycosylated surface on a polyacrylonitrile-based film is described. Construction and protein adsorption processes were monitored in situ using a QCM. A PANCHEMA film was deposited on the gold surface of the quartz crystal, and the glycosylated surface was then constructed through surface modification. Con A and BSA were used as probes to study the specificity of this surface to proteins. It can recognize Con A, while showing no specific interaction with BSA. The binding affinity indicates the presence of strong multivalent interactions between Con A and the glucose residues (cluster glycoside effect). Reproducibility and repeatability of the glycosylated polymer surface are sufficient to allow specific adsorption of Con A.

Detecting the molecular basis of protein-protein recognition is an essential element in understanding protein function because their ability to form specific complexes with other proteins underlies most cellular processes. The use of labels has limitations, such as changes to the binding kinetics due to the alterations in structure and function that occur with label addition, difficulty in detecting biochemical activities and the need for additional steps in assay development. These issues have driven the development of label-free formats for identifying the full range of biochemical activities.Although optical-based systems dominate the label-free biosensor market, electrochemical, piezoelectric and acoustic devices represent similar but significantly less expensive alternatives. Acoustic biosensors have been employed in the label-free detection of an incredibly broad range of analytes, from interfacial chemistries and lipid membranes, to small molecules and whole cells.Resonant acoustic profiling (RAP) technology offers label-free, real-time analysis of biomolecular interactions and offers an efficient way to optimize the development and production process of recombinant proteins. RAP measures only the physical binding events and is insensitive to refractive index and colour changes. This enables direct measurement in undiluted crude and complex samples, such as cell culture media or periplasmic extracts, without intensive assay calibration. This advantage simplifies experimental design and eliminates expensive time-consuming purification of often limited material, while delivering high content information. In this respect RAP technology reduces costs and increases the throughput and the density of information to optimize and control the processes more effectively. Copyright (c) 2010 John Wiley & Sons, Ltd.

Supported lipid bilayers (SLBs) mimic biological membranes and are a versatile platform for a wide range of biophysical research fields including lipid-protein interactions, protein-protein interactions and membrane-based biosensors. The quartz crystal microbalance with dissipation monitoring (QCM-D) has had a pivotal role in understanding SLB formation on various substrates. As shown by its real-time kinetic monitoring of SLB formation, QCM-D can probe the dynamics of biomacromolecular interactions. We present a protocol for constructing zwitterionic SLBs supported on silicon oxide and titanium oxide, and discuss technical issues that need to be considered when working with charged lipid compositions. Furthermore, we explain a recently developed strategy that uses an amphipathic, alpha-helical (AH) peptide to form SLBs on gold and titanium oxide substrates. The protocols can be completed in less than 3 h.

Early determination of the metastatic potential of cancer cells is a crucial step for successful oncological treatment. Besides the remarkable progress in molecular genomics- or proteomics-based diagnostics, there is a great demand for in vitro biosensor devices that allow rapid and selective detection of the invasive properties of tumor cells. Here, the classical cancer cell motility in vitro assays for migration and invasion relying on Boyden chambers are compared to a real-time biosensor that analyzes the dynamic properties of adherent cells electro-acoustically with a time resolution on the order of seconds. The sensor relies on the well-established quartz crystal microbalance technique (QCM) that measures the shift in resonance frequency and damping of an oscillating quartz crystal when adsorption, desorption or changes in material properties close to the quartz surface occur. In addition, the QCM is capable of detecting the rather subtle fluctuations of the cell bodies as an indicator for their micromotility. QCM-based micromotility readings of three different cancer cell lines (HT-29, HSC-4, FaDu) are compared with the well-known electrical cell-substrate impedance sensing (ECIS) revealing collective stochastic motion that corresponds to the malignancy of the cells.

Based on specific chemical reactions, chemoselective gas detection of aldehydes, ketones and amines in real time was efficiently achieved on QCM chips thin-coated with sensing ionic liquids (SILs).

We describe multivalent biorecognition of adsorbed lectin layers by biomimetic sensing nanoplatforms based on dynamic glycovesicles in a continuous flow QCM setup.

Membrane proteins are essential in the exchange processes of cells. In spite of great breakthrough in soluble proteins studies, membrane proteins structures, functions and interactions are still a challenge because of the difficulties related to their hydrophobic properties. Most of the experiments are performed with detergent-solubilized membrane proteins. However widely used micellar systems are far from the biological two-dimensions membrane. The development of new biomimetic membrane systems is fundamental to tackle this issue.We present an original approach that combines the Fluorescence Recovery After fringe Pattern Photobleaching technique and the use of a versatile sponge phase that makes it possible to extract crucial informations about interactions between membrane proteins embedded in the bilayers of a sponge phase. The clear advantage lies in the ability to adjust at will the spacing between two adjacent bilayers. When the membranes are far apart, the only possible interactions occur laterally between proteins embedded within the same bilayer, whereas when membranes get closer to each other, interactions between proteins embedded in facing membranes may occur as well.After validating our approach on the streptavidin-biotinylated peptide complex, we study the interactions between two membrane proteins, MexA and OprM, from a Pseudomonas aeruginosa efflux pump. The mode of interaction, the size of the protein complex and its potential stoichiometry are determined. In particular, we demonstrate that: MexA is effectively embedded in the bilayer; MexA and OprM do not interact laterally but can form a complex if they are embedded in opposite bilayers; the population of bound proteins is at its maximum for bilayers separated by a distance of about 200 A, which is the periplasmic thickness of Pseudomonas aeruginosa. We also show that the MexA-OprM association is enhanced when the position and orientation of the protein is restricted by the bilayers. We extract a stoichiometry for the complex that exhibits a strong pH dependance: from 2 to 6 MexA per OprM trimer when the pH decreases from 7.5 to 5.5.Our technique allows to study membrane protein associations in a membrane environment. It provides some challenging information about complexes such as geometry and stoichiometry.

Surface Plasmon Resonance (SPR) biosensor technology has been successfully used for the detection of various analytes such as proteins, drugs, DNA, and microorganisms. SPR-based immunosensors that coupled with a specific antigen-antibody reaction, have become a promising tool for the quantification of bacteria as it offers sensitive, specific, rapid, and label-free detection. In this paper, we review the important issues in the development of SPR-based immunoassays for bacteria detection, concentrating on instrumentation, surface functionalization, liquid handling, and surface regeneration. In addition, this review touches on the recent advances in SPR biosensing for sensitivity enhancement.

In this work, electroacoustic impedance analysis based on a modified Butterworth-Van Dyke (BVD) model is used to complement resonance frequency measurements of piezoelectric crystal sensors for the identification and removal of interfering signals. This approach enables the accurate use of the Sauerbrey correlation to establish a direct relationship between mass deposited at the sensor surface and measured frequency variations. Kinetic models can thus be evaluated and binding constants estimated directly from the measured data. We further demonstrate the usefulness of this approach by applying it to the study of the formation of 11-hydroxy-1-undecanothiol self-assembled monolayers (SAM) as well as to the binding of streptavidin to immobilized biotin. Kinetic and equilibrium parameters were estimated from transient analysis, adsorption isotherms, Scatchard and Hill plots obtained from the frequency data for both the alkanethiol and streptavidin films.This strategy based on electroacoustic impedance assisted quartz-crystal microbalance (QCM) biosensors is expected to be a major contribution for the use of these piezoelectric devices as a reliable and cheap detection system that can easily be integrated into analytical techniques. Copyright (c) 2008 John Wiley & Sons, Ltd.

This review presents an overview of 20 years of worldwide development in the field of biosensors based on special types of surface acoustic wave (SAW) devices that permit the highly sensitive detection of biorelevant molecules in liquid media (such as water or aqueous buffer solutions). 1987 saw the first approaches, which used either horizontally polarized shear waves (HPSW) in a delay line configuration on lithium tantalate (LiTaO(3)) substrates or SAW resonator structures on quartz or LiTaO(3) with periodic mass gratings. The latter are termed "surface transverse waves"(STW), and they have comparatively low attenuation values when operated in liquids. Later Love wave devices were developed, which used a film resonance effect to significantly reduce attenuation. All of these sensor approaches were accompanied by the development of appropriate sensing films. First attempts used simple layers of adsorbed antibodies. Later approaches used various types of covalently bound layers, for example those utilizing intermediate hydrogel layers. Recent approaches involve SAW biosensor devices inserted into compact systems with integrated fluidics for sample handling. To achieve this, the SAW biosensors can be embedded into micromachined polymer housings. Combining these two features will extend the system to create versatile biosensor arrays for generic lab use or for diagnostic purposes. Figure Monitoring and verification.

Several neuroendocrine signals of the hypothalamic-pituitary-adrenal (HPA) axis are released following exposure to stressful events. It has long been proposed that the signals in this cascade each act to modify ongoing and future behavior. In this study we investigated whether blocking glucocorticoid synthesis, corticotropin-releasing factor (CRF)-1 receptors, or CRF-2 receptors during social defeat would alter subsequent behavioral responses. We used a conditioned defeat model in Syrian hamsters in which social defeat results in a dramatic shift from territorial aggression to increased submissive and defensive behavior in future social encounters. We found that intracerebroventricular administration of anti-sauvagine-30, a CRF-2 receptor antagonist, prior to social defeat training reduced the acquisition of conditioned defeat. In contrast, the acquisition of conditioned defeat was not altered by the CRF-1 receptor antagonist CP-154,526 or the glucocorticoid synthesis inhibitor metyrapone. Our results suggest that CRF, and perhaps related neuropeptides such as urocortins, act at CRF-2 receptors to promote the development of defeat-induced changes in social behavior, whereas signaling at CRF-1 and glucocorticoid receptors plays a negligible role in this process.

Citropin 1.1, Maculatin 1.1 and Caerin 1.1 are short antibacterial cationic peptides from the skin glands of the Australian tree frog Litoria species. Several analogues have been synthesized to give a better insight into the relationship between the structure of the peptides and their antibacterial and haemolytic activity. Binding studies using a surface plasmon resonance (SPR) biosensor together with a vesicle-capture sensor chip have been used to investigate selectivity of the peptides and their analogues for 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles, as well as for vesicles made from lipid extracts from Escherichia coli and bovine brain. Data obtained for membrane selectivity using natural lipid extracts show better correlation with MIC values against Gram-positive bacteria and haemolytic activity than that obtained using synthetic DMPG and DMPC. Electron microscopy and membrane leakage studies using Gram-positive bacteria gave further insight into the membrane disruption properties of the peptides. For Maculatin 1.1, it was found that the central proline residue, which is responsible for a bend in the alpha-helical structure, is essential not only for the antibacterial activity, but also for binding to, and perturbation of membranes. The Caerin analogues showed only small variations in their MIC values and membrane binding. In contrast, for Citropin 1.1, the analogue replacing the aspartate with a lysine showed the lowest MIC against Gram-positive bacteria and best membrane binding to E. coli lipid extracts, coinciding with an increased hydrophobic moment of the peptide. These data give further insight into these anti-microbial natural products, towards the development and evaluation of these and other analogues as potential antibiotics.(c) 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2010.

By specifically binding derivatized colloidal particles and physisorbing nonderivatized particles to the surface of a quartz crystal microbalance (QCM), we have observed positive shifts of frequency, Deltaf, in contrast to the negative frequency shifts typically found in adsorption experiments. Evidently, the Sauerbrey relation does not apply to this situation. A comparison of frequencies shifts and bandwidths on different overtones reveals a coupled resonance: at low overtones, Deltaf is negative, whereas it is positive at high overtones, with maximal resonance bandwidth observed at the crossover point. As predicted by the Dybwad model,(1) the spheres bound to the surface form resonating systems on their own. A composite resonator is formed, consisting of a large crystal with resonance frequency omega and the adsorbed spheres with resonance frequency omega(S). In the case in which the resonance frequency of the small spheres (firmly attached to crystal), omega(S), is higher than the resonance frequency of the crystal, omega, Deltaf of the composite system is negative (leading to the Sauerbrey limit). In the opposite limit (that is, in the case of large adsorbed particles bound to the sensor surface via a sufficiently weak bridge) Deltaf is positive. Such a behavior is known from sphere-plate contacts in the dry state. Finite element calculation demonstrates that this phenomena is also plausible in liquid phase media, with Deltaf critically dependent on the strength of the sphere-plate contact. Operated in this mode, the QCM most likely probes the contact strength, rather than the mass of the particle.

Functionalised thiols presenting peptides found in the peptidoglycan of vancomycin-sensitive and -resistant bacteria were synthesised and used to form self-assembled monolayers (SAMs) on gold surfaces. This model bacterial cell-wall surface mimic was used to study binding interactions with vancomycin. Force spectroscopy, using the atomic force microscope (AFM), was used to investigate the specific rupture of interfacial vancomycin dimer complexes formed between pairs of vancomycin molecules bound to peptide-coated AFM probe and substrate surfaces. Clear adhesive contacts were observed between the vancomycin-sensitive peptide surfaces when vancomycin was present in solution, and the adhesion force demonstrated a clear dependence on antibiotic concentration.

Poly(oligo(ethylene glycol) methacrylate)(POEGMA) brushes are extremely protein resistant polymer coatings that can reduce nonspecific adsorption of proteins from complex mixtures such as blood, sera and plasma. These coatings can be prepared via atom transfer radical polymerization with excellent control of their thickness and grafting density. We studied their direct functionalization with streptavidin and developed an assay for determining which coupling conditions afford the highest streptavidin loading efficiency. Disuccinimidyl carbonate was found to be the most efficient activating agent for covalent capture of the receptor. Using infrared and X-ray photoelectron spectroscopy, fluorescence microscopy, surface plasmon resonance, and ellipsometry, we examined how structural parameters such as the length of the oligo(ethylene glycol) side chain affect streptavidin functionalization, but also immobilization of biotinylated antibodies, subsequent selective secondary recognition and nonspecific binding of proteins. We found evidence that large macromolecules cannot infiltrate dense polymer brushes and that bulky antibody recognition occurs in the upper part of these coatings.

Knowledge of the way in which ligands modulate cellular responses via membrane-associated receptors is of central importance to drug discovery and elucidation of signal transduction pathways. Biophysical label-free methods can be used to characterize ligand and drug candidate interactions with neurotransmitters, cytokine receptors, tyrosine kinase receptors, ligand- and voltage-gated ion channels, G protein-coupled receptors (GPCRs), and antibody receptors. Ligand or drug candidate screening typically involves selecting ligands or subsets of a compound library for analysis, transfecting a cell line overexpressing the target receptor, then monitoring one or two downstream reporters of receptor activation such as Ca(2+), cAMP, inositol phosphate, etc. Inevitably, this process leads to a data set predicated by these selections. In contrast, label-free screening techniques allow a holistic, pathway-independent screening strategy to provide a functional or phenotypic readout of receptor activation. Detection techniques that measure changes in cell conductance, viscoelastic properties, refractive index, and other optical parameters that are modulated as a consequence of receptor activation are reviewed.

A rapid and sensitive detection of staphylococcal enterotoxin B (SEB) was developed using a novel acoustic sensing technique: Resonant Acoustic Profiling (RAP), which utilizes high-frequency piezoelectric quartz resonators for monitoring biomolecular interactions. An automated four-channel instrument consisting of acoustic sensors covalently conjugated with anti-SEB antibodies was used. As the samples flowed across control and active sensors simultaneously, binding was measured as a change in the resonant frequency. The lower limit of detection (LLOD) for the label free direct format was 25 ng/mL. Detection sensitivity was increased by adding mass sequentially to the captured SEB on the sensor in the form of sandwich antibodies and biotin-avidin-based gold nanoparticles. The LLOD for the mass enhanced formats were 5 and 0.5 ng/mL of SEB, respectively. The lowest sensitivity corresponds to 1.3 fM in a 75 muL sample. The total assay time including the enhancement steps was less than 10 min. SEB was detected in both neat urine and PBS buffer-spiked samples, with linear correlations between resonant frequency signals and SEB concentrations (R(2) of 0.999 and 0.998, respectively). No significant cross-reactivity was observed with homologue toxins SEA, SED, and TSST, but some cross-reactivity was observed with the closely related toxin SEC(1) when we used a polyclonal antibody in the assay. SEC(1) cross-reactivity was not observed when a SEB-specific monoclonal antibody was employed in the assay. Thus the specificity of the assay presented here was dependent on the quality of the antibodies used. In addition to detection, we evaluated RAP's ability to measure the toxin in unknown samples rapidly by measuring the initial binding rate of the interaction, thereby further shortening the assay time to 6 min.

The alarming growth of the antibiotic-resistant superbugs methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) is driving the development of new technologies to investigate antibiotics and their modes of action. We report the label-free detection of vancomycin binding to bacterial cell wall precursor analogues (mucopeptides) on cantilever arrays, with 10 nM sensitivity and at clinically relevant concentrations in blood serum. Differential measurements have quantified binding constants for vancomycin-sensitive and vancomycin-resistant mucopeptide analogues. Moreover, by systematically modifying the mucopeptide density we gain new insights into the origin of surface stress. We propose that stress is a product of a local chemical binding factor and a geometrical factor describing the mechanical connectivity of regions activated by local binding in terms of a percolation process. Our findings place BioMEMS devices in a new class of percolative systems. The percolation concept will underpin the design of devices and coatings to significantly lower the drug detection limit and may also have an impact on our understanding of antibiotic drug action in bacteria.

In animal models, serotonin (5-HT) activity contributes to stress-induced changes in behavior. Syrian hamsters (Mesocricetus auratus) exhibit a stress-induced change in behavior in which social defeat results in increased submissive and defensive behavior and a complete loss of normal territorial aggression directed toward a novel, non-aggressive opponent. We refer to this defeat-induced change in agonistic behavior as conditioned defeat. In this study we tested the hypothesis that 5-HT activity in the dorsal raphe nucleus (DRN) contributes to the acquisition and expression of conditioned defeat. We investigated whether injection of the selective 5-HT1A agonist flesinoxan (200ng, 400ng, or 800ng in 200nl saline) into the DRN would reduce the acquisition and expression of conditioned defeat. Additionally, we investigated whether injection of the selective 5-HT1A antagonist WAY 100635 (400ng in 200nl saline) into the DRN would enhance the acquisition and expression of conditioned defeat following a sub-optimal social defeat experience. We found that injection of flesinoxan into the DRN before exposure to a 15-min social defeat reduced the amount of submissive and defensive behavior shown at testing. We also found that injection of flesinoxan into the DRN before testing similarly reduced submissive and defensive behavior. In addition, we found that WAY 100635 enhanced conditioned defeat when injected either before social defeat or before testing. These data support the hypothesis that the activity of 5-HT cells in the DRN, as regulated by 5-HT1A autoreceptors, contributes to the formation and display of conditioned defeat. Further, our results suggest that 5-HT release in DRN projection regions augments defeat-induced changes in social behavior.

We describe the detection of specific, conserved DNA sequences of herpes simplex virus (HSV) type 1 by means of a novel, high sensitivity acoustic biosensor. Repeated assays on planar and polymeric carboxylic acid- and biotin-presenting surface chemistries enabled statistical comparison of assay specificity and sensitivity and evaluation of assay Z-factor scores. Using a three minute hybridisation with NeutrAvidin capture for signal enhancement, it was possible to detect HSV viral nucleic acids at 5.2 x 10(-11) M concentration.

Surface plasmon resonance (SPR) is a robust method to detect and quantify macromolecular interactions; however, to measure binding interactions, one component must be immobilized on a sensor surface. This is typically achieved using covalent immobilization via free amines or thiols, or noncovalent immobilization using high-affinity interactions such as biotin/streptavidin or antibody/antigen. In this chapter we describe a robust method to covalently immobilize His(6) fusion proteins on the sensor surface for SPR analysis.

Surface plasmon resonance (SPR) analysis is rather unique in that it allows assay of binding constants (affinity) and kinetic analysis of binding phenomena. This introductory chapter deals with some specific features that are relevant to many diverse applications. The role and impact of kinetics in biomolecular interactions is highlighted. A concise description of the physical principles of the SPR phenomenon is given from a practical point of view, such that some possibilities and limitations of the method can be rationalized, e.g., depth of the evanescent field. A specific condition that may come forward in kinetic analysis is mass transport limitation (MTL). A practical model is presented, which allows estimation of the extent of MTL. Based on this model it can be rationalized whether MTL can be avoided by experimental design. In this framework also rules are presented to convert SPR signals (RU or millidegree) to mass/surface unit. The chapter concludes with an overview of commercially available SPR equipment.

Nowadays, high-throughput analysis of biological events is a great challenge which could take benefit of the recent development of microarray devices. The great potential of such technology is related to the availability of a chip bearing a large set of probes, stable and easy to obtain, and suitable for ligand-binding detection. Here, we describe a new method based on polypyrrole chemistry, allowing the covalent immobilization of peptides in a microarray format and on a gold surface compatible with the use of surface plasmon resonance. This technique is then illustrated by the detection and characterization of antibodies induced by hepatitis C virus and present in patients' serums.

Surface plasmon resonance imaging (SPRI) is a useful tool for the study of surface biomolecular interactions allowing for label-free detection and elegant instrumentation. SPRI imaging system is described in this review with an emphasis on recent applications with examples of different biological interactions and high throughput analysis. Signal amplification in SPRI using nanoparticle and waveguide-based optical coupling is introduced. Finally the detection sensitivity of the SPRI system is examined in terms of other competitive methods.

The quartz crystal microbalance (QCM) was used to monitor the immobilization of tyrosinase on polycationic and polyanionic precursor assemblies in situ and in real-time. The resulting enzymatic surfaces were then exposed to various flavonoids, and the degree of binding was measured using QCM. We show that enzyme activity is retained when immobilized on polycationic films (flavonoid binding observed), while the active site is blocked when assembled on a polyanionic film (no flavonoid binding to the enzyme). We rationalize these observations by considering a combination of interlayer interpenetration and strong electrostatic interactions between the polyelectrolyte and tyrosinase's dicopper 2(+)center. Ion-pair formation between anionic moieties of the polyanion and the metal-coordinated active site is suggested as the dominant mechanism leading to the deactivation of tyrosinase. We are currently working to expand this research to achieve a more general theory of how various metal-coordinated enzymes react with polyelectrolyte surfaces of varying structural morphology, charge density, and chemical composition.

An analysis is developed for the frequency response of a quartz crystal resonator (often referred to as a quartz crystal microbalance) that is modified with a grafted solvent-swollen polymer brush and placed in contact with a membrane capping layer. The shear wave generated at the resonator surface couples into the membrane layer with an efficiency that is strongly dependent on the thickness of the swollen brush layer. As a result, the resonant frequency changes by a maximum amount that is closely approximated by the Sauerbrey shift for the capping layer. The calculated shift substantially decreases for increases in the brush thickness of approximately 10 nm, which gives a net frequency response that is extremely sensitive to the degree of swelling of the polymer brush. An optimum capping layer thickness is determined by balancing the Sauerbrey shift against dissipative effects that weaken the crystal resonance. This optimum membrane thickness depends only weakly on the properties of the membrane material and is in the micron range. Detailed multilayer calculations are presented for the specific case of a poly(ethylene glycol) brush swollen with water and brought into contact with an elastomeric water-permeable membrane. These calculations confirm that the method is sensitive to the properties of the brush layer in the experimentally relevant thickness regime. Connections are also made to conceptually simpler two and three layer models of the acoustic impedance of the material systems that are brought into contact with the resonator.

The most popular bulk acoustic wave (BAW) sensor is the quartz crystal microbalance (QCM), which has electrodes on both the top and bottom surfaces of an AT-cut quartz wafer. In the QCM, the exciting electric field is primarily perpendicular to the crystal surface, resulting in a thickness field excitation (TFE) of a resonant temperature compensated transverse shear mode (TSM). The TSM, however, can also be excited by lateral field excitation (LFE) in which electrodes are placed on one side of the wafer leaving a bare sensing surface exposed directly to a liquid or a chemi/bio selective layer allowing the detection of both mechanical and electrical property changes caused by a target analyte. The use of LFE sensors has motivated an investigation to identify other piezoelectric crystal orientations that can support temperature-compensated TSMs and operate efficiently at high frequencies resulting in increased sensitivity. In this work, theoretical search and experimental measurements are performed to identify the existence of high-frequency temperature-compensated TSMs in LiTaO(3). Prototype LFE LiTaO(3) sensors were fabricated and found to operate at frequencies in excess of 1 GHz and sensitively detect viscosity, conductivity, and dielectric constant changes in liquids.

We develop a new multifunctional optical biochip system that integrates an ellipsometer with a surface plasmon resonance (SPR) feature. This newly developed biochip biosensor, which we call ESPR for an ellipsometric SPR, provides us with a system to retrieve detailed information such as the optical properties of immobilized biomolecular monolayers, surface concentration variations of biomedical reactions, and kinetic affinity between biomolecules required for further biotech analysis. Our ESPR can also serve as both a research and development tool and a manufacturing tool for various biomedical applications.

Acoustic wave biosensors are a real-time, label-free biosensor technology, which have been exploited for the detection of proteins and cells. One of the conventional biosensor approaches involves the immobilization of a monolayer of antibodies onto the surface of the acoustic wave device for the detection of a specific analyte. The method described within includes at least two immobilizations of two different antibodies onto the surfaces of two separate acoustic wave devices for the detection of several analogous analytes. The chemical specificity of the molecular recognition event is achieved by virtue of the extremely high (nM to pM) binding affinity between the antibody and its antigen. In a standard ELISA (Enzyme-Linked ImmunoSorbent Assay) test, there are multiple steps and the end result is a measure of what is bound so tightly that it does not wash away easily. The fact that this "gold standard" is very much not real time, masks the dance that is the molecular recognition event. X-Ray Crystallographer, Ian Wilson, demonstrated more than a decade ago that antibodies undergo conformational change during a binding event[1, 2]. Further, it is known in the arena of immunochemistry that some antibodies exhibit significant cross-reactivity and this is widely termed antibody promiscuity. A third piece of the puzzle that we will exploit in our system of acoustic wave biosensors is the notion of chemical orthogonality. These three biochemical constructs, the dance, antibody promiscuity and chemical orthogonality will be combined in this paper with the notions of in-phase (I) and quadrature (Q) signals from digital radio to manifest an approach to molecular recognition that allows a level of discrimination and analysis unobtainable without the aggregate. As an example we present experimental data on the detection of TNT, RDX, C4, ammonium nitrate and musk oil from a system of antibody-coated acoustic wave sensors.

The efficient and rapid detection of bioactive compounds in complex matrices of different origins (natural or synthetic) is a key step in the discovery of molecules with potential application in therapy. Among them, molecules able to interact with nucleic acids can represent important targets. In this study, an optical DNA biosensor, based on surface plasmon resonance (SPR) transduction, has been studied in its potential application as new analytical device for drug screening. This device was applied to the analysis of pure synthetic or natural molecules and also to some fractions obtained by chromatographic separation of an extract of Chelidonium majus L.(great celandine), a plant containing benzo[c]phenanthridinium alkaloids having intercalating properties. The ability of these molecules to interact with the double stranded nucleic acid (dsDNA) immobilised on the sensor surface has been investigated. The optical sensing relies on the SPR-based bench instrument Biacore Xtrade mark and represents an example of multiuse sensor. The results obtained demonstrate the potential application of this device for the rapid screening of bioeffective compounds. The characteristics of the biosensor offer the possibility to be coupled to chemical analysis as in hyphenated technologies.