A contactless surface acoustic wave biosensor.

Surface acoustic wave sensors operating in liquid generally cause problems resulting from wire bonding. The authors present an approach for a biosensor where the need for bonding wires is eliminated by utilizing inductive coupling of the sensor device to the RF circuitry. Protection of the electrodes from the liquid is achieved by coating the device surface with a SiO2 layer, resulting in a simplified handling of the devices. The first measurements with a sensor operating at 420 MHz are presented, demonstrating the potential of this operating principle for biosensing.     

Rapid detection of human papilloma virus using a novel leaky surface acoustic wave peptide nucleic acid biosensor

A novel leaky surface acoustic wave (LSAW) bis-peptide nucleic acid (bis-PNA) biosensor with double two-port resonators has been constructed successfully for the quantitative detection of human papilloma virus (HPV). The bis-PNA probe can directly detect HPV genomic DNA without polymerase chain reaction (PCR) amplification, and it can bind to the target DNA sequences more effectively and specifically than a DNA probe. When the concentrations varied from 1 pg/L to 1000 μg/L, with 100 μg/L being the optimal, a typical linearity was found between the quantity of target and the phase shifts. The detection limit was 1.21 pg/L and the clinical specificity was 97.22% of that of real-time PCR. The bis-PNA probe was able to distinguish sequences that differ only in one base. Both the intraassay and interassay coefficients of variance (CVs) were <10%, and the biosensor can be regenerated for ten times without appreciable loss of activity. Therefore, this technical platform of LSAW biosensor can be applied to clinical samples for direct HPV detection.     

Detection of viral bioagents using a shear horizontal surface acoustic wave biosensor

Viruses are of high medical and biodefense concern and their detection at concentrations well below the threshold necessary to cause health hazards continues to be a challenge with respect to sensitivity, specificity, and selectivity. Ideally, assays for accurate and real time detection of viral agents would not necessitate any pre-processing of the analyte, which would make them applicable for example to bodily fluids (blood, sputum) and man-made as well as naturally occurring bodies of water (pools, rivers). We describe herein a robust biosensor that combines the sensitivity of surface acoustic waves (SAW) generated at a frequency of 325MHz with the specificity provided by antibodies for the detection of viral agents. A lithium tantalate-based SAW transducer with silicon dioxide waveguide sensor platform featuring three test and one reference delay lines was used to adsorb antibodies directed against either Coxsackie virus B4 or the category A bioagent Sin Nombre virus (SNV), a member of the genus Hantavirus, family Bunyaviridae, negative-stranded RNA viruses. Rapid detection (within seconds) of increasing concentrations of viral particles was linear over a range of order of magnitude for both viruses, although the sensor was approximately 5 x 10(5)-fold more sensitive for the detection of SNV. For both pathogens, the sensor's selectivity for its target was not compromised by the presence of confounding Herpes Simplex virus type 1. The biosensor was able to detect SNV at doses lower than the load of virus typically found in a human patient suffering from hantavirus cardiopulmonary syndrome (HCPS). Further, in a proof-of-principle real world application, the SAW biosensor was capable to selectively detect SNV agents in complex solutions, such as naturally occurring bodies of water (river, sewage effluent) without analyte pre-processing. This is the first study that reports on the detection of viral agents using an antibody-based SAW biosensor that has the potential to be used as a hand-held and self-contained device for rapid viral detection in the field.     

Integration of a surface acoustic wave biosensor in a microfluidic polymer chip

SAW devices based on horizontally polarized surface shear waves (HPSSW) enable label-free, sensitive and cost-effective detection of biomolecules in real time. It is known that small sampling volumes with low inner surface areas and minimal mechanical stress arising from sealing elements of miniaturized sampling chambers are important in this field. Here, we present a new approach to integrate SAW devices with sampling chamber. The sensor device is encapsulated within a polymer chip containing fluid channel and contact points for fluidic and electric connections. The chip volume is only 0.9 microl. The polymeric encapsulation was performed tailor-made by Rapid Micro Product Development 3Dimensional Chip-Size-Packaging (RMPD 3D-CSP), a 3D photopolymerisation process. The polymer housing serves as tight and durable package for HPSSW biosensors and allows the use of the complete chips as disposables. Preliminary experiments with these microfluidic chips are shown to characterise the performance for their future applications as generic bioanalytical micro devices.     

Kinetic studies of small molecule interactions with protein kinases using biosensor technology

Protein kinases are among the most commonly targeted groups of molecules in drug discovery today. Despite this, there are few examples of using surface plasmon resonance (SPR) for kinase inhibitor interaction studies, probably reflecting the need for better developed assays for these proteins. In this article, we present a general methodology that uses biosensor technology to study small molecule binding to eight different serine/threonine and tyrosine kinases. Mild immobilization conditions and a carefully composed assay buffer were identified as key success factors. The methodology package consists of direct binding studies of compounds to immobilized kinases, kinase activity assays to confirm inhibitory effects, detailed kinetic analyses of inhibitor binding, and competition assays with ATP for identification of competitive inhibitors. The kinetic assays resolve affinity into the rates of inhibitor binding and dissociation. Therefore, more detailed information on the relation between inhibitor structure and function is obtained. This might be of key importance for the development of effective kinase inhibitors.     

Potential of biosensor technology for the characterization of interactions by quantitative affinity chromatography

This review places the characterization of interactions by biosensor technology in the broader context of their study by quantitative affinity chromatography. The general reluctance to consider biosensor-based characterization as a form of quantitative affinity chromatography on the grounds of a difference in aims of the two techniques reflects a mistaken belief that BIAcore and IAsys studies characterize the kinetics of the chemical reaction responsible for biospecific adsorption of a soluble reactant to an immobilized form of its affinity partner. It now transpires that the association and dissociation rate constants thereby determined refer to thermodynamic characterization of biospecific adsorption in terms of a single-phase model in which affinity sites are distributed uniformly throughout the liquid-phase volume accessible to the partitioning reactant—the model used for characterization of biospecific adsorption by quantitative affinity chromatography. In that light the most important attribute of biosensor technology is its potential for thermodynamic characterization of biospecific adsorption by virtue of its ability to monitor complex formation directly; and hence its potential for the characterization of interactions with affinities that are too strong for study by forms of quantitative affinity chromatography that monitor complex formation on the basis of reactant depletion from the liquid phase. Kinetic as well as thermodynamic analyses of biosensor data are described for attainment of that potential.     

Kinetic characterization of carboxypeptidase-Y-catalyzed peptide semisynthesis Prediction of yields

Summary Carboxypeptidase-Y-catalyzed peptide semisynthesis has been characterized at pH 7.5, 25°C from initial rate steady state kinetic and progress reaction studies of hydrolysis and aminolysis of-N-benzoyl-L-tyrosine 4-nitro-anilide using the natural L-amino acids and their amides as nucleophiles. The reaction mechanism previously shown to account for carboxypeptidase-Y-catalyzed aminolysis reactions (Christensen et al., 1992) was found also to account for all of the reactions studied here. It involves in addition to the classical serine proteinase mechanism: i) complex formation between the free enzyme and the nucleophile, an interaction characterized by the competitive inhibition constant,K _i, and ii) reaction of the nucleophile with the acylated enzyme forming a complex of enzyme and aminolysis product, characterized by the aminolysis kinetic parameter,K _N.A competitive inhibitory effect showing binding to the free enzyme is seen mainly with large hydrophobic amino acids and their amides i.e. the same residues as those preferred on either side of the scissile bond in carboxypeptidase-Y substrates. The stoichiometry of the inhibition is 1 : 1 and the actual binding position most likely is that of the leaving group of substrates,S ₁.Aminolysis effects are obtained with a wide range of amino acids and amino acid amides, exceptions are Pro and, probably due to their low solubility, Tyr, Trp, Asp and Glu. TheK _N-values show relatively little dependence on the chemical nature of the side groups, but a marked difference between the amino acid and its amide. The amides interact more strongly. The kinetic parameter,k _c/K_m, of the hydrolysis of the aminolysis products is another important factor in peptide semisynthesis. Thek _c/K_m-values obtained of the amidated aminolysis products are much less than those of the products formed with free amino acids. All in all this leads to rather efficient aminolysis with the L-amino acid amides and poor aminolysis with the L-amino acids.Abbreviations BzTyrNHPhNO₂ -N-benzoyl-L-tyrosinyl 4-nitro-aniline - Xaa L-amino acids - Xaaa L-amino acid amides - Z-Phe Carbobenzoxy-L-phenylalanine - Z-Met Carbobenzoxy-L-methionine - BzTyr -N-benzoyl-L-tyrosine - AlaVal L-alanyl-L-valine - ValAla L-valyl-L-alanine     

An acoustic wave biosensor for human low-density lipoprotein particles: construction of selective coatings

Cholesterol is found in four major classes of blood particles including chylomicrons, very low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). The most studied fraction is LDL as it is most closely associated with heart disease. The challenge in current methods of analysis is the determination of the cholesterol in the individual lipoprotein fractions. Accordingly, the critical step in any analysis is the complete separation of the lipoprotein fractions. In this work, enhanced selectivity for the LDL fraction was achieved by the covalent binding of dextran sulfate (DS) to the gold surface of a thickness shear-mode acoustic wave sensor. The thickness and surface concentration of the DS layer was estimated by in situ ellipsometry to be 219 A and 0.8 ng/mm(2), respectively, but it was difficult to construct the sensing layer reproducibly. The DS coated sensor was ten times more responsive to LDL than the other lipoprotein (LP) fractions. The sensor was a main component in a flow injection analysis system that exposed LDL, VLDL and HDL to not only the DS layer, but also to the underlayers used in the construction of the DS layer. A possible regeneration solution was found which would rinse the LDL from the layer, restoring the sensor for repeated use. Frequency shifts from LP absorption into the DS layer were corrected for dissipative losses through the DS layer using an oscillator circuit equipped with an automatic gain control feature.     

An interleukin-6 ZnO/SiO(2)/Si surface acoustic wave biosensor

A novel high sensitivity ZnO/SiO(2)/Si Love mode surface acoustic wave (SAW) biosensor for the detection of interleukin-6 (IL-6), is reported. The biosensors operating at 747.7MHz and 1.586GHz were functionalized by immobilizing the monoclonal IL-6 antibody onto the ZnO biosensor surface both through direct surface adsorption and through covalent binding on gluteraldehyde. The morphology of the IL-6 antibody-protein complex was studied using scanning electron microscopy (SEM), and the mass of the IL-6 protein immobilized on the surface was measured from the frequency shift of the SAW resonator biosensor. The biosensor was shown to have extended linearity, which was observed to improve with higher sensor frequency and for IL-6 immobilization through the monoclonal antibody. Preliminary results of biosensor measurements of low levels of IL-6 in normal human serum are reported. The biosensor can be fully integrated with CMOS Si chips and developed as a portable real time detection system for the interleukin family of proteins in human serum.     

Structural characterization of peptide hormone/receptor interactions by NMR spectroscopy.

The structural characterization of peptide hormones and their interaction with G-protein (guanine nucleotide-binding regulatory protein) coupled receptors by high-resolution nmr is described. The general approaches utilized can be categorized into three different classes based on their target: the ligand, the receptor, and the ligand/receptor complex. Examples of these different approaches, aimed at facilitating the rational design of peptides and peptidomimetics with improved pharmacological profiles, based on work carried out in our own laboratory, are given. In the ligand-based approach, the high-resolution structures of bradykinin analogues allowing for the development of a structure-activity relationship for activation of the B1 receptor are described. Studies targeting the receptor are to a large extent theoretical, based on computational molecular modeling. However, experimentally based structural features provided by high-resolution nmr can be used to great advantage, providing insight into the mechanism of receptor function, as illustrated here with results from parathyroid hormone. A similar combination of theoretical methods, supplemented by high-resolution structures from nmr has been utilized to probe the formation and stabilization of the ligand/receptor complex both for parathyroid hormone and cholecystokinin. In each of these three approaches, the importance of well-designed peptide mimetics and accurate structural analysis by high-resolution nmr, will be highlighted.     

Shear acoustic wave biosensor for detecting DNA intrinsic viscosity and conformation: a study with QCM-D

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.     

Kinetic measurements of molecular interactions by spectrofluorometry

The kinetics of antibody–antigen interactions are reviewed in terms of general trends observed in both polyclonal and monoclonal antibody populations. Anti-fluorescein antibodies are featured in the review as model proteins to explore fluorescence-based kinetic measurements. Since the fluorescence of the fluorescein ligand is significantly quenched upon interaction with both polyclonal and monoclonal anti-fluorescein antibodies, the quenching parameter can be advantageously employed in measuring the rates of association (k₁) and dissociation (k₂). The near diffusion-limited k₁ rates and the prolonged k₂ rates are discussed in terms of antibody affinity and mechanisms involved in ligand binding. Specific prolongation effects of reagents, such as anti-metatype antibodies, on the dissociation rate are discussed in terms of antibody dynamics and conformational substates.     

EPR spectroscopic analysis of TAR RNA-metal ion interactions

Metal ion-induced changes in HIV-1 TAR RNA internal dynamics were determined by the changes in EPR spectral width for TAR RNAs containing spin-labeled nucleotides (U23, U25, U38, and U40). This gave a dynamic signature for each of 10 metal ions studied, which fell into one of three distinct groups. While Li(+) and K(+) had little effect on TAR RNA internal dynamics, Na(+) unexpectedly had a dynamic signature that was similar to Ca(2+) and Sr(2+), with a decrease in mobility at U23 and U38, little or no change at U25, and an increase in mobility at U40. Mg(2+), Co(2+), Ni(2+), Zn(2+), and Ba(2+) had similar effects on U23, U38, and U40, but the mobility of U25 was markedly increased. Our results show that RNA dynamics change upon metal binding to the TAR RNA bulge, indicating that RNA structure adapts to accommodate metal ions of different size and coordination properties.     

光纤倏逝波生物传感器及其应用

介绍光纤倏逝波生物传感器的基本原理、常用试验方法、基本仪器构建及应用进展。光纤倏逝波生物传感器是基于光波在光纤内以全反射方式传输时产生倏逝波的原理,以生物分子作为敏感元件进行检测的一类新兴传感器。光纤倏逝波生物传感器有望应用于环境监控、食品卫生监控、临床疾病监测、DNA检测和生物战剂检测。     

The analysis of heparin-protein interactions using evanescent wave biosensor with regioselectively desulfated heparins as the ligands

Evanescent wave biosensor has been recently employed as a powerful tool for analyses of macromolecular interactions. In the present study, evanescent wave biosensor analysis was developed to analyze the heparin-protein interaction using as ligands a series of heparin derivatives regioselectively desulfated by chemical methods, particularly to evaluate the effect of each sulfate group of heparin. The method for immobilizing heparin on the cuvette of the evanescent wave biosensor equipment was optimized to obtain the high response required for accurate measurement. The best result was achieved when the amino group introduced at the reducing end of heparin was coupled with carboxymethyl dextran on the surface of the cuvette using glycolchitosan as a multivalent linker. The established system appeared to describe well the interactions of heparin with such proteins as acidic and basic fibroblast growth factors and tissue factor pathway inhibitor.     

Isolation and characterization of three phosphoamido-neomycins and their conversion into neomycin by Streptomyces fradiae

Some amino acids, particularly glycine and serine, favour the accumulation in the fermentation broth of three phosphorylated amino sugar compounds that are intermediates in the pathway of neomycin biosynthesis by Streptomyces fradiae 3535. The compounds were separated and purified further by Amberlite IRC-50 (NH(4) (+) form). The intermediates were characterized by physicochemical methods as neomycin B pyrophosphate (C(23)H(48)N(6)O(19)P(2),3H(2)O), neomycin C pyrophosphate (C(23)H(48)N(6)O(19)P(2),3H(2)O) and neomycin C dipyrophosphate complex (C(24)H(66)N(8)O(33)P(4)).