Different approaches for the detection of thrombin by an electrochemical aptamer-based assay coupled to magnetic beads

Different assay formats based on the coupling of magnetic beads with electrochemical transduction were compared here for the detection of thrombin by using a thrombin specific aptamer. By using the thrombin-binding aptamer, a direct and an indirect competitive assay for thrombin have been developed by immobilising the aptamer or the protein, respectively. Moreover, another strategy was based on the direct measurement of the enzymatic product of thrombin captured by the immobilised aptamer. All the assays were developed by coupling the electrochemical transduction with the innovative and advantageous use of magnetic beads.

The assays based on the immobilisation of the protein were not successful since no binding was recorded between thrombin and its aptamer. With the direct competitive assay, when the aptamer was immobilised onto the magnetic beads, a detection limit of 430 nM for thrombin was achieved. A lower detection limit for the protein (175 nM) was instead obtained by detecting the product of the enzymatic reaction catalysed by thrombin. All these assays were finally compared with a sandwich assay which reached a detection limit of 0.45 nM of thrombin demonstrating the best analytical performances.

With this comparison the importance of a deep study on the different analytical approaches for thrombin detection to reach the performances of the best assay configuration has been demonstrated.     

Using liquid crystals for the label-free detection of catalase at aqueous-LC interfaces

In this study, we developed a simple label-free method for the detection of catalase (CAT) using liquid crystals (LCs). The optical appearance of LCs changed from bright to dark when hydrogen peroxide was in contact with the dodecanal-doped nematic LC, 4-cyano-4′-pentylbiphenyl (5CB). Since hydrogen peroxide can oxidize aldehyde into carboxylic acid, an orientational transition of the LC from the planar to homeotropic state was induced by the self-assembled carboxylate monolayer formed at the aqueous/LC interface. The optical response of LCs exhibited a higher sensitivity to the presence of hydrogen peroxide in an alkaline solution. A new type of LC-based sensor was developed to monitor the presence of CAT in the aqueous phase. Due to the enzymatically catalytic hydrolysis of hydrogen peroxide, the bright-to-dark shift in the optical signal did not take place in the aqueous mixture of hydrogen peroxide and catalase. In contrast, the optical response changed from bright to dark when the mixture in the optical cell was replaced with an aqueous solution of hydrogen peroxide. Considering the optical response of LCs related to the absence and presence of hydrogen peroxide, the aldehyde-doped 5CB might have potential utility in real-time recognition and detection of chemical and biological events associated with hydrogen peroxide.     

Molecular dynamics study of thrombin capture by aptamers TBA26 and TBA29 coupled to a DNA origami

The fabrication of DNA origamis is one of the very few possibilities to create nanostructures with precise atomistic-tailored geometries in a variety of shapes. In addition, these origamis can be functionalized or be impregnated with specialised aptamers in order to convert them into nanosensors or to tune them with pre-specified properties simply by impregnating single-stranded DNA or RNA chains (aptamers) via the precise features of DNA pairing. We performed molecular dynamics simulations to determine the relative energetics associated with the capture of thrombin by two aptamers TBA26 and TBA29 attached to a rectangular DNA origami. The molecular simulations provided detailed structural information of aptamer–enzyme interactions which are crucial for the efficient design of aptamer-based biosensors. In addition, the simulations showed a remarkable selectivity of the biosensor assembly for thrombin. The detection, capture, and sensing of enzymes is of great significance in biomedicine. In particular, the detection of thrombin is a major task in cardiovascular diagnostics and therapeutics. On the other hand, our simulations can be extended to detect biotoxins or any other chemical or biological agent by simply choosing proper aptamers. Finally, the problems due to the large number of atoms involved as well as the quality of the approximations are also discussed.     

A Strong to Fragile Transition in a Model of Liquid Silica

Abstract

The transport properties of an ionic model for liquid silica [1] at high temperatures and pressure are investigated using molecular dynamics simulations. With increasing pressure, a clear change from “strong” to “fragile” behaviour (according to Angell's classification of glass-forming liquids) is observed, albeit only on the small viscosity range that can be explored in MD simulations. This change is related to structural changes, from an almost perfect four-fold coordination to an imperfect five or six-fold coordination.     

Use of laurdan fluorescence intensity and polarization to distinguish between changes in membrane fluidity and phospholipid order

Laurdan is a fluorescent probe that detects changes in membrane phase properties through its sensitivity to the polarity of its environment in the bilayer. Variations in membrane water content cause shifts in the laurdan emission spectrum, which are quantified by calculating the generalized polarization (GP). We tested whether laurdan fluorescence could be used to distinguish differences in phospholipid order from changes in membrane fluidity by examining the temperature dependence of laurdan GP and fluorescence anisotropy in dipalmitoylphosphatidylcholine (DPPC) vesicles. The phase transition from the solid ordered phase to the liquid disordered phase was observed as a decrease in laurdan GP values from 0.7 to −0.14 and a reduction in anisotropy from 0.25 to 0.12. Inclusion of various amounts of cholesterol in the membranes to generate a liquid ordered phase caused an increase in the apparent melting temperature detected by laurdan GP. In contrast, cholesterol decreased the apparent melting temperature estimated from anisotropy measurements. Based on these results, it appeared that laurdan anisotropy detected changes in membrane fluidity while laurdan GP sensed changes in phospholipid order. Thus, the same fluorescent probe can be used to distinguish effects of perturbations on membrane order and fluidity by comparing the results of fluorescence emission and anisotropy measurements.     

A chronocoulometric aptasensor based on gold nanoparticles as a signal amplification strategy for detection of thrombin

A sensitive chronocoulometric aptasensor for the detection of thrombin has been developed based on gold nanoparticle amplification. The functional gold nanoparticles, loaded with link DNA (LDNA) and report DNA (RDNA), were immobilized on an electrode by thrombin aptamers performing as a recognition element and capture probe. LDNA was complementary to the thrombin aptamers and RDNA was noncomplementary, but could combine with [Ru(NH₃)₆]³⁺ (RuHex) cations. Electrochemical signals obtained by RuHex that bound quantitatively to the negatively charged phosphate backbone of DNA via electrostatic interactions were measured by chronocoulometry. In the presence of thrombin, the combination of thrombin and thrombin aptamers and the release of the functional gold nanoparticles could induce a significant decrease in chronocoulometric signal. The incorporation of gold nanoparticles in the chronocoulometric aptasensor significantly enhanced the sensitivity. The performance of the aptasensor was further increased by the optimization of the surface density of aptamers. Under optimum conditions, the chronocoulometric aptasensor exhibited a wide linear response range of 0.1–18.5 nM with a detection limit of 30 pM. The results demonstrated that this nanoparticle-based amplification strategy offers a simple and effective approach to detect thrombin.     

Probing interactions between the coagulants thrombin, Factor XIII, and fibrin(ogen)

Thrombin cleaves fibrinopeptides A and B from fibrinogen leading to the formation of a fibrin network that is later covalently crosslinked by Factor XIII (FXIII). Thrombin helps activate FXIII by catalyzing hydrolysis of the FXIII activation peptides (AP). In the current work, the role of exosites in the ternary thrombin-FXIII-fibrin(ogen) complex was further explored. Hydrolysis studies indicate that thrombin predominantly utilizes its active site region to bind extended Factor XIII AP (FXIII AP 33-64 and 28-56) leaving the anion-binding exosites for fibrin(ogen) binding. The presence of fibrin-I leads to improvements in the K(m) for hydrolysis of FXIII AP (28-41), whereas peptides based on the cardioprotective FXIII V34L sequence exhibit less reliance on this cofactor. Surface plasmon resonance measurements reveal that d-Phe-Pro-Arg-chloromethylketone-thrombin binds to fibrinogen faster than to FXIII a(2) and dissociates from fibrinogen more slowly than from FXIII a(2). This system of thrombin exosite interactions with differing affinities promotes efficient clot formation.     

Interaction between phorbol esters and phospholipid in a monolayer model membrane

The possible molecular interaction between phorbol esters and a phospholipid was examined in monolayer films at an air-water interface. The surface pressure isotherms indicated that, at close-to-physiological pressure, there existed a repulsive interaction between the phospholipid and biologically active phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol 12,13-didecanoate (PDD). There was a close parallelism between the relative biological potency of different phorbol esters, as tumour promoters, and the magnitude of their repulsive interaction with the phospholipid. These findings raise the possibility that a biophysical interference of phorbol esters with the phospholipid domain of biological membranes may represent an important determinant of their biological actions.     

Liquid chromatography-inductively coupled plasma-based metallomic approaches to probe health-relevant interactions between xenobiotics and mammalian organisms

In mammals, the transport of essential elements from the gastrointestinal tract to organs is orchestrated by biochemical mechanisms which have evolved over millions of years. The subsequent organ-based assembly of sufficient amounts of metalloproteins is a prerequisite to maintain mammalian health and well-being. The chronic exposure of various human populations to environmentally abundant toxic metals/metalloid compounds and/or the deliberate administration of medicinal drugs, however, can adversely affect these processes which may eventually result in disease. A better understanding of the perturbation of these processes has the potential to advance human health, but their visualization poses a major problem. Nonetheless, liquid chromatography-inductively coupled plasma-based 'metallomics' methods, however, can provide much needed insight. Size-exclusion chromatography-inductively coupled plasma atomic emission spectrometry, for example, can be used to visualize changes that toxic metals/medicinal drugs exert at the metalloprotein level when they are added to plasma in vitro. In addition, size-exclusion chromatography-inductively coupled plasma mass spectrometry can be employed to analyze organs from toxic metal/medicinal drug-exposed organisms for metalloproteins to gain insight into the biochemical changes that are associated with their acute or chronic toxicity. The execution of such studies-from the selection of an appropriate model organism to the generation of accurate analytical data-is littered with potential pitfalls that may result in artifacts. Drawing on recent lessons that were learned by two research groups, this tutorial review is intended to provide relevant information with regard to the experimental design and the practical application of these aforementioned metallomics tools in applied health research.     

Synthesis of CC,CN coupled novel substituted dibutyl benzothiazepinone derivatives and evaluation of their thrombin inhibitory activity

The formation of a thrombus is a key event in thromboembolic disorders, that contribute to high mortality and morbidity in affected patients. In the present study, we synthesized a library of novel substituted 3,3-dibutyl-8-methoxy-2,3-dihydrobenzo [b] [1,4] thiazepin-4(5H)-one derivatives which were tested for their platelet aggregation and thrombin inhibitory activity. Among the tested compounds, 3,3-dibutyl-7-(2-chlorophenyl)-8-methoxy-2,3-dihydrobenzo[b] [1,4]thiazepin-4(5H)-one (DCT) displayed the maximum thrombin inhibition with an IC₅₀ value of 3.85 μM and thus DCT was chosen for further studies. Next, the effect of DCT on primary hemostasis was evaluated using agonist-induced platelet aggregation model. The lead compound inhibited the collagen- or ADP- or thrombin-induced platelet aggregation in a dose-dependent manner. Furthermore, DCT prolonged the process of clot formation when evaluating plasma re-calcification time (320 ± 11 sec at 5 µg DCT), activated partial thromboplastin time (58.0 ± 0.01 sec at 2 µg), and prothrombin time (14.7 ± 0.01 sec at 5 µg). Molecular docking studies suggested that the benzothiazepinones evaluated here consistently display hydrogen bonding with Ser214 of thrombin, which is similar to that of the co-crystallized ligand (1-(2R)-2-amino-3-phenyl-propanoyl-N-(2,5dichlorophenyl)methylpyrrolidine-2-carboxamide). DCT displayed additional hydrogen bonding to Ser195 and π-π interactions between its methoxyphenyl groups and Trp60, thereby providing a structural rationale for the observed biological effect.     

Design, synthesis and SAR of a series of 1,3,5-trisubstituted benzenes as thrombin inhibitors

A novel 1,3,5-trisubstituted benzamide thrombin inhibitor template was designed via hybridization of a known aminopyridinoneacetamide and a known 1,3,5-trisubstituted phenyl ether. Optimization of this lead afforded a novel potent series of biaryl 1,3,5-trisubstituted benzenes with excellent functional anticoagulant potency.     

Determination of melagatran, a novel, direct thrombin inhibitor, in human plasma and urine by liquid chromatography-mass spectrometry

Analytical methods for the determination of melagatran (H 319/68) in biological samples by liquid chromatography (LC)-positive electrospray ionization mass spectrometry using multiple reaction monitoring are described. Melagatran in plasma was isolated by solid-phase extraction on octylsilica, either in separate extraction tubes or in 96-well plates. Absolute recovery of melagatran from plasma was >92%. Melagatran and the internal standard, H 319/68 D2 13C2, were separated from other sample components by LC utilizing a C18 stationary phase and a mobile phase comprising 35% acetonitrile and 0.08% formic acid in 0.0013 mol/l ammonium acetate solution. After dilution, urine was injected directly onto the LC column and subjected to gradient LC. The relative standard deviation was 1-5% for concentrations above the limit of quantification, which was estimated for plasma at 10 or 25 nmol/l for sample volumes of 500 or 200 microl, respectively, and 100 nmol/l for urine.     

The long term immunological response of swine after two exposures to a salmon thrombin and fibrinogen hemostatic bandage

Experimental salmon thrombin/fibrinogen dressings have been shown to provide effective hemostasis in severe hemorrhage situations. The hypothesis for this study was that swine would still remain healthy without coagulopathy six months after exposure to salmon thrombin/fibrinogen dressings. Initial exposure was by insertion of the salmon dressing into the peritoneal cavity. Three months after the initial exposure, the same animals were subjected to two full thickness dermal wounds on the dorsal surface. One wound was bandaged with the salmon thrombin/fibrinogen bandage and the other wound was dressed with a standard bandage. The animals were monitored for an additional three months. Blood was drawn every 14 days over the six months for immunological and coagulation function analysis. All of the animals (8 pigs) remained healthy during the six month period and the dermal wounds healed without incidence. Lymph nodes and spleen showed signs of normal immune response and Western blots showed development of antibodies against salmon fibrinogen, but none of the animals made antibodies that recognized any species of thrombin. Coagulation parameters (fibrinogen concentration, thrombin time, PT and aPTT) and hematological parameters remained normal over the course of the study when compared to initial values of the subject swine.     

Near infrared spectroscopy compared to liquid chromatography coupled to mass spectrometry and capillary electrophoresis as a detection tool for peptide reaction monitoring

Peptide interaction is normally monitored by liquid chromatography (LC), liquid chromatography coupled to mass spectrometry (LC-MS), mass spectrometric (MS) methods such as MALDI-TOF/MS or capillary electrophoresis (CE). These analytical techniques need to apply either high pressure or high voltages, which can cause cleavage of newly formed bondages. Therefore, near infrared reflectance spectroscopy (NIRS) is presented as a rapid alternative to monitor the interaction of glutathione and oxytocin, simulating physiological conditions. Thereby, glutathione can act as a nucleophile with oxytocin forming four new conjugates via a disulphide bondage. Liquid chromatography coupled to UV (LC-UV) and mass spectrometry via an electrospray ionisation interface (LC-ESI-MS) resulted in a 82% and a 78% degradation of oxytocin at pH 3 and a 5% and a 7% degradation at pH 6.5. Capillary electrophoresis employing UV-detection (CE-UV) showed a 44% degradation of oxytocin. LC and CE in addition to the NIRS are found to be authentic tools for quantitative analysis. Nevertheless, NIRS proved to be highly suitable for the detection of newly formed conjugates after separating them on a thin layer chromatography (TLC) plate. The recorded fingerprint in the near infrared region allows for a selective distinct qualitative identification of conjugates without the need for expensive instrumentation such as quadrupole or MALDI-TOF mass spectrometers. The performance of the established NIRS method is compared to LC and CE; its advantages are discussed in detail.     

Electrochemical modeling of electron and proton transfer to ubiquinone-10 in a self-assembled phospholipid monolayer.

Ubiquinone-10 (UQ) was incorporated at concentrations ranging from 0.5 to 2 mol% in a self-assembled monolayer of dioleoylphosphatidylcholine (DOPC) deposited on a mercury drop electrode, and its electroreduction to ubiquinol (UQH2) was investigated in phosphate and borate buffers over the pH range from 7 to 9.5 by a computerized chronocoulometric technique. The dependence of the applied potential for a constant value of the faradaic charge due to UQ reduction upon the electrolysis time t at constant pH and upon pH at constant t was examined on the basis of a general kinetion approach. This permitted us to conclude that the reduction of UQ to UQH2 in DOPC monolayers takes place via the reversible uptake of one electron with the formation of the semiubiquinone radical anion UQ.-, followed by the rate-determining protonation of this anion with UQH. formation; this neutral radical is more easily reduced than UQ, yielding the ubiquinol UQH2. In spite of the very low concentration of hydrogen ions as compared with that of the acidic component of the buffer, the only effective proton donor is the proton itself; this strongly suggests that the protonation step takes place inside the polar head region of the DOPC monolayer, which is only accessible to protons.     

Video fluorescence microscopy studies of phospholipid vesicle fusion with a planar phospholipid membrane. Nature of membrane-membrane interactions and detection of release of contents

Video fluorescence microscopy was used to study adsorption and fusion of unilamellar phospholipid vesicles to solvent-free planar bilayer membranes. Large unilamellar vesicles (2-10 microns diam) were loaded with 200 mM of the membrane-impermeant fluorescent dye calcein. Vesicles were ejected from a pipette brought to within 10 microns of the planar membrane, thereby minimizing background fluorescence and diffusion times through the unstirred layer. Vesicle binding to the planar membrane reached a maximum at 20 mM calcium. The vesicles fused when they were osmotically swollen by dissipating a KCl gradient across the vesicular membrane with the channel-forming antibiotic nystatin or, alternatively, by making the cis compartment hyperosmotic. Osmotically induced ruptures appeared as bright flashes of light that lasted several video fields (each 1/60 s). Flashes of light, and therefore swelling, occurred only when channels were present in the vesicular membrane. The flashes were observed when nystatin was added to the cis compartment but not when added to the trans. This demonstrates that the vesicular and planar membranes remain individual bilayers in the region of contact, rather than melding into a single bilayer. Measurements of flash duration in the presence of cobalt (a quencher of calcein fluorescence) were used to determine the side of the planar membrane to which dye was released. In the presence of 20 mM calcium, 50% of the vesicle ruptures were found to result in fusion with the planar membrane. In 100 mM calcium, nearly 70% of the vesicle ruptures resulted in fusion. The methods of this study can be used to increase significantly the efficiency of reconstitution of channels into planar membranes by fusion techniques.     

Antithrombotic effects of LB30870, a potent,orally active,selective and direct thrombin inhibitor,and pharmacokinetics of its prodrug

Antithrombotic activity and bleeding complication of a new potent, selective, and direct thrombin inhibitor, LB30870, were evaluated in comparison with other anticoagulants. In order to improve oral absorption of LB30870, pharmacokinetics of LB30889, which is a double prodrug with blocking groups in both amidine and carboxyl groups, was studied in rats and dogs. LB30870 was more potent than melagatran or argatroban with thrombin inhibition constants of 0.02, 1.3 and 4.5 nM, respectively. All three direct thrombin inhibitors were selective towards other serine proteases with selectivity ratio greater than 1000, except for trypsin. Thrombin binding kinetics of LB30870 showed rapid association and slow dissociation rate constants, demonstrating its potential as anticoagulant. LB30870 was more effective than melagatran or argatroban in plasma clot-bound thrombin inhibition. In the rat venous stasis model of the caval vein, LB30870 reduced wet clot weights in a dose dependent manner after the intravenous bolus with infusion administration. The ED₅₀ of LB30870, melagatran and enoxaparin were 50 μg/kg + 2 μg/kg/min, 35 μg/kg + 1.4 μg/kg/min and 200 μg/kg + 8.3 μg/kg/min, respectively. No significant bleeding problem was observed with LB30870 at the dose up to two times ED₈₀ in rats. LB30889, a double prodrug of LB30870, showed species difference in pharmacokinetics. Its oral bioavailability in rats or dogs was not better than that of LB30870. In conclusion, LB30870 has the potential to be useful as an effective oral anticoagulant for the prevention and treatment of thromboembolic diseases.     

Criteria to optimize designs for detection and estimation of linkage between marker loci from segregating populations containing several families

Summary Construction of a genome map of highly polymorphic markers has become possible in the past decade. Establishing a complete marker map is an enormous task. Therefore, designs to map molecular markers should be optimal. Designs to detect and estimate linkage between markers from segregating populations were studied. Two measures of design quality were used. The expectation of the maximum lod score indicates the possibility of designs to detect linkage. The accuracy of estimating recombination rate was measured as the probability that the true recombination rate is in a specified internal given the estimate. Accurate approximate methods were developed for rapid evaluation of designs. Seven family types (e.g., double backcross) can be distinguished that describe all families in a segregating population. The family type influences the expected maximum lod score and the accuracy of estimation. The frequency of favorable family types increased with increasing marker polymorphism. At a true recombination rate of 0.20,27 observations on offspring when five alleles were segregating, and 55 observations on offspring when two alleles were segregating, were necessary to obtain an expected maximum lod score of 3. The probability that the true recombination rate was between 0.15 and 0.25, given an estimate of 0.20, was about 0.85 for a design with 40 families with ten offspring and two alleles segregating and for a design with ten families with ten offspring and six alleles segregating. For smaller designs, accuracies were less, approximate evaluation of accuracy was not justified and, on average, true recombination rates were much greater than estimated given a specified value for the estimated recombination rate.