Acoustic Wave-Driven Functionalized Particles for Aptamer-Based Target Biomolecule Separation.

We developed a hybrid microfluidic device that utilized acoustic waves to drive functionalized microparticles inside a continuous flow microchannel and to separate particle-conjugated target proteins from a complex fluid. The acoustofluidic device is composed of an interdigitated transducer that produces high-frequency surface acoustic waves (SAW) and a polydimethylsiloxane (PDMS) microfluidic channel. The SAW interacted with the sample fluid inside the microchannel and deflected particles from their original streamlines to achieve separation. Streptavidin-functionalized polystyrene (PS) microparticles were used to capture aptamer (single-stranded DNA) labeled at one end with a biotin molecule. The free end of the customized aptamer15 (apt15), which was attached to the microparticles via streptavidin-biotin linkage to form the PS-apt15 conjugate, was used to capture the model target protein, thrombin (th), by binding at exosite I to form the PS-apt15-th complex. We demonstrated that the PS-apt15 conjugate selectively captured thrombin molecules in a complex fluid. After the PS-apt15-th complex was formed, the sample fluid was pumped through a PDMS microchannel along with two buffer sheath flows that hydrodynamically focused the sample flow prior to SAW exposure for PS-apt15-th separation from the non-target proteins. We successfully separated thrombin from mCardinal2 and human serum using the proposed acoustofluidic device.

Online transient isotachophoresis concentration by the pseudo-terminating electrolyte buffer for the separation of DNA-aptamer and its thrombin complex in poly(methyl methacrylate) microchip.

Online automatic transient isotachophoresis concentration of DNA-aptamer and its thrombin complex by using one kind of pseudo-terminating electrolyte buffer in a cross-channel poly(methyl methacrylate) microchip is reported. Sample injection, transient concentration and separation were done continuously and controlled by a sequential voltage switching program, time-consuming steps and complicated chip design were not required. Peak resolution between DNA-aptamer and its thrombin complex was influenced by this novel pseudo-terminating electrolyte buffer, which was prepared by the addition of chemical component with slow mobility into the same buffer as leading electrolyte buffer. 1100-fold signal enhancement of thrombin complex was achieved by this transient isotachophoresis on a standard cross-form microchip. The concentration effect or standing time of transient isotachophoresis was proved to be influenced by the concentration of leading electrolyte ion and the concentration of pseudo-terminating electrolyte buffer ion (glycine). The transient concentration was followed by on-chip nondenaturing gel electrophoresis in methylcellulose solution for the size-based separation. The detection limit, taken as the lowest thrombin concentration at threefold S/N, was determined to be 0.5 amol in mass by this method.

Evaluation of bone growth using artificial bone substitute (osteoset) and platelet gel mixtures: a preliminarily study in dogs.

Platelet gels (PG), activated by bovine thrombin (BT), have increasingly been used in orthopedic surgery. However, BT may induce immunological reactions and carry potential viral and prion risks. To avoid these side effects, thrombin derived from human plasma (human thrombin, HT) is becoming the preferred platelet activator to prepare PG. However, limited experience and data on the clinical benefits of HT-generated PG (HTPG) in orthopedic surgery is reported. Consequently, we designed and performed a series of studies in dogs to compare the impacts of promotion of bone growth by an artificial bone substitute (Osteoset) in combination with HTPG or without it in the spinal repair experiments. X-ray observations and histological studies were performed at predetermined periods post-operation. The preliminary results revealed the preparation of HTPG was easy and required less than 30 minutes. HTPG was capable of embedding the artificial bone substitute Osteoset to prepare a sticky and easily manipulated composite for the application into spinal defect. We found HTPG exhibited enhancement of grafting capacity in consolidation of bone mass. After 12 weeks, tissue reconstruction reached approximately 80% of the injury defects when treated by HTPG/Osteoset combination, but only 30 approximately 40% in the absence of HTPG. The physiological activity of artificial bone substitute combined with PG activated by HT may therefore open beneficial prospects for more successful and safer bone formation in spine procedures in the near future.

Highly efficient electrogenerated chemiluminescence quenching of PEI enhanced Ru(bpy)3²âº nanocomposite by hemin and Au@CeO2 nanoparticles.

In this work, a new signal amplified strategy based on the quenching effect of hemin and Au nanoparticles decorated CeO2 nanoparticles (Au@CeO2 NPs) for ultrasensitive detection of thrombin (TB) is reported for the first time. Herein, the poly(ethylenimine) (PEI) enhanced Ru(bpy)3(2+) nanocomposite was implemented by direct chemical polymerization, which could provide the desirable enhanced initial ECL signal. Furthermore, the detection aptamer of thrombin (TBA 2) was immobilized on Au@CeO2 NPs to form TBA 2/Au@CeO2 conjugates. Then, the G-rich DNA of TBA 2 sequence could fold into a G-quadruplex structure to embed hemin to obtain the quenching probe of hemin/TBA 2/Au@CeO2 conjugates. In the presence of target TB, the sandwiched structure could be formed between capture aptamer (TBA 1), TB and hemin/TBA 2/Au@CeO2 conjugates, thereby resulting in a proportional quenching in ECL response with TB, due to the quenching of both hemin and Au@CeO2 NPs. As a result, the signal-off aptasensor showed a wider linear range response from 10(-13) to 10(-8) M with lower detection limit of 0.03 pM.

Amidolytic detection of prothrombin activation products after SDS-gel electrophoresis.

In this paper we report a method via which enzymatically active products formed during prothrombin activation can be detected by simple photographic means after SDS-gel electrophoresis, blotting onto a nitrocellulose membrane and visualization with the chromogenic substrate, S2238. After amidolytic detection the same nitrocellulose membrane can also be used for immunologic detection of prothrombin activation products, thus allowing a complete description of product formation during prothrombin activation. The detection limit of the so-called "amidoblot" is approximately 3 ng thrombin per gel sample which is comparable to the sensitivity of immunoblotting. It is further shown that the amidoblot technique can also be applied to other coagulation factors for which a suitable chromogenic substrate is available (factor XIIa, kallikrein, factor XIa, factor Xa, plasmin and activated protein C).

Binding of various thrombin fractions to fibrin and the influence of AT-III on their adsorption.

Human thrombin with high affinity for fibrin was obtained by subjecting purified thrombin to affinity chromatography on Sepharose insolubilized fibrin monomers, after addition of a radioiodinated subsample of thrombin, molar ratio 1:600. As judged by radioprofiling of the electrophoretic distribution of high-affinity thrombin on 10 per cent polyacrylamide gel containing urea/SDS, the preparation consisted of 70 per cent alpha-thrombin, 28 per cent beta-thrombin and only 2 per cent gamma-thrombin. Although alpha-thrombin was bound more strongly to insolubilized fibrin monomers than the other subfractions, complete separation of the individual components could not be achieved. High-affinity thrombin was employed for studies on thrombin adsorption to polymerized fibrin, assuming equal behaviour of labelled and unlabelled thrombin. To avoid passive entrapment of thrombin within the fibrin meshwork at physiological pH, ionic strength and calcium concentration, the optimal fibrinogen concentration was found to be 2.94 umol/l. During such conditions, adsorption of thrombin to polymerized fibrin did not exceed 65 per cent of added thrombin, despite an increasing availability of fibrin-related thrombin binding domains obtained by reducing the thrombin concentration. Adsorption of thrombin to polymerized fibrin increased by 25 per cent when the ionic strength was reduced to 0.05 mol/l. These findings suggest the presence of thrombin subfractions with different affinities for polymerized fibrin. Aggregates of high-affinity thrombin formed during its preparation by affinity chromatography, but were prevented by adding polyethylene glycol (m.w. 6,000, final conc. 6.6 g/l). Such aggregates were not inactivated by AT-III, but could still adsorb to polymerized fibrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibrin sealant in the United States: clinical use at the University of Virginia.

Fibrin sealant use in the United States has been limited because of the lack of a commercially available, government approved product. However, the significant need and usefulness of this material at the time of surgical operations has stimulated alternative sources of production. At the University of Virginia, the Blood Bank produces concentrated single donor or autologous fibrinogen for use with commercially available bovine thrombin in order to make fibrin sealant. The hemostatic and adhesive properties of fibrin sealant have been used since 1985 at this Center in a wide variety of operations with over 90% effectiveness. This chapter will review the development and role of this material in modern clinical surgery at this institution. The advantages, disadvantages, and never applications of this substance in various forms will be assessed. The role of fibrin sealant is increasing in this country and additional developmental work is continuing. The era of a commercially available product in the United States appears to be on the horizon.

Beware of commercial human thrombins used to stimulate cultured endothelial cells.

In the field of in vitro biocompatibility testing, the investigation of cell response at the interface with a biomaterial is of great importance; there is a need for standard conditions and thus of well-defined and reliable sources of materials for an objective evaluation of cellular function. Thrombin is often used in vitro as a stimulating agent to check the specific functions of cultured endothelial cells. In the present work, and in order to select a thrombin of commercial origin, two criteria were borne in mind: purity towards the presence of the von Willebrand factor (vWF) and effectiveness towards vWF release by human umbilical venous endothelial cells (HUVEC) that have been submitted to four human commercial thrombins. We detected the presence of vWF in some thrombin solutions that have not yet been in contact with HUVEC. The different thrombins contained vWF antigen ranging from less than 0.1 mUnit per NIH unit of thrombin (from Diagnostica Stago and Sigma Chemical Co.) to 10-20 mUnit per NIH unit of Fibrindex thrombin (from Ortho Diagnostic Systems). Thus, if vWF is present in commercial thrombins, it contributes to and overestimates the vWF appearance in the media resulting from cell stimulation. Consequently, we fixed on thrombin from Diagnostica Stago for further studies involving HUVEC on biomaterials.

Isolation, subunit structure, and proteolytic modification of bovine factor VIII.

A new method has been described for the isolation of factor VIII. The method results in a high yield of factor VIII that is homogeneous by several different criteria. The purified protein is very stable and is not dissociated in the presence of 1 M NaCl or 0.25 M CaCl2. The highly purified protein is readily activated and inactivated by various proteolytic enzymes, such as thrombin, plasmin, and trypsin. The molecular events that lead to the activation reaction, however, have not been established.

Preparation of fibrin glue from single-donor fresh-frozen plasma.

Fibrin glue is used widely in Europe as a tissue sealant and hemostatic agent. The European glue is prepared commercially from pooled human blood. It is not available in this country because of the risk of transmission of hepatitis B, acquired immune deficiency syndrome, and other blood-transmitted diseases. We describe a cryoprecipitation technique for preparation of fibrin glue from single-donor fresh-frozen plasma. This technique enables the glue to be made in large quantities with no greater risk of disease transmission than with that from the transfusion of single-unit fresh-frozen plasma. We have found that the glue is a useful tool in surgery. By helping to control difficult bleeding, its use can decrease the need for blood transfusions and shorten operating room time. It also is effective as a means to pretreat highly porous vascular prostheses that currently are used infrequently because of bleeding. These porous grafts offer potential advantages in handling, suturing, and long-term patency. This new technique of fibrin glue preparation may make this useful surgical adjunct as readily available in this country as it is in Europe.

Fibrin glue from stored human plasma. An inexpensive and efficient method for local blood bank preparation.

European surgeons have used fibrin glue extensively during thoracic, cardiovascular, and general surgical operations. Until now, however, it has been available only as a commercial preparation made from pooled human plasma, and it has not been approved by the U.S. Food and Drug Administration for use in the United States because of a high associated risk of hepatitis and acquired immune deficiency syndrome. Methods of obtaining fibrinogen, an essential component of fibrin glue, from cryoprecipitate or fresh frozen plasma have been published recently. However, the cryoprecipitate method results in relatively low concentrations of fibrinogen, which can reduce glue effectiveness. The fresh frozen plasma method is more expensive and does not meet the standards of the American Association of Blood Banks for the "closed" system required for safe handling and management of blood component products. Both the cryoprecipitate and the fresh frozen plasma methods result in waste of unstable clotting factors. These factors are necessary to replace human plasma clotting deficiencies but are not necessary for the production of fibrin glue. The authors have developed an efficient, high-concentration blood bank method for producing and maintaining a local supply of a safer and less expensive but equally effective material derived from stored human plasma. This material is produced using approved blood bank techniques for a "closed" system in blood component production, thus reducing the risks of contamination and infection, and its fibrinogen concentration is higher than that of standard cryoprecipitate. The cost of 1 unit of this fibrin glue is comparable to that for 1 unit of cryoprecipitate and less than that for 1 unit of fresh frozen plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Bioinspired affinity DNA polymers on nanoparticles for drug sequestration and detoxification.

Nanomaterials with the ability of sequestering target molecules hold great potential for a variety of applications. To ensure the stable sequestration, most of these nanomaterials have been traditionally designed with a clear boundary or compact structures and behave as closed systems. While this feature is beneficial to applications such as drug delivery, it may pose a challenge to applications where fast molecular transport from the environment to nanomaterials is critical. Thus, this study was aimed at exploring a nanomaterial with affinity DNA polymers and nanoparticles as an open system with function similar to jellyfish tentacles in sequestering target molecules from surroundings. The results show that this nanomaterial can effectively and rapidly sequester both small molecule drugs and large molecule biologics and resultantly mitigate their biological effects. Thus, this nanomaterial holds potential as a universal nanoscale antidote for drug removal and detoxification. While this nanomaterial was evaluated by using drug removal and detoxification as a model, the synthesis of periodically oriented affinity polymers on a nanoparticle with the capability of sequestering target molecules may be tuned for broad applications such as separation, sensing, imaging and drug delivery.

Thrombin and plasmin-like activities in the latices of Cryptostegia grandiflora and Plumeria rubra.

Latex proteins have drawn attention because they have shown several pharmacological activities. Herein, the fibrin(ogen)olytic activity of Cryptostegia grandiflora (CgLP) and Plumeria rubra (PrLP) latices were evaluated and characterized. Ion-exchange chromatography separated CgLP in proteolytic (CgLP PI) and nonproteolytic proteins (CgLP PII). CgLP and CgLP PI hydrolyzed azocasein in a dose-dependent manner, whereas CgLP PII and PrLP showed negligible activities. CgLP and CgLP PI accelerated plasmatic clot formation and digested all fibrinogen chains in a time/dose-dependent manner, though in a nonspecific way. CgLP and CgLP PI did not fully hydrolyze the subunits of the fibrin clot since fibrin α-chain showed resistance to proteolysis. No fibrinogenolytic activity was noticed after incubation of CgLP and CgLP PI with E-64. These results suggested that fibrinogenolytic and procoagulant activities of C. grandiflora were performed by cysteine proteases and confirm the activity of latex cysteine proteases as thrombin and plasmin-like proteins.

Electrogenerated trisbipyridyl Ru(II)-/nitrilotriacetic-polypyrene copolymer for the easy fabrication of label-free photoelectrochemical immunosensor and aptasensor: application to the determination of thrombin and anti-cholera toxin antibody.

A bifunctional copolymer was electrogenerated, which allows efficient bioreceptor immobilization and transduction of the biorecognition event. This copolymer was formed using pyrenebutyric acid Nα',Nα-bis(carboxymethyl)-L-lysine amide (NTA-pyrene) and [tris-(2,2'-bipyridine) (4,4'-bis(4-pyrenyl-1-ylbutyloxy)-2,2'-bipyridine] ruthenium(II) hexafluorophosphate (Ru(II)-pyrene) complex. The pyrene groups, present in both compounds, undergo oxidative electropolymerization on platinum electrodes. The resulting copolymer contains NTA moieties, which were used as a versatile immobilization system for biotin- and histidine-tagged biomolecules, while Ru(II)-pyrene was employed as a photoelectrochemical transducing molecule. The efficiency of this copolymer for biomolecule anchoring was investigated with biotin- and histidine- tagged glucose oxidases, biotin-tagged cholera toxin and a histidine-tagged thrombin aptamer. The constructed enzyme electrodes exhibited an amperometric response toward glucose at 0.6 V vs SCE, demonstrating the anchoring of this enzyme via two coordination systems. An immunosensor configuration based on the immobilization of biotin-tagged cholera toxin was applied to the detection of anti-cholera antibody while the aptasensor based on the immobilization of histidine-tagged thrombin aptamer was tested for thrombin determination. The biorecognition events were monitored via the evolution of the photocurrent intensity generated by the polymerized Ru(II)-pyrene in the presence of visible light and a sacrificial donor (ascorbate). The binding of the targets hinders the diffusion of the sacrificial donor, inducing thus a photocurrent decrease. The constructed immunosensor presents a specific label-free photoelectrochemical response to anti-cholera antibody without labeling step, the detection limit being 0.2 µg mL⁻¹. The label-free photoelectrochemical response of the aptasensor varies linearly with thrombin concentrations up to 10 pmol L⁻¹, the detection limit being 1×10⁻¹³ mol L⁻¹.

Mesoporous carbon microparticles as a novel fluorescent sensing platform for thrombin detection.

The present paper presents the novel use of MC microparticles (MCMPs) as a novel fluorescent sensing platform for thrombin detection. The MCMPs were prepared by a nanocasting method using mesoporous silica (MS) NPs as a hard template. The general concept used in this approach lies in the facts that the non-covalent adsorption of the dye-labeled TA on MCMP driven by π-π stacking of DNA bases on MCMP leads to substantial quenching of dye fluorescence due to their very close proximity. However, the presence of target TB results in the change of TA conformation to quadruplex due to the quadruplex-TB complex formation. Because the binding between the complex and MCMP is not strong enough to guarantee the close proximity of dyes to MCMP surface, fluorescence quenching is suppressed. This sensing system has a low detection limit down to 0.25 nM and exhibits excellent selectivity. We also demonstrate its application in human blood serum system.