聚乙烯表面接枝聚合改性及抗凝血性的研究

聚乙烯(PE)膜经Ar等离子体预处理,无光引发剂紫外光照接枝甲基丙烯酸缩水甘油酯(GMA),然后进行肝素化处理,以改善PE的抗凝血性能。用正交实验确定接枝反应的最优条件。通过X-射线光电子能谱(XPS)、衰减全反射红外光谱(ATR-FTIR)、扫描电子显微镜(SEM)和接触角测定PE膜接枝GMA前后表面性能和表面形貌。用复钙时间、凝血酶原时间、部分凝血活酶时间、凝血酶时间和血小板粘附实验对其抗凝血性能进行评价,结果表明,被修饰PE膜的抗凝血性能显著提高。     

Single-walled carbon nanotube biosensors using aptamers as molecular recognition elements

We report the real-time detection of protein using SWNT-FET-based biosensors comprising DNA aptamers as molecular recognition elements. Anti-thrombin aptamers that are highly specific to serine protein thrombin were immobilized on the sidewall of a SWNT-FET using CDI-Tween linking molecules. The binding of thrombin aptamers to SWNT-FETs causes a rightward shift of the threshold gate voltages, presumably due to the negatively charged backbone of the DNA aptamers. While the addition of thrombin solution causes an abrupt decrease in the conductance of the thrombin aptamer immobilized SWNT-FET, no noticeable change was observed with elastase.     

Study on an electrochemical biosensor for thrombin recognition based on aptamers and nano particles

This paper presents a high specific, sensitive electrochemical biosensor for recognition of protein such as thrombin based on aptamers and nano particles. Two different aptamers were chosen to construct a sandwich manner for detecting thrombin. Aptamer I was immobilized on nano magnetic particle for capturing thrombin, and aptamer II labled with nano gold was used for detection. The electrical current generated from gold after the formation of the complex of magnetic particle, thrombin and nano gold, and then an electrochemical cell designed by ourselves was used for separating, gathering, and electrochemical detecting. Through magnetic separation, high specific and sensitive detection of the target protein, thrombin, was achieved. Linear response was observed over the range 5.6×10-12―1.12×10-9 mol/L, with a detection limit of 1.42×10-12 mol/L. The presence of other protein as BSA did not affect the detection, which indicates that high selective recognition of thrombin can be achieved in complex biological samples such as human plasma.     

Thrombin allostery

Thrombin is a Na(+)-activated, allosteric serine protease that plays multiple functional roles in blood pathophysiology. Binding of Na(+) is the major driving force behind the procoagulant, prothrombotic and signaling functions of the enzyme. This review summarizes our current understanding of the molecular basis of thrombin allostery with special emphasis on the kinetic aspects of Na(+) activation. The molecular mechanism of thrombin allostery is a remarkable example of long-range communication that offers a paradigm for many other biological systems.     

Anticoagulant action of vanadate.

Sodium orthovanadate (vanadate) prolonged the clotting time of normal human plasma in a dose-dependent manner. The prolongation of clotting time by vanadate linearly decreased with an increase in the concentration of amiloride. Vanadate also was completely additive to prolongation by heparin. When factor Xa or thrombin was incubated with vanadate, the amidolytic activity of each decreased in a dose-dependent manner with vanadate. Amiloride protected the decrease of amidolytic activity of both factor Xa and thrombin by vanadate. The amidolytic activity of trypsin also was inhibited by vanadate, but that of alpha-chymotrypsin was not inhibited, suggesting that vanadate preferentially inhibits the amidolytic activity of trypsin and trypsin-like enzymes. These results show that vanadate prolongs the clotting time of plasma through mechanisms involving in part the inhibition of the activity of both factor Xa and thrombin.     

Surface plasmon resonance spectroscopy study of interfacial binding of thrombin to antithrombin DNA aptamers

We have applied surface plasmon resonance (SPR) spectroscopy, in combination with one-step direct binding, competition, and sandwiched assay schemes, to study thrombin binding to its DNA aptamers, with the aim to further the understanding of their interfacial binding characteristics. Using a 15-mer aptamer that binds thrombin primarily at the fibrinogen-recognition exosite as a model, we have demonstrated that introducing a DNA spacer in the aptamer enhances thrombin-binding capacity and stability, as similarly reported for hydrocarbon linkers. The bindings are aptamer surface coverage and salt concentration dependent. When free aptamers or DNA sequences complementary to the immobilized aptamer are applied after the formation of thrombin/aptamer complexes, bound thrombin is displaced to a certain extent, depending on the stability of the complexes formed under different conditions. When the 29-mer aptamer (specific to thrombin's heparin-binding exosite) is immobilized on the surface, its affinity to thrombin appears to be lower than the immobilized 15-mer aptamer, although the 29-mer aptamer is known to have a higher affinity in the solution phase. These findings underline the importance of aptamers' ability to fold into intermolecular structures and their accessibility for target capture. Using a sandwiched assay scheme followed by an additional signaling step involving biotin-streptavidin chemistry, we have observed the simultaneous binding of the 15- and 29-mer aptamers to thrombin protein at different exosites and have found that one aptamer depletes thrombin's affinity to the other when they bind together. We believe that these findings are invaluable for developing DNA aptamer-based biochips and biosensors.     

Simultaneously fluorescence detecting thrombin and lysozyme based on magnetic nanoparticle condensation

In this protocol, a fluorescent aptasensor based on magnetic separation for simultaneous detection thrombin and lysozyme was proposed. Firstly, one of the anti-thrombin aptamer and the anti-lysozyme aptamer were individually immobilized onto magnetic nanoparticles, acting as the protein captor. The other anti-thrombin aptamer was labeled with rhodamine B and the anti-lysozyme aptamer was labeled with fluorescein, employing as the protein report. By applying the sandwich detection strategy, the fluorescence response at 515 nm and 578 nm were respectively corresponding to lysozyme and thrombin with high selectivity and sensitivities. The fluorescence intensity was individually linear with the concentration of thrombin and lysozyme in the range of 0.13-4 nM and 0.56-12.3 nM, and the detection limits were 0.06 nM of thrombin and 0.2 nM of lysozyme, respectively. The preliminary study on simultaneous detection of thrombin and lysozyme in real plasma samples was also performed. It shows that the proposed approach has the good character for simultaneous multiple protein detection.     

An Optical Biosensing System Based on Interference-Enhanced Reflection with Aptameric Enzyme Subunits of Thrombin

The aptameric enzyme subunit (AES) is an artificial enzyme subunit that can allosterically control partner enzyme activity. By means of an AES, target molecules can be detected by measurement of enzymatic activity in a homogeneous solution. We have developed a thrombin aptamer-based AES that can detect several targets by measuring clotting time in a fibrinogen solution. Measurement of the clotting activity in the fibrinogen solution is not suitable for a convenient biosensor. However, kinetics measurement of clotting activity is suitable for convenient detection of thrombin activity. The interference-enhanced reflection (IER) method is a simple, real-time technique to detect the thickness and/or refractive index of a thin film formed on a glass substrate surface. We demonstrated that clotting activity on a glass substrate with immobilized thrombin can be monitored in real time by IER. By means of the IER method, we were able to detect the target molecules of an AES. IER-based sensors have already been commercialized as portable sensors for the detection of organic compounds. Thus, portable biosensors could be developed by combination of the IER and AES technologies.     

A bifunctional electrochemical aptasensor based on AuNPs-coated ERGO nanosheets for sensitive detection of adenosine and thrombin

A simple and sensitive bifunctional electrochemical aptasensor for detection of adenosine and thrombin has been developed using gold nanoparticles–electrochemically reduced graphene oxide (AuNPs-ERGO) composite film-modified electrode. Firstly, the reduced graphene oxide film and AuNPs were sequentially immobilized on glassy carbon electrode (GCE) surface. Secondly, thrombin aptamer was immobilized on the modified electrode. Finally, adenosine aptamer was hybridized with it to serve as a recognition element and methylene blue (MB) as electrochemical signal indicator. In the presence of adenosine or thrombin, the sensor recognized it and a conformational change was induced in aptamer, resulting in decrease of the peak current of MB. The linear relation between concentration of adenosine or thrombin and peak current of MB allowed quantification of them. Thanks to the special electronic characteristic of AuNPs-ERGO composite film, sensitivity of sensor was greatly improved. Under optimal conditions, the proposed aptasensor presented an excellent performance in a linear range of 25 nM to 750 nM for adenosine and 0.5 nM to 10 nM for thrombin. Detection limits were estimated to be 8.3 nM for adenosine and 0.17 nM for thrombin, respectively. Moreover, dual-analyte detection of adenosine and thrombin was achieved without potentially increasing the complexity and cost of the assay.

     

Plasma-assisted immobilization of heparin onto low-density polyethylene surface

In this study heparin was covalently immobilized onto LDPE-VEMAC sheet fabricated by the introduction of carboxyl groups to the surface of low-density polyethylene (LDPE) using a plasma technique. The plasma irradiation time influenced the density of carboxyl groups on the LDPE-VEMAC sheet. Heparin was immobilized on the LDPE-VEMAC sheet using a condensation reagent, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC). We confirmed the immobilization of heparin from the ATR-FT-IR spectrum of the sheet obtained. Where heparin was directly immobilized on the LDPE-VEMAC sheet, the density of the immobilized heparin depended on that of the carboxyl groups. Heparin was also immobilized with a spacer, hexamethylene diamine, and the density of such heparin was about 1.6 times that of the directly immobilized heparin. This result suggests that the introduction of a spacer may be an effective way to increase the density of immobilized heparin.     

Poly(methyl methacrylate) microchip affinity capillary gel electrophoresis of aptamer-protein complexes for the analysis of thrombin in plasma

Thrombin generation in blood serves as an important marker for various hemostasis-related diseases and conditions. Analytical techniques currently utilized for determining the thrombin potential of patients rely primarily on the enzymatic activity of thrombin. Microfluidic-based ACE using fluorescently labeled aptamers as affinity probes could provide a simple and efficient technique for the real-time analysis of thrombin levels in plasma. In this study, aptamers were used for the analysis of thrombin by affinity microchip CGE. The CGE used a poly(methyl methacrylate) (PMMA) microfluidic device for the sorting of the affinity complexes with a linear polyacrylamide (LPA) serving as the sieving matrix. Due to the fact that the assay was run under nonequilibrium electrophoresis conditions, the presence of the sieving gel was found to stabilize the affinity complex, providing improved electrophoretic performance compared to free-solution electrophoresis. Two fluorescently labeled aptamer affinity probes, HD1 and HD22, which bind to exosites I and II, respectively, of thrombin were investigated. With an electric field strength of 300 V/cm, two well-resolved peaks corresponding to free aptamer and the thrombin-aptamer complex were obtained in less than 1 min of separation time with a run-to-run and chip-to-chip reproducibility (RSD) of migration times <10% using both aptamers. HD22 affinity assays of thrombin produced baseline-resolved peaks with favorable efficiency due to its higher binding affinity, whereas HD1 assays showed poorer resolution of the free aptamer and complex peaks. HD22 was used in determining the level of thrombin in human plasma. Assays were performed directly on plasma that was diluted to 10% v/v. Thrombin was successfully analyzed by microchip CGE at a concentration level of 543.5 nM for the human plasma sample.     

Effect of the immobilisation of DNA aptamers on the detection of thrombin by means of surface plasmon resonance

We report a multichannel surface plasmon resonance (SPR) sensor for detection of thrombin via DNA aptamers immobilized on the SPR sensor surface. A detailed investigation of the effect of the immobilisation method on the interaction between thrombin and DNA aptamers is presented. Three basic approaches to the immobilisation of aptamers on the surface of the SPR sensor are examined: (i) immobilisation based on chemisorption of aptamers modified with SH groups, (ii) immobilisation of biotin-tagged aptamers via previously immobilized avidin, neutravidin or streptavidin molecular linkers, and (iii) immobilisation employing dendrimers as a support layer for subsequent immobilisation of aptamers. A level of nonspecific binding of thrombin to immobilized human serum albumin (HSA) for each of the immobilisation methods is determined. Immobilisation of aptamers by means of the streptavidin–biotin system yields the best results both in terms of sensor specificity and sensitivity.     

PHOTOCOAGULATION OF HUMAN PLASMA: ACYL SERINE PROTEINASE PHOTOCHEMISTRY

Human alpha-thrombin or bovine Factor Xa was acylated at the active site serine hydroxyl with alpha-methyl-2-hydroxy-4-diethylaminocinnamic acid. These modified serine proteinase enzymes showed no plasma coagulation biological activity in the absence of light. Photolysis of the acyl serine proteinase enzymes in plasma for 1-35 s with monochromatic 366 nm light isolated from a high pressure mercury arc results in coagulation of the plasma. For example, photolysis of 3 NIH U of the acyl human alpha-thrombin for 5 s in human plasma results in a clot in 23 s. For comparison, 1 NIH U of unmodified human alpha-thrombin gave a clot in 21 s under the conditions of the assay but without photolysis. Appropriate controls showed that the coagulation is the result of the formation of active thrombin due to photodeacylation of the enzymes. The photoinduced clotting time measured is dependent on acyl thrombin concentration and photolysis time. Thus higher concentrations of acyl thrombin and longer photolysis times give a shorter clotting time. A kinetic scheme based upon Lineweaver-Burke analysis of the clotting process is developed.     

Studies of the binding mechanism between aptamers and thrombin by circular dichroism, surface plasmon resonance and isothermal titration calorimetry

Thrombin, a multifunctional serine protease, has both procoagulant and anticoagulant functions in human blood. Thrombin has two electropositive exosites. One is the fibrinogen-binding site and the other is the heparin-binding site. Over the past decade, two thrombin-binding aptamers (15-mer and 29-mer) were reported by SELEX technique. Recently, many studies examined the interactions between the 15-mer aptamer and thrombin extensively, but the data on the difference of these two aptamers binding to thrombin are still lacking and worth investigating for fundamental understanding. In the present study, we combined conformational data from circular dichroism (CD), kinetics and thermodynamics information from surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) to compare the binding mechanism between the two aptamers with thrombin. Special attentions were paid to the formation of G-quadruplex and the effects of ions on the aptamer conformation on the binding and the kinetics discrimination between specific and nonspecific interactions of the binding. The results indicated reasonably that the 15-mer aptamer bound to fibrinogen-binding site of thrombin using a G-quadruplex structure and was dominated by electrostatic interactions, while the 29-mer aptamer bound to heparin-binding site thrombin using a duplex structure and was driven mainly by hydrophobic effects.     

Fluorescence correlation spectroscopy of gold nanoparticles,and its application to an aptamer-based homogeneous thrombin assay

We have studied the fluorescence properties and diffusion behaviors of gold nanoparticles (GNPs) in solution by using fluorescence correlation spectroscopy (FCS) at single molecule level. The GNPs display a high photo-saturation feature. Under illumination with strong laser light, they display higher brightness per particle (BPP) despite their low quantum yields. Based on the unique fluorescence properties and diffusion behaviors of GNPs, we have developed a sensitive and homogenous thrombin assay. It is based on a sandwich strategy and is making use of GNPs to which two different aptamers are conjugated. When the differently aptamer-labeled GNPs are mixed with solutions containing thrombin, the affinity reaction causes the GNPs to form dimers or oligomers. This leads to an increase in the diffusion time of the GNPs in the detection volume that is seen in FCS. The FCS method enables sensitive detection of the change in the characteristic diffusion time of the GNPs before and after the affinity reaction. Quantitative analysis of thrombin is based on the measurement of the change in the diffusion time. Under optimal conditions, the calibration plot is linear in the 0.5 nM to 110 nM thrombin concentration range, and the detection limit is 0.5 nM. The method was successfully applied to the direct determination of thrombin in human plasma.

Aptasensor for Thrombin Based on Carbon Nanotubes‐Methylene Blue Composites

The amperometric and EQCM aptasensors based on DNA aptamers immobilized by avidin‐biotin method or by electrostatic adsorption onto multiwalled carbon nanotube layer contained methylene blue (MB) have been developed and examined for thrombin detection in buffer and in spiked blood serum. The presence of MB increases the binding capacity of the surface layer and enhances the range of thrombin concentrations to be determined. This results in significant improvement of analytical characteristics of thrombin detection. The EQCM aptasensors allowed us to detect 0.3–100 nM and amperometric aptasensors 10–1000 nM of thrombin.     

利用互补核酸杂交富集金胶实现信号扩增的电化学凝血酶蛋白生物传感器研究

介绍了一种利用互补核酸杂交富集金胶实现信号扩增的蛋白质生物传感器. 以凝血酶蛋白为研究对象, 利用凝血酶蛋白相对应的两段核酸适配体, 将适配体Ⅰ固定在磁性颗粒上, 用于特异性地捕获蛋白, 将适配体Ⅱ标记金胶作为检测信标. 由凝血酶蛋白和相对应的两段核酸适配体构建三明治结构的凝血酶蛋白生物传感器. 另外, 再通过信标金胶上过剩的核酸适配体链与另一段标记有金胶的互补核酸进一步杂交, 获得金胶的选择性聚集, 实现了信号扩增. 通过信号扩增, 使此传感器的灵敏度大大提高, 对凝血酶蛋白的检测下限可达到4.52×10-15 mol/L. 平行测定浓度为7.47×10-14 mol/L的凝血酶8次, 其RSD为3.0%. 该生物传感器对凝血酶蛋白有很好的特异性, 其它蛋白如溶菌酶和牛血清白蛋白的存在对于检测没有影响.     

适配体传感器检测凝血酶的研究进展

适配体是通过指数富集配体系统进化技术(SELEX)筛选得到的,能与多种目标物质高特异性、高选择性结合的寡核苷酸序列.适配体因合成简单、稳定性高、设计多样化、修饰方便、成本低等优点被用作为识别探针,并与不同的转导技术相结合,构建了多种类型的适配体传感器,并借助纳米材料独特的光、电特性,提高适配体传感器的性能.凝血酶是一种...     

A novel chitosan-based sponge coated with self-assembled thrombin/tannic acid multilayer films as a hemostatic dressing

In order to prepare a novel hemostatic dressing for uncontrolled hemorrhage, a porous chitosan sponge was coated with self-assembled(thrombin/tannic acid)n films, which were based on hydrogen bonding interactions between thrombin and tannic acid at physiologic p H. According to the whole blood clotting test, the coated chitosan sponges showed a significantly high rate of blood clotting due to the addition of thrombin. On the other hand, the storable half-life of immobilized thrombin is extended to 66.9 days at room temperature, which is 8.5 times longer than unfixed thrombin. It is because of the immobilization effect of, not only the porous structure of chitosan sponge but also the interactions between thrombin and tannic acid. In addition, the tannic acid has similar antibacterial effect to chitosan. Therefore, it is an excellent combination of chitosan, thrombin and tannic acid. Besides, all of materials in this research have been approved by the United States Food and Drug Administration(FDA). So the chitosan-based sponge is a promising candidate dressing for uncontrolled hemorrhage due to its storable, bio-safe and highly effective hemostatic properties.     

Polarographic Behavior of Human Plasma Thrombin and Changes in Its Anticoagulation Activity Through Various Conformations

Thrombin plays a pivotal role in the regulation of blood fluidity and therefore constitutes a primary target in the treatment of various hemostatic disorders. For this differential pulse polarography has been developed in an appropriate supporting electrolyte, which produced well‐defined DPP peaks. The average prothrombin time (PT) in citrated plasma samples was determined, which indicated decrease of average PT with rise of thrombin and albumin concentrations, while PT resulted differently (increase) with heparin, thus confirming their role in anticoagulation activity. The PT of thrombin‐heparin and thrombin‐albumin conformations showed sharp increased and steady decreased respectively. Thus, two opposite actions of thrombin, coagulation (thrombin‐albumin) and anticoagulation (thrombin‐heparin) may prove fruitful in clinical approach for thrombotic disorders.