Synthesis of functionally diverse bicyclic sulfonamides as constrained proline analogues and application to the design of potential thrombin inhibitors

Bicyclic sulfonamides were synthesized from 4-alkenyl N-alkenylsulfonyl l-prolines using a ring-closure metathesis reaction. Three types of bicyclic sulfonamides varying in the size of the second ring (5,5; 5,6; 5,7) were synthesized. A sulfonamide counterpart of an indolizidinone 2-carboxylic acid was synthesized and evaluated for its activity against the enzyme thrombin.     

Computer based screening of compound databases: 1. Preselection of benzamidine-based thrombin inhibitors

We present a computational protocol which uses the known three-dimensional structure of a target enzyme to identify possible ligands from databases of compounds with low molecular weight. This is accomplished by first mapping the essential interactions in the binding site with the program GRID. The resulting regions of favorable interaction between target and ligand are translated into a database query, and with UNITY a flexible 3D database search is performed. The feasibility of this approach is calibrated with thrombin as the target. Our results show that the resulting hit lists are enriched with thrombin inhibitors compared to the total database.     

非标记凝血酶阵列电化学适体传感器的研究

基于自制的集成化三阵列金膜电极,构建了一个简单、灵敏、非标记的凝血酶阵列电化学适体传感器。以聚乙烯不干胶掩膜版法结合金属溅射沉积技术,在FR-4玻璃纤维板上制作了由3个金膜工作电极、1个大面积金膜对电极和1个厚膜Ag/AgCl参比电极构成的集成化金膜阵列电极系统。以集成化金膜阵列电极作为基础电极,采用巯基自组装技术将带巯基的凝血酶适体固定在3个金工作电极表面,巯基己醇封闭后获得三阵列凝血酶适体传感器,以电活性物质铁氰化钾作为电化学探针,基于凝血酶适体和凝血酶结合前后铁氰化钾在电极表面传质的不同导致电流变化进行凝血酶的测定。采用方波脉冲伏安法,铁氰化钾氧化峰电流的变化值与凝血酶浓度在 1.52~63 nmol/L范围内呈良好的线性关系,检出限(S/N=3)为0.92 nmol/L。     

两种表征核酸适配体-靶蛋白亲和作用的毛细管电泳方法比较

适配体-靶分子间的亲和作用表征是理解和应用核酸适配体发挥特异亲和作用的基本前提,CE技术则为上述表征提供了多模式的简捷途径,但多种模式体系间的结果往往存在差异,导致CE亲和评价可靠性和进一步应用受到限制,亟须建立多CE方法测定适配体-靶分子间亲和作用的系统比较研究。该研究以凝血酶及其特异性作用于肝素结合位点的适配体29mer为模型体系,基于CE-激光诱导荧光检测,引入CE-迎头分析（FA）评价方法,并比较其与预平衡-毛细管区带电泳（PE-CZE）的异同。首先进行了CE-FA方法分离条件的优化,37 ℃、0.5 h孵育完全后进样,进样时间为30 s,在较低工作温度（15 ℃）、较短毛细管长度（30 cm）及生物相容性好的缓冲体系2×TG（Tris-甘氨酸缓冲液,pH 8.5）条件下,经15 kV分离时,得到了稳定的荧光标记29mer（F29mer）-凝血酶复合物及游离F29mer平台峰。加入1 g/L牛血清白蛋白（BSA）,有效提高了CE-FA平台峰高及迁移时间的重复性。详细讨论了两种方法下6种拟合方式的结果及特点。针对CE-FA和PE-CZE法,以结合适配体/游离适配体的浓度比对游离适配体浓度非线性拟合、平台峰高变化对浓度间的非线性拟合、平衡混合物的非平衡CE（NECEEM）计算等进行拟合。结果表明,6种拟合结果中5种不存在显著性差异,得到的解离常数（K_d）值均介于24~64 nmol/L范围。CE-FA法中的3种拟合结果符合度较好,说明CE-FA法易于在非平衡的CE分离体系下保持适配体-复合物间的结合-解离平衡,所测得K_d准确度较高。PE-CZE法中,借由降低的游离F29mer峰高对凝血酶浓度间的非线性拟合所得K_d值偏差过大;选择凝血酶浓度为F29mer浓度的0.5~2倍,且观察到明显的两峰间"指数桥"为前提,可经NECEEM法进行数据计算求解得到较为准确的K_d值。CE-FA法可与PE-CZE法互为印证,提高了亲和作用评价的可靠性。首选推荐使用多浓度平台峰高变化进行非线性拟合的CE-FA法,可相对有效克服高压电场对复合物稳定性的影响,具有适用范围广、方法稳定、结果拟合简便准确等特点。     

Beyond labels: A review of the application of quantum dots as integrated components of assays, bioprobes, and biosensors utilizing optical transduction

A comprehensive review of the development of assays, bioprobes, and biosensors using quantum dots (QDs) as integrated components is presented. In contrast to a QD that is selectively introduced as a label, an integrated QD is one that is present in a system throughout a bioanalysis, and simultaneously has a role in transduction and as a scaffold for biorecognition. Through a diverse array of coatings and bioconjugation strategies, it is possible to use QDs as a scaffold for biorecognition events. The modulation of QD luminescence provides the opportunity for the transduction of these events via fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), charge transfer quenching, and electrochemiluminescence (ECL). An overview of the basic concepts and principles underlying the use of QDs with each of these transduction methods is provided, along with many examples of their application in biological sensing. The latter include: the detection of small molecules using enzyme-linked methods, or using aptamers as affinity probes; the detection of proteins via immunoassays or aptamers; nucleic acid hybridization assays; and assays for protease or nuclease activity. Strategies for multiplexed detection are highlighted among these examples. Although the majority of developments to date have been in vitro, QD-based methods for ex vivo biological sensing are emerging. Some special attention is given to the development of solid-phase assays, which offer certain advantages over their solution-phase counterparts.     

Multianalyte Electrochemical Biosensor Based on Aptamer‐ and Nanoparticle‐Integrated Bio‐Barcode Amplification

In the present work, a signal‐on electrochemical sensing strategy for the simultaneous detection of adenosine and thrombin is developed based on switching structures of aptamers. An Au electrode as the sensing surface is modified with two kinds of thiolated capture probes complementary to the linker DNA that contains either an adenosine aptamer or thrombin aptamer. The capture probes hybridize with their corresponding linker DNA, which has prehybridized with the reporter DNA loaded onto the gold nanoparticles (AuNPs). The AuNP contained two kinds of bio‐barcode DNA: one is complementary to the linker DNA (reporter), whereas the other is not (signal) and is tagged with different metal sulfide nanoparticles. Thus a “sandwich‐type” sensing interface is fabricated for adenosine and thrombin. With the introduction of adenosine and thrombin, the aptamer parts bind with their targets and fold to form the complex structures. As a result, the bio‐barcoded AuNPs are released into solution. The metal sulfide nanoparticles are measured by anodic stripping voltammetry (ASV), and the concentrations of adenosine and thrombin are proportional to the signal of either metal ion. With the dual amplification of the bio‐barcoded AuNP and the preconcentration of metal ions through ASV technology, detection limits as low as 6.6×10^?12 M for adenosine and 1.0×10^?12 M for thrombin are achieved. The sensor exhibits excellent selectivity and detectability in biological samples.     

Solid phase synthesis of benzamidine-derived sulfonamide libraries

Using solid phase synthesis, a library has been constructed of benzamidine-derived sulfonamides which have strong inhibitory activity against blood coagulant thrombin. The library compounds were obtained in good yield and high purity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Interactions of DNA with graphene and sensing applications of graphene field-effect transistor devices: A review

Graphene field-effect transistors (GFET) have emerged as powerful detection platforms enabled by the advent of chemical vapor deposition (CVD) production of the unique atomically thin 2D material on a large scale. DNA aptamers, short target-specific oligonucleotides, are excellent sensor moieties for GFETs due to their strong affinity to graphene, relatively short chain-length, selectivity, and a high degree of analyte variability. However, the interaction between DNA and graphene is not fully understood, leading to questions about the structure of surface-bound DNA, including the morphology of DNA nanostructures and the nature of the electronic response seen from analyte binding. This review critically evaluates recent insights into the nature of the DNA graphene interaction and its affect on sensor viability for DNA, small molecules, and proteins with respect to previously established sensing methods. We first discuss the sorption of DNA to graphene to introduce the interactions and forces acting in DNA based GFET devices and how these forces can potentially affect the performance of increasingly popular DNA aptamers and even future DNA nanostructures as sensor substrates. Next, we discuss the novel use of GFETs to detect DNA and the underlying electronic phenomena that are typically used as benchmarks for characterizing the analyte response of these devices. Finally, we address the use of DNA aptamers to increase the selectivity of GFET sensors for small molecules and proteins and compare them with other, state of the art, detection methods.     

Highly efficient control of thrombin activity by multivalent nanoparticles

We have demonstrated that the incorporation of sulfated galactose acid (sulf-Gal) into thrombin-binding-aptamer (TBA)-conjugated gold nanoparticles (TBA-AuNPs) enables highly effective inhibition of thrombin activity toward fibrinogen. AuNP bioconjugates (TBA(15)/TBA(29)/sulf-Gal-AuNPs) were prepared from 13 nm AuNPs, 15-mer thrombin-binding aptamer (TBA(15)), 29-mer thrombin-binding aptamer (TBA(29)), and sulf-Gal. The numbers of TBA and sulf-Gal molecules per AuNP proved to have a strong impact on inhibitory potency. The best results were observed for 15-TBA(15)/TBA(29)/sulf-Gal-AuNPs (with 15 TBA(15) and 15 TBA(29) molecules per AuNP), which, because of their particularly flexible conformation and multivalency, exhibited ultrahigh binding affinity toward thrombin (K(d)=3.4×10(-12) M) and thus extremely high anticoagulant (inhibitory) potency. Compared to the case without inhibitors (the "normal" value), their measured thrombin clotting time (TCT) was 91 times longer, whereas for TBA(15) alone it was only 7.2 times longer. Their anticoagulant activity was suppressed by TBA-complementary-sequence (cTBA)-modified AuNPs (cTBA(15)/cTBA(29)-AuNPs) at a rate that was 20 times faster than that of free cTBA(15)/cTBA(29). Thus, easily prepared, low-cost, multivalent AuNPs show great potential for biomedical control of blood clotting.