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.     

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.     

Aptamer-based electrochemical sensors that are not based on the target binding-induced conformational change of aptamers

This study describes a new kind of aptamer-based electrochemical sensor that is not based on the target binding-induced conformational change of the aptamers by using a 15-mer thrombin-binding aptamer (5'-GGTTGGTGTGGTTGG-3') as the model oligonucleotide. The sensors are developed by first self-assembling the aptamer (i.e. a thrombin-binding aptamer) onto an Au electrode and then hybridizing the assembled aptamer with a ferrocene (Fc)-labeled short aptamer-complementary DNA oligonucleotide to form an electroactive double-stranded DNA (ds-DNA) oligonucleotide onto the Au electrode. The binding of the target (i.e. thrombin) towards the aptamer essentially destroys the Watson-Crick helix structure of the ds-DNA oligonucleotide assembled onto the electrode and leads to the dissociation of the Fc-labeled short complementary DNA oligonucleotide from the electrode surface to the solution, resulting in a decrease in the current signal obtained at the electrode, which can be used for the determination of the target. With the thrombin-binding aptamer as the model oligonucleotide, the current decrease obtained with the aptamer-based electrochemical sensors is linear with the concentration of thrombin within the concentration range from 0 to 10 nM (DeltaI/nA = 6.7C(thrombin)/nM + 2.8, gamma = 0.975). Unlike most kinds of existing aptamer-based electrochemical sensor, the electrochemical aptasensors demonstrated here are not based on the conformational change of the aptamers induced by the specific target binding. Moreover, the aptasensors are essentially label-free and are very responsive toward the targets. This study may pave a facile and general way to the development of aptamer-based electrochemical sensors.     

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.     

Colorimetric detection of DNA by modulation of thrombin activity on gold nanoparticles

A colorimetric, non-cross-linking aggregation-based gold-nanoparticle (AuNP) probe has been developed for the detection of DNA and the analysis of single-nucleotide polymorphism (SNP). The probe acts by modulating the enzyme activity of thrombin relative to fibrinogen. A thrombin-binding aptamer with a 29-base-long oligonucleotide (TBA(29)) assembled on the nanoparticles (TBA(29)-AuNPs) through sandwich DNA hybridization was found to possess ultra-high anticoagulant potency. The enzyme inhibition of thrombin was determined by thrombin-induced aggregation of fibrinogen-functionalized 56 nm AuNPs (Fib-AuNPs). The potency of the inhibition of TBA(29)-AuNPs relative to thrombin--and thus the degree of aggregation of the Fib-AuNPs--is highly dependent on the concentration of perfectly matched DNA (DNA(pm)). Under optimal conditions [Tris-HCl (20 mM, pH 7.4), KCl (5 mM), MgCl(2) (1 mM), CaCl(2) (1 mM), NaCl (150 mM), thrombin (10 pM), and TBA(29)-AuNPs (20 pM)], the new TBA(29)-AuNP/Fib-AuNP probe shows linear sensitivity to DNA(pm) in the concentration range 20-500 pM with a correlation coefficient of 0.96. The limit of detection for DNA(pm) was experimentally determined to be 12 pM, based on a signal-to-noise ratio (S/N) of 3. The new probe was successfully applied to the analysis of an SNP that is responsible for sickle cell anemia. Relative to conventional molecular-beacon-based probes, the new probe offers the advantages of higher sensitivity and selectivity towards DNA and lower cost, showing its great potential for practical studies of SNPs.     

Au Nanoparticle-DNAzyme Dual Catalyst System for Sensitively Colorimetric Detection of Thrombin

A novel colorimetric aptasensor was developed for thrombin detection with high sensitivity and specificity. The assay takes the advantage of Au nanoparticles-DNAzyme as a dual catalytic system for signal amplification. Au nanparticles were modified with peroxidase mimicking DNAzyme sequence as well as thrombin binding aptamer. And then the thrombin binding aptamer hybridized with its complementary sequence which was immobilized on the surface of the magnetic nanoparticles to construct the colorimetric aptasensor. In the presence of thrombin, the target-induced displacement takes place, resulting in the dissociation of the aptasensor. The DNAzyme functioned Au NPs are released due to the combine ability of thrombin binding aptamer with thrombin. The released Au NPs are capable of catalyzing the colorless 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid(ABTS) conversion into a blue-green product by H₂O₂-mediated oxidation, thus can amplify the colorimetric readout signals of thrombin detection. Such a device can serve as a novel selectivity sensor for thrombin with a detection limit of 0.6 nmol/L.     

Chemiluminescent and Colorimetric Aptamer-Based Assays of Human α-Thrombin

Abstract

A microplate-based sandwich assay for the determination of α-human thrombin (HTb) was developed. Fluorescein-modified 29-mer thrombin binding aptamer (FAM-TBA29) and biotinylated 15-mer thrombin binding aptamer (Bio-TBA15) reacting with different exosites of HTb were used as the biorecognition components in the assay. FAM-TBA29 (capture aptamer) was immobilized using its interaction with anti-fluorescein antibody adsorbed on the microplate surface. As a sensitive signaling system, a combination of Bio-TBA15 and streptavidin-polyHRP conjugate was used. Under the optimized conditions, the detection limit for HTb was 1.4?nM; this value was the same in both the colorimetric and the chemiluminescent assays. The replacement of colorimetry for HRP measurement with chemiluminescence increased the assay sensitivity from 0.06 to 1.7?×?10⁶?nM^?1 that clearly demonstrated advantage of the latter approach.     

Label-free fluorescent detection of thrombin using G-quadruplex-based DNAzyme as sensing platform

We report herein a label-free and sensitive fluorescent method for detection of thrombin using a G-quadruplex-based DNAzyme as the sensing platform. The thrombin-binding aptamer (TBA) is able to bind hemin to form the G-quadruplex-based DNAzyme, and thrombin can significantly enhance the activity of the G-quadruplex-based DNAzyme. The G-quadruplex-based DNAzyme is found to effectively catalyze the H(2)O(2)-mediated oxidation of thiamine, giving rise to fluorescence emission. This allows us to utilize the H(2)O(2)-thiamine fluorescent system for the quantitative analysis of thrombin. The assay shows a linear toward thrombin concentration in the range of 0.01-0.12 nM. The present limit of detection for thrombin is 1 pM, and the sensitivity for analyzing thrombin is improved by about 10,000-fold as compared with the reported colorimetric counterpart. The work also demonstrates that thiamine is an excellent substrate for the fluorescence assay using the G-quadruplex-based DNAzyme as the sensing platform.     

A label-free and high sensitive aptamer biosensor based on hyperbranched polyester microspheres for thrombin detection

In this paper, we have synthesized hyperbranched polyester microspheres with carboxylic acid functional groups (HBPE-CA) and developed a label-free electrochemical aptamer biosensor using thrombin-binding aptamer (TBA) as receptor for the measurement of thrombin in whole blood. The indium tin oxide (ITO) electrode surface modified with HBPE-CA microspheres was grafted with TBA, which has excellent binding affinity and selectivity for thrombin. Binding of the thrombin at the modified ITO electrode surface greatly restrained access of electrons for a redox probe of [Fe(CN)₆]^3−/4−. Moreover, the aptamer biosensor could be used for detection of thrombin in whole blood, a wide detection range (10 fM–100 nM) and a detection limit on the order of 0.90 fM were demonstrated. Control experiments were also carried out by using bull serum albumin (BSA) and lysozyme in the absence of thrombin. The good stability and repeatability of this aptamer biosensor were also proved. We expect that this demonstration will lead to the development of highly sensitive label-free sensors based on aptamer with lower cost than current technology. The integration of the technologies, which include anticoagulant, sensor and nanoscience, will bring significant input to high-performance biosensors relevant to diagnostics and therapy of interest for human health.     

A novel dot-blot DNAzyme-linked aptamer assay for protein detection

In this work, a novel dot-blot DNAzyme-linked aptamer assay (DLAA) for protein detection is developed with thrombin as a model protein. A peroxidase-like DNAzyme which serves as the catalytic label is tethered to a 15-mer thrombin-binding aptamer to form a label-free DNAzyme-linked aptamer probe. Based on specific interaction of the aptamer with target protein immobilized on nitrocellulose membrane, a DNAzyme layer is introduced onto the membrane. The DNAzyme can catalyze the H₂O₂-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine to produce a colored insoluble product that is apt to be adsorbed onto the nitrocellulose membrane. As a result, blue dots appear on the membrane, in contrast to the colorless background. As the concentration of thrombin increases, the color of dots gets deep. Such a protein concentration-dependent color change can be quantified via an image-processing software, with a detection limit of 0.6 μM. Furthermore, this assay has been applied successfully to the detection of thrombin in biological samples (e.g., human serum), indicating its practicality for bioanalysis.     

Interaction of viologens with nucleic acid G-tetrades

The interaction between the tetrade-forming oligonucleotide 5'-d(T 4G 4T 4) and monoalkylated bipyridinium salts, such as 1-ethyl-4-pyridin-4-ylpyridinium bromide, is reported. The oligomer forms tetrades in the presence of K+ ions but not with Li+. Additionally, the interaction of the thrombin-binding aptamer 5'-d(GGTTGGTGTGGTTGG) (TBA) with a dialkylated bipyridinium salt, viologen, was studied by cyclic voltammetry. This was performed either on a TiO2 electrode, derivatized with 3-aminopropyltriethoxysilane (APS), using [Fe(CN)6](4-) as a marker ion or without a marker ion on an electrostatically TiO2-bound amino-ferrocenyl derivative. Both experiments proof a strong interaction between the immobilized aptamer and the viologen. Third, the electrochemical response of the specific thrombin binding to the immobilized aptamer was studied.     

Stereoselective and Chiroselective Surface Plasmon Resonance (SPR) Analysis of Amino Acids by Molecularly Imprinted Au‐Nanoparticle Composites

Au nanoparticles (NPs) functionalized with thioaniline and cysteine are used to assemble bis‐aniline‐bridged Au‐NP composites on Au surfaces using an electropolymerization process. During the polymerization of the functionalized Au NPs in the presence of different amino acids, for example, L ‐glutamic acid, L ‐aspartic acid, L ‐histidine, and L ‐phenylalanine, zwitterionic interactions between the amino acids and the cysteine units linked to the particles lead to the formation of molecularly imprinted sites in the electropolymerized Au‐NP composites. Following the elimination of the template amino acid molecules, the electropolymerized matrices reveal selective recognition and binding capabilities toward the imprinted amino acid. Furthermore, by imprinting of L ‐glutamic or D ‐glutamic acids, chiroselective imprinted sites are generated in the Au‐NP composites. The binding of amino acids to the imprinted recognition sites was followed by surface plasmon resonance spectroscopy. The refractive index changes occurring upon the binding of the amino acids to the imprinted sites are amplified by the coupling between the localized plasmon associated with the Au NPs and the surface plasmon wave.     

An electrochemical aptasensor for thrombin detection based on the recycling of exonuclease III and double-stranded DNA-templated copper nanoparticles assisted signal amplification

In this paper, we report an improved electrochemical aptasensor based on exonuclease III and double-stranded DNA (dsDNA)-templated copper nanoparticles (CuNPs) assisted signal amplification. In this sensor, duplex DNA from the hybridization of ligated thrombin-binding aptamer (TBA) subunits and probe DNA can act as an effective template for the formation of CuNPs on the electrode surface, so copper ions released from acid-dissolution of CuNPs may catalyze the oxidation of ο-phenylenediamine to produce an amplified electrochemical response. In the presence of thrombin, a short duplex domain with four complementary base pairs can be stabilized by the binding of TBA subunits with thrombin, in which TBA subunit 2 can be partially digested from 3′ terminal with the cycle of exonuclease III, so the ligation of TBA subunits and the subsequent formation of CuNPs can be inhibited. By electrochemical characterization of dsDNA-templated CuNPs on the electrode surface, our aptasensor can display excellent performances for the detection of thrombin in a broad linear range from 100 fM to 1 nM with a low detection limit of 20.3 fM, which can also specially distinguish thrombin in both PBS and serum samples. Therefore, our aptasensor might have great potential for clinical diagnosis of biomarkers in the future.     

High-sensitive determination of human alpha-thrombin by its 29-mer aptamer in affinity probe capillary electrophoresis

ACE technique provides an effective tool for the separation and identification of disease-related biomarkers in clinical analysis. In recent years, a couple of synthetic DNA or RNA oligonucleotides, known as aptamers, rival the specificity and affinity for targets to antibodies and are employed as one kind of powerful affinity probe in ACE. In this work, based on high affinity between antithrombin aptamer and thrombin (their dissociation constant is 0.5 nM), a carboxyfluorescein-labeled 29-nucleotide (nt) aptamer (F29-mer) was used and an aptamer-based affinity probe CE (aptamer-based APCE) method was successfully established for high-sensitive detection and quantitative analysis of thrombin. Experimental conditions including incubation temperature and time, buffer composition, and concentration of cations were investigated and optimized. Under the optimized condition, the linear range was from 0 to 400 nM and the LOD was 2 nM (74 ng/mL, S/N = 3), i.e., 40 amol, both in running buffer and in 5% v/v human serum. This LOD is the lowest one than those achieved by the previous APCE methods but based on a 15-mer aptamer. This approach offers a promising method for the rapid, selective, and sensitive detection of thrombin in practical utility. Further binding experiments using one carboxyfluorescein-labeled aptamer and the other nonlabeled aptamer or vice versa were carried out to deduce the formation of ternary complex when these two aptamers coexisted in the free solution with thrombin.     

采用纳米金和荧光标记的适配体检测凝血酶

将荧光染料分子标记的含29个碱基的可识别凝血酶的DNA适配体非特异吸附到纳米金表面,荧光发生猝灭,加入凝血酶后,凝血酶与适配体特异性结合,使适配体空间结构发生改变,荧光染料分子远离纳米金表面,荧光恢复,因此可以实现对凝血酶的检测。实验结果表明,这种检测方法简便、快速、特异性强,检出限为0.54 nmol/L(对应样品体积为200μL)。     

Time-resolved fluorescence aptamer-based sandwich assay for thrombin detection

In the present study, the authors report a novel sensitive method for the detection of thrombin using time-resolved fluorescence sensing platform based on two different thrombin aptamers. The thrombin 15-mer aptamer as a capture probe was covalently attached to the surface of glass slide, and the thrombin 29-mer aptamer was fluorescently labeled as a detection probe. A bifunctional europium complex was used as the fluorescent label. The introduction of thrombin triggers the two different thrombin aptamers and thrombin to form a sandwich structure. The fluorescence intensity is proportional to the thrombin concentration. The present sensing system could provide both a wide linear dynamic range and a low detection limit. The proposed sensing system also presented satisfactory specificity and selectivity. Results showed that thrombin was retained at the aptamer-modified glass surface while nonspecific proteins were removed by rinsing with buffer solution. This approach successfully showed the suitability of aptamers as low molecular weight receptors on glass slides for sensitive and specific protein detection.     

A near-infrared fluorescent bioassay for thrombin using aptamer-modified CuInS2 quantum dots

We describe a near-infrared (NIR) fluorescent thrombin assay using a thrombin-binding aptamer (TBA) and Zn(II)-activated CuInS₂ quantum dots (Q-dots). The fluorescence of Zn(II)-activated Q-dots is quenched by the TBA via photoinduced electron transfer, but if thrombin is added, it will bind to TBA to form G-quadruplexes and the Q-dots are released. As a result, the fluorescence intensity of the system is restored. This effect was exploited to design an assay for thrombin whose calibration plot, under optimum conditions, is linear in the 0.034 to 102 nmol L⁻¹ concentration range, with a 12 pmol L⁻¹ detection limit. The method is fairly simple, fast, and due to its picomolar detection limits holds great potential in the diagnosis of diseases associated with coagulation abnormalities and certain kinds of cancer.

We developed a simple near-infrared fluorescence assay using thrombin binding aptamer (TBA) and Zn(II)-activated CuInS₂ quantum dots for the highly selective and sensitive detection of thrombin.

     

Determination of the platelet-derived growth factor BB by a competitive thrombin-linked aptamer-based Fluorometric assay

The authors describe a competitive aptamer based assay for detection of the platelet-derived growth factor BB (PDGF-BB; used as a model protein). The assay is making use of thrombin (a serine protease) as an enzyme label for reporting signals. It is taking advantage of a highly selective aptamer and of the fairly specific enzymatic activity of thrombin in terms of cleaving artificial fluorogenic peptide substrates. In a first step, the surface of wells of microplates is coated with PDGF-BB. On addition of a sample containing PDGF-BB, free and bound PDGF-BB compete with each other for binding to a DNA probe that consists of an aptamer sequence for PDGF-BB and a 29-mer aptamer sequence for thrombin. After washing, thrombin is added and will attach to the DNA probe that bound to the PDGF-BB on the microplates. Following addition of a fluorogenic peptide substrate, the bound thrombin will catalyze the cleavage of the substrate to generate a fluorescent product whose fluorescence intensity is measured at excitation/emission wavelengths of 370/440 nm. Fluorescence intensity decreases with increasing PDGF-BB concentration in the sample because less thrombin will bind to the PDGF-BB coated surface of the microplate. Under optimal conditions, PDGF-BB can be quantified in the 0.125 to 3 nM concentration range. This assay was successfully applied to the determination of PDGF-BB in spiked 100-fold diluted human serum.

Open image in new window

Graphical abstract In a competitive thrombin-linked aptamer assay, free platelet-derived growth factor BB (PDGF-BB) sample competes with the PDGF-BB coated on microplates for binding to a DNA probe containing PDGF-BB-binding aptamer and thrombin-binding aptamer. The labeled thrombin cleaves substrate into product, achieving signal generation.

     

Detection of mercury(II) based on Hg2+ -DNA complexes inducing the aggregation of gold nanoparticles

A DNA-Au NP probe for sensing Hg2+ using the formation of DNA-Hg2+ complexes through thymidine (T)-Hg2+ -T coordination to control the negative charge density of the DNA strands-thereby varying their structures-adsorbed onto Au NPs.     

Aptamer sandwich assays: label-free and fluorescence investigations of heterogeneous binding events

We studied aptamer binding events in a heterogeneous format using label-free and fluorescence measurements for the purpose of developing an aptamer-based sandwich assay on a standard microtiter plate platform. The approach allowed visualization of the underlying aptamer immobilization and target binding events rather than relying on only an endpoint determination for method optimization. This allowed for a better understanding of these multi-step assays and optimal conditions specific to aptamers. α-thrombin was chosen as a prototypical analyte as two well-studied aptamers (15 and 29-mer) binding distinct epitopes are available. The Corning Epic? system, which utilizes a resonance waveguide diffraction grating in a 384-well microtiter plate format, was employed to measure relative immobilization and binding levels for various modified aptamers. Parameters investigated included the effects of aptamer orientation, label orientation, spacer length, spacer type, immobilization concentration, and binding buffer. Most notably, the 15-mer aptamer was preferable for capture over the 29-mer aptamer and aptamers with increasing poly(dT) spacer length between the biotin modification and the aptamer yielded decreased immobilization levels. This decreased immobilization resulted in increased α-thrombin binding ability for 15-mer aptamers with the poly(dT) spacer. Fluorescence measurements of fluorescein-labeled 29-mer aptamers with varying spacers were used to visualize sandwich complex formation. Using both label-free and traditional fluorescence measurements, an in-depth understanding of the overall assay was obtained, thus the inclusion of label-free measurements is recommended for future method development.