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 detection of thrombin by acoustic method using DNA tetrahedrons as immobilisation platform

The thickness shear mode acoustic method was used to study the binding of thrombin to DNA aptamers immobilised on the gold surface covered by DNA tetrahedrons. The binding of thrombin to conventional aptamers sensitive to fibrinogen (FBT) and heparin (HPT) exosites as well as to HPT in a loop configuration (HPTloop) made it possible to determine the constant of dissociation (K_D) and the limit of detection (LOD). The sensing system composed of a HPTloop was characterised by K_D = (66.7 ± 22.7) nM, which was almost twice as low as that of FBT and HPT. For this biosensor, a lower LOD of 5.2 nM compared with 17 nM for conventional HPT aptamers was also obtained. Less sensitive sensors based on FBT aptamers revealed an LOD of 30 nM which is in agreement with the lower affinity of these aptamers to thrombin in comparison with that of HPT. The surface concentration of DNA tetrahedrons was determined by the electrochemical method using [Ru(NH₃)₆]³⁺ as a redox probe. These experiments confirmed that the “step by step” method of forming the sensing layer, consisting first in chemisorption of DNA tetrahedrons onto a gold surface and then in hybridisation of the aptamer-supporting part with complementary oligos at the top of the tetrahedron, is preferable. In addition, atomic force microscopy was applied to analyse the topography of the gold layers modified stepwise by DNA tetrahedrons, DNA aptamers and thrombin. The height profiles of the layers were in reasonable agreement with the dimensions of the adsorbed molecules. The results indicate that DNA tetrahedrons represent an efficient platform for immobilisation of aptamers.     

Development of surface chemistry for surface plasmon resonance based sensors for the detection of proteins and DNA molecules

The immobilisation of biological recognition elements onto a sensor chip surface is a crucial step for the construction of biosensors. While some of the optical biosensors utilise silicon dioxide as the sensor surface, most of the biosensor surfaces are coated with metals for transduction of the signal. Biological recognition elements such as proteins can be adsorbed spontaneously on metal or silicon dioxide substrates but this may denature the molecule and can result in either activity reduction or loss. Self assembled monolayers (SAMs) provide an effective method to protect the biological recognition elements from the sensor surface, thereby providing ligand immobilisation that enables the repeated binding and regeneration cycles to be performed without losing the immobilised ligand, as well as additionally helping to minimise non-specific adsorption. Therefore, in this study different surface chemistries were constructed on SPR sensor chips to investigate protein and DNA immobilisation on Au surfaces. A cysteamine surface and 1%, 10% and 100% mercaptoundecanoic acid (MUDA) coatings with or without dendrimer modification were utilised to construct the various sensor surfaces used in this investigation. A higher response was obtained for NeutrAvidin immobilisation on dendrimer modified surfaces compared to MUDA and cysteamine layers, however, protein or DNA capture responses on the immobilised NeutrAvidin did not show a similar higher response when dendrimer modified surfaces were used.     

On-chip graphene oxide aptasensor for multiple protein detection

The versatility of an on-chip graphene oxide (GO) aptasensor was successfully confirmed by the detection of three different proteins, namely, thrombin (TB), prostate specific antigen (PSA), and hemagglutinin (HA), simply by changing the aptamers but with the sensor composition remaining the same. The results indicate that both DNA and RNA aptamers immobilized on the GO surface are sufficiently active to realize an on-chip aptasensor. Molecular selectivity and concentration dependence were investigated in relation to TB and PSA detection by using a dual, triple, and quintuple microchannel configuration. The multiple target detection of TB and PSA on a single chip was also demonstrated by using a 2 × 3 linear-array GO aptasensor. This work enables us to apply this sensor to the development of a multicomponent analysis system for a wide variety of targets by choosing appropriate aptamers.     

RNA Aptamers That Reversibly Bind Photoresponsive Azobenzene‐Containing Peptides

Modulation of biological networks assembled by diverse interactions among biologically active molecules has provided a platform for innovative biotechnologies. Here, we report RNA aptamers that bind to a photoresponsive peptide (KRAzR; Lys‐Arg‐azobenzene‐Arg) containing azobenzene chromophore, which can change its structure by photoirradiation. Aptamers were identified after 10 cycles of an in vitro selection procedure starting with a DNA library containing a 70 nt random region. Surface plasmon resonance (SPR) analysis demonstrated that interactions between aptamers and KRAzR were fully controlled by appropriate photoirradiation to the SPR sensor chip. Upon irradiation of 360 nm on the KRAzR‐immobilized surface, the binding of each aptamer to the surface was significantly decreased. Subsequent photoirradiation of the same surface with 430 nm restored the aptamer binding to the surface. We also observed that direct photoirradiation of the aptamer–peptide complex on a gold surface actively promoted dissociation of the complex. Furthermore, a doped reselection method was applied to acquire structural and sequence information of aptamer 66. From a data analysis of the conserved region and the mutation frequency, we were able to select a plausible secondary structure among three candidates predicted by computational folding simulation.     

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.     

Direct immobilisation of DNA probes for the development of affinity biosensors

An immobilisation procedure based on the direct coupling of thiolated probes (Probe-C6-SH) to bare gold sensor surfaces has been compared with a reference immobilisation method, based on the coupling of biotinylated probes onto a streptavidin-coated dextran-modified surface. The instrumentations used were a quartz crystal microbalance (QCM) and the optical instruments Biacore X and Spreeta based on surface plasmon resonance (SPR). The performances of the DNA-based sensors resulting from direct coupling of thiolated DNA probes onto electrodes of quartz crystals or gold SPR-chips have been studied in terms of the main analytical parameters, i.e. selectivity, sensitivity, reproducibility, etc. In particular, the two immobilisation approaches have been applied to the analysis of oligonucleotides, DNA amplified by polymerase chain reaction (PCR) and genomic DNA enzymatically digested.     

A electrochemiluminescence aptasensor for detection of thrombin incorporating the capture aptamer labeled with gold nanoparticles immobilized onto the thio-silanized ITO electrode

A novel electrochemiluminescence (ECL) aptasensor was proposed for sensitive and cost-effective detection of the target thrombin adopted an aptamer-based sandwich format. To detect thrombin, capture aptamers labeled with gold nanoparticles (AuNPs) were first immobilized onto the thio-silanized ITO electrode surface through strong Au-S bonds. After catching the target thrombin, signal aptamers tagged with ECL labels were attached to the assembled electrode surface. As a result, an AuNPs-capture-aptamer/thrombin/ECL-tagged-signal-aptamer sandwich type was formed. Treating the resulting electrode surface with tri-n-propylamine (TPA) and applying a swept potential to the electrode, ECL response was generated which realized the detection of target protein. Spectroscopy and electrochemical impedance techniques were used to characterize and confirm the fabrication of the ECL aptasensor. AuNPs amplification and smart sensor fabrication art were implemented for the sensitive and cost-effective detection purpose. Signal-to-dose curve excellently followed a sandwich format equation and could be used to quantify the protein, and the detection limit was estimated to be 10 nM. Other forms of thrombin such as β- and γ-thrombins had negligible response, which indicated a high specificity of α-thrombin detection. The aptasensor opened up new fields of aptamer applications in ECL domain, a highly sensitive technique, and had a promising perspective to be applied in microarray analysis.     

Optimization of electrochemical aptamer-based sensors via optimization of probe packing density and surface chemistry

Electrochemical, aptamer-based (E-AB) sensors, which are comprised of an electrode modified with surface immobilized, redox-tagged DNA aptamers, have emerged as a promising new biosensor platform. In order to further improve this technology we have systematically studied the effects of probe (aptamer) packing density, the AC frequency used to interrogate the sensor, and the nature of the self-assembled monolayer (SAM) used to passivate the electrode on the performance of representative E-AB sensors directed against the small molecule cocaine and the protein thrombin. We find that, by controlling the concentration of aptamer employed during sensor fabrication, we can control the density of probe DNA molecules on the electrode surface over an order of magnitude range. Over this range, the gain of the cocaine sensor varies from 60% to 200%, with maximum gain observed near the lowest probe densities. In contrast, over a similar range, the signal change of the thrombin sensor varies from 16% to 42% and optimal signaling is observed at intermediate densities. Above cut-offs at low hertz frequencies, neither sensor displays any significant dependence on the frequency of the alternating potential employed in their interrogation. Finally, we find that E-AB signal gain is sensitive to the nature of the alkanethiol SAM employed to passivate the interrogating electrode; while thinner SAMs lead to higher absolute sensor currents, reducing the length of the SAM from 6-carbons to 2-carbons reduces the observed signal gain of our cocaine sensor 10-fold. We demonstrate that fabrication and operational parameters can be varied to achieve optimal sensor performance and that these can serve as a basic outline for future sensor fabrication.     

Binding kinetics of human cellular prion detection by DNA aptamers immobilized on a conducting polypyrrole

We developed a biosensor based on the surface plasmon resonance (SPR) method for the study of the binding kinetics and detection of human cellular prions (PrP^C) using DNA aptamers as bioreceptors. The biosensor was formed by immobilization of various biotinylated DNA aptamers on a surface of conducting polypyrrole modified by streptavidin. We demonstrated that PrP^C interaction with DNA aptamers could be followed by measuring the variation of the resonance angle. This was studied using DNA aptamers of various configurations, including conventional single-stranded aptamers that contained a rigid double-stranded supporting part and aptamer dimers containing two binding sites. The kinetic constants determined by the SPR method suggest strong interaction of PrP^C with various DNA aptamers depending on their configuration. SPR aptasensors have a high selectivity to PrP^C and were regenerable by a brief wash in 0.1 M NaOH. The best limit of detection (4 nM) has been achieved with this biosensor based on DNA aptamers with one binding site but containing a double-stranded supporting part.

Au NPs-aptamer conjugates as a powerful competitive reagent for ultrasensitive detection of small molecules by surface plasmon resonance spectroscopy

Features of Au NPs-aptamer conjugates as a powerful competitive reagent to substitute antibody in enhancing surface plasmon resonance spectroscopy (SPR) signal for the detection of small molecule are explored for the first time. In order to evaluate the sensing ability of Au NPs-aptamer conjugates as a competitive reagent, a novel SPR sensor based on indirect competitive inhibition assay (ICIA) for the detection of adenosine is constructed by employing the competitive reaction between antiadenosine aptamer with adenosine and antiadenosine aptamer with its partial complementary ss-DNA. The partial complementary ss-DNA of antiadenosine aptamer is firstly immobilized on SPR gold film as sensing surface. When the Au NPs-antiadenosine aptamer conjugates solution is added to SPR cell in the absence of adenosine, Au NPs-antiadenosine aptamer conjugates is adsorbed to SPR sensor by the DNA hybridization reaction, and results in a large change of SPR signal. However, the change of SPR signal is decreased when the mixing solution of adenosine with Au NPs-antiadenosine aptamer conjugates is added. This is because adenosine reacts with antiadenosine aptamer in Au NPs-antiadenosine aptamer conjugates and changes its structure from ss-DNA to tertiary structure, which cannot hybridize with its partial complementary ss-DNA immobilized on SPR gold surface. Based on this principle, a SPR sensor for indirect detection of adenosine can be developed. The experimental results confirm that the SPR sensor possesses a good sensitivity and a high selectivity for adenosine, which indirectly confirms that Au NPs-aptamer conjugates is a powerful competitive reagent. More significantly, it can be used to develop other SPR sensors based on ICIA to detect different targets by changing the corresponding type of aptamer in Au NPs-aptamer conjugates.     

Effects of DNA mismatches on binding affinity and kinetics of polymerase-DNA complexes as revealed by surface plasmon resonance biosensor

In this study, surface plasmon resonance (SPR) biosensor techniques were used to obtain quantitative information on the kinetics of the DNA and polymerase I (Klenow fragment) interaction. DNA duplexes containing different base compositions at the binding site were immobilized on the SPR sensor surface via biotin-streptavidin chemistry and the subsequent binding of the polymerase was measured in real time. Various kinetic models were tested and a translocation model was shown to provide the best fit for the binding and dissociation profiles. The results revealed that the enzyme binds to DNA at both the polymerase and the exonuclease domains with different association and dissociation rates as well as affinity constants, depending on the presence of mismatches near the primer 3'-end. Introduction of unpaired bases increases the DNA binding affinity towards the exonuclease domain and promotes the translocation of DNA from the polymerase site to the exonuclease site. The results also demonstrated that SPR biosensors may be used as a sensitive technique for studying molecular recognition events such as single-base discrimination involved in protein-DNA interaction.     

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.     

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.     

Aptamer-based electrochemical approach to the detection of thrombin by modification of gold nanoparticles

This paper presents a simple electrochemical approach for the detection of thrombin, using aptamer-modified electrodes. The use of gold nanoparticles results in significant signal enhancement for subsequent detection. 1,6-Hexanedithiol was used as the medium to link Au nanoparticles to a bare gold electrode. Anti-thrombin aptamers were immobilized on the gold nanoparticles’ surfaces by self-assembly. The packing density of aptamers was determined by cyclic voltammetric (CV) studies of redox cations (e.g., [Ru(NH₃)₆]³⁺) which were electrostatically bound to the DNA phosphate backbones. The results indicate that the total amount of aptamer probes immobilized on the gold nanoparticle surface is sixfold higher than that on the bare electrode, leading to increased sensitivity of the aptasensor and a detection limit of 1 pmol L⁻¹. Based on the Langmuir model, the sensor signal displayed an almost perfect linear relationship over the range of 1 pmol L⁻¹ to 30 nmol L⁻¹. Moreover, the proposed aptasensor is highly selective and stable. In summary, this biosensor is simple, highly sensitive, and selective, which is beneficial to the ever-growing interest in fabricating portable bio-analytical devices with simple electrical readout procedures.     

Single-Round DNA Aptamer Selection by Combined Use of Capillary Electrophoresis and Next Generation Sequencing: An Aptaomics Approach for Identifying Unique Functional Protein-Binding DNA Aptamers

In DNA aptamer selection, existing methods do not discriminate aptamer sequences based on their binding affinity and function and the reproducibility of the selection is often poor, even for the selection of well-known aptamers like those that bind the commonly used model protein thrombin. In the present study, a novel single-round selection method (SR-CE selection) was developed by combining capillary electrophoresis (CE) with next generation sequencing. Using SR-CE selection, a successful semi-quantitative and semi-comprehensive aptamer selection for thrombin was demonstrated with high reproducibility for the first time. Selection rules based on dissociation equilibria and kinetics were devised to obtain families of analogous sequences. Selected sequences of the same family were shown to bind thrombin with high affinity. Furthermore, data acquired from SR-CE selection was mined by creating sub-libraries that were categorized by the functionality of the aptamers (e. g., pre-organized aptamers versus structure-induced aptamers). Using this approach, a novel fluorescent molecular recognition sensor for thrombin with nanomolar detection limits was discovered. Thus, in this proof-of-concept report, we have demonstrated the potential of a “DNA Aptaomics” approach to systematically design functional aptamers as well as to obtain high affinity aptamers.     

Fe3O4 nanoparticles-enhanced SPR sensing for ultrasensitive sandwich bio-assay

Magnetic nanoparticles (MNPs) have been receiving increasing attention because of its great potentials in bioseparation. However, the separation products are difficult to be detected by general method due to their extremely small size. Here, we demonstrate that MNPs can greatly enhance the signal of surface plasmon resonance spectroscopy (SPR). Features of MNPs-aptamer conjugates as a powerful amplification reagent for ultrasensitive immunoassay are reported in this work for the first time. In order to evaluate the sensing ability of MNPs-aptamer conjugates as an amplification reagent, a sandwich SPR sensor is constructed by using thrombin as model analyte. Thrombin, captured by immobilized anti-thrombin aptamer on SPR gold film, is sensitively detected by SPR spectroscopy with a lowest detection limit of 0.017 nM after MNPs-aptamer conjugates is used as amplification reagent. At the same time, the excellent selectivity of the present biosensor is also confirmed by using three kinds of proteins (BSA, human IgM and human IgE) as controls. These results confirm that MNPs is a powerful sandwich element and an excellent amplification reagent for SPR based sandwich immunoassay and SPR has a great potential for the detection of MNPs-based bioseparation products.     

Electrochemical Aptasensor Based on ZnO Modified Gold Electrode

We developed an electrochemical thrombin aptasensor based on ZnO nanorods functionalized by electrostatically adsorption of 30‐mer DNA aptamers. The sensor surface was characterized by AFM and SEM. The surface layer assembling was optimized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) with ferricyanide ions as redox markers. The peak current of the ferricyanide and the charge transfer resistance gradually decreased with increasing concentration of thrombin in the range from 3 pM to 100 nM due to formation of aptamer‐thrombin complexes and slower diffusion of the marker ions through the surface layer. At optimal conditions, a limit of detection (LOD) of 3 pM for EIS measurements and 10 pM for CV response was calculated from the S/N=3.     

Target-Dependent Protection of DNA Aptamers against Nucleolytic Digestion Enables Signal-On Biosensing with Toehold-Mediated Rolling Circle Amplification

We report a generalizable strategy for biosensing that takes advantage of the resistance of DNA aptamers against nuclease digestion when bound with their targets, coupled with toehold mediated strand displacement (TMSD) and rolling circle amplification (RCA). A DNA aptamer containing a toehold extension at its 5′-end protects it from 3′-exonuclease digestion by phi29 DNA polymerase (phi29 DP) in a concentration-dependent manner. The protected aptamer can participate in RCA in the presence of a circular template that is designed to free the aptamer from its target via TMSD. The absence of the target leads to aptamer digestion, and thus no RCA product is produced, resulting in a turn-on sensor. Using two different DNA aptamers, we demonstrate rapid and quantitative real-time fluorescence detection of two human proteins: platelet-derived growth factor (PDGF) and thrombin. Sensitive detection of PDGF was also achieved in human serum and human plasma, demonstrating the selectivity of the assay.     

Effects of solute-matrix interaction on monitoring the conformational changes of immobilized proteins by surface plasmon resonance sensor

A real-time and labeling-free surface plasmon resonance (SPR) sensor was used to monitor the conformational changes of immobilized globule proteins (RNase A and lysozyme) in chemical unfolding and refolding. The effects of chemical denaturants on the protein structures were investigated. The methodology in protein conformational study on the solid surface is refined through the theoretic calculations and the conformational information of native/denatured proteins in solution. Additionally, our observation illustrates that the ambient buffer solution is merit to influence the refractive index of immobilized protein films and directly be observed from the SPR resonance angle shifts.