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.     

Surface plasmon resonance imaging for affinity analysis of aptamer–protein interactions with PDMS microfluidic chips

We report on the use of PDMS multichannels for affinity studies of DNA aptamer–human Immunoglobulin E (IgE) interactions by surface plasmon resonance imaging (SPRi). The sensing surface was prepared with thiol-terminated aptamers through a self-assembling process in the PDMS channels defined on a gold substrate. Cysteamine was codeposited with the thiol aptamers to promote proper spatial arrangement of the aptamers and thus maintain their optimal binding efficiencies. Four aptamers with different nucleic acid sequences were studied to test their interaction affinity toward IgE, and the results confirmed that aptamer I (5′-SH-GGG GCA CGT TTA TCC GTC CCT CCT AGT GGC GTG CCC C-3′) has the strongest binding affinity. Control experiments were conducted with a PEG-functionalized surface and IgG was used to replace IgE in order to verify the selective binding of aptamer I to the IgE molecules. A linear concentration-dependent relationship between IgE and aptamer I was obtained, and a 2-nM detection limit was achieved. SPRi data were further analyzed by global fitting, and the dissociation constant of aptamer I–IgE complex was found to be 2.7 × 10⁻⁷ M, which agrees relatively well with the values reported in the literature. Aptamer affinity screening by SPR imaging demonstrates marked advantages over competing methods because it does not require labeling, can be used in real-time, and is potentially high-throughput. The ability to provide both qualitative and quantitative results on a multichannel chip further establishes SPRi as a powerful tool for the study of biological interactions in a multiplexed format. Figure The SPRi sensograms and thier global fits for aptamer I and IgE interactions. Insert in the difference image obtained with the PDMS microchannel flow cell for aptamer IV, III, and I (from left to right     

Influence of ionic strength, pH and aptamer configuration for binding affinity to thrombin

We used the methods of electrochemical indicators and the quartz crystal microbalance (QCM) for detection of thrombin-aptamer interactions. We analyzed how the method of immobilization of aptamer to a solid support, the aptamer configuration as well as variation in ionic strength and pH will affect the binding of thrombin to the aptamer. The immobilization of aptamer by means of avidin-biotin technology revealed best results in sensitivity in comparison with immobilization utilizing dendrimers of first generation and in comparison with chemisorption of aptamer to a gold surface. Linear and molecular beacon aptamers of similar structure of binding site revealed similar binding properties to thrombin. Increased concentration of NaCl resulted in weakening of the binding of thrombin to the aptamers, probably due to shielding effect of Na(+) ions. The binding of the thrombin to the aptamer depends on electrolyte pH, which is presumably connected with maintaining the three dimensional aptamer configuration, optimal for binding the protein.     

Biorecognition on Graphene: Physical,Covalent, and Affinity Immobilization Methods Exhibiting Dramatic Differences

The preparation of biorecognition layers on the surface of a sensing platform is a very crucial step for the development of sensitive and selective biosensors. Different protocols have been used thus far for the immobilization of biomolecules onto various electrode surfaces. In this work, we investigate how the protocol followed for the immobilization of a DNA aptamer affects the performance of the fabricated thrombin aptasensor. Specifically, the differences in selectivity and optimum amount of immobilized aptamer of the fabricated aptasensors adopting either physical, covalent, or affinity immobilization were compared. It was discovered that while all three methods of immobilization uniformly show a similar optimum amount of immobilized aptamer, physical, and covalent immobilization methods exhibit higher selectivity than affinity immobilization. Hence, it is believed that our findings are very important in order to optimize and improve the performance of graphene‐based aptasensors.     

Streptavidin binding bifunctional aptamers and their interaction with low molecular weight ligands

This paper describes the measurement of the binding affinities of two bifunctional RNA aptamers to their respective ligands. The aptamers comprise either a theophylline or malachite green binding sequence fused to a streptavidin binding sequence. These bifunctional aptamers are shown to bind simultaneously to both the small ligand and to streptavidin whether in free solution or on gold surfaces. Binding isotherms for both interactions were measured by different physiochemical techniques: surface plasmon resonance, fluorescence spectroscopy and dynamic light scattering. Both qualitatively and quantitatively there is little difference in binding affinities between the bifunctional aptamers and their monofunctional components. The respective K_d values for streptavidin binding in the monofunctional aptamer and in the theophylline bifunctional aptamer were 12 nM and 65 nM, respectively whilst the K_d values for theophylline binding in the monofunctional aptamer and the streptavidin bifunctional aptamer were 300 nM and 120 nM. These results are consistent with treating each aptamer sequence as a module that can be combined with others without significant loss of function. This allows for the use of streptavidin based immobilization strategies without either the cost of biotinylated dNTPs or the variable yields associated with the chemical biotinylation of RNA.     

Aptasensor for ampicillin using gold nanoparticle based dual fluorescence–colorimetric methods

A gold nanoparticle based dual fluorescence–colorimetric method was developed as an aptasensor to detect ampicillin using its single-stranded DNA (ssDNA) aptamer, which was discovered by a magnetic bead-based SELEX technique. The selected aptamers, AMP4 (5′-CACGGCATGGTGGGCGTCGTG-3′), AMP17 (5′-GCGGGCGGTTGTATAGCGG-3′), and AMP18 (5′-TTAGTTGGGGTTCAGTTGG-3′), were confirmed to have high sensitivity and specificity to ampicillin (K _d, AMP7 = 9.4 nM, AMP17 = 13.4 nM, and AMP18 = 9.8 nM, respectively). The 5′-fluorescein amidite (FAM)-modified aptamer was used as a dual probe for observing fluorescence differences and color changes simultaneously. The lower limits of detection for this dual method were a 2 ng/mL by fluorescence and a 10 ng/mL by colorimetry for ampicillin in the milk as well as in distilled water. Because these detection limits were below the maximum residue limit of ampicillin, this aptasensor was sensitive enough to detect antibiotics in food products, such as milk and animal tissues. In addition, this dual aptasensor will be a more accurate method for antibiotics in food products as it concurrently uses two detection methods: fluorescence and colorimetry.     

Aptamers as Recognition Elements for Electrochemical Detection of Exosomes

Exosome analysis is emerging as an attractive noninvasive approach for disease diagnosis and treatment monitoring in the field of liquid biopsy. Aptamer is considered as a promising molecular probe for exosomes detection because of the high binding affinity, remarkable specificity, and low cost. Recently, many approaches have been developed to further improve the performance of electrochemical aptamer based(E-AB) sensors with a lower limit of detection. In this review, we focus on the development of using aptamer as a specific recognition element for exosomes detection in electrochemical sensors. We first introduce recent advances in evolving aptamers against exosomes. Then, we review methods of immobilization aptamers on electrode surfaces, followed by a summary of the main strategies of signal amplification. Finally, we present the insights of the challenges and future directions of E-AB sensors for exosomes analysis.     

Finding the Optimal Surface Density of Aptamer Monolayers by SPR Imaging Detection‐based Aptamer Microarrays

Surface plasmon resonance imaging (SPRi) by enabling label‐free, real time assessment of biomolecular interactions in multiplexed manner is one of the methods of choice for high throughput characterization of large pools of DNA aptamer candidates following in vitro selection. Moreover, with major advances in in situ amplification methods SPRi became also a viable detection platform for aptamer microarrays. In case of aptamer microarrays, commonly prepared by microspotting, the direct assessment of the surface density of aptamer probes, which is essential for both kinetic and sensing measurements is not possible. Therefore, here we introduce a methodology for simple, one‐step determination of surface densities of thiol labelled aptamer monolayers microspotted on gold SPRi chips. Based on this methodology we investigated in detail the effect of the surface density of aptamers on target binding through two aptamer‐target systems, i. e. human immunoglobulin E (hIgE) and six histidine tag 6xHis‐tag. We found that the surface density of the aptamers is indeed critical and shows a sharp maximum in terms of target binding efficiency, which is largely determined by the size of the target. The optimal aptamer surface densities determined, the immobilization chemistry (shared by many detection platforms, e. g., electrochemical, surface acoustic) and the trends identified may be used for rapid rational optimization of aptamer‐target assays.     

RNA aptamer-based optical nanostructured sensor for highly sensitive and label-free detection of antigen–antibody reactions

Developments of optical protein sensors with nanostructure based on the noble metals have currently received great attention for their high efficiency and simultaneous analysis of various important biomolecules from proteomics to genetics. In this study, we exploited the absorbance spectra of gold-capped nanoparticles substrate for label-free detections of antigen–antibody reactions using a specific thiolated RNA aptamer. These synthesized RNA aptamers have been optimized to bind to the Fc portion of the human IgG1 subclass, due to their ability to orient antibodies direction on the gold surface. After attaching the anti-fibrinogen antibodies on the surface via these linkers, our thiolated RNA aptamer-based nanostructured sensors were easily applicable to specific detections of fibrinogen with a limit of detection of 0.1 ng/mL. These nanostructured sensor-based models will open a way to display numerous immunosensors as well as to develop other functionally similar sensors which could then be expanded into multi-arrays assay systems.     

Covalently bonded DNA aptamer chiral stationary phase for the chromatographic resolution of adenosine

In this work, a target-specific aptamer chiral stationary phase (CSP) based on the oligonucleotidic selector binding to silica particles through a covalent linkage was developed. An anti-d-adenosine aptamer was coupled, using an in-situ method, by way of an amide bond to macroporous carboxylic acid based silica. Frontal chromatography analysis was performed to evaluate the column properties, i.e., determination of the stationary phase binding capacity and the dissociation constant of the target-immobilized aptamer complex. It was found that such covalent immobilization was able to maintain the aptamer binding properties at a convenient level for an efficient enantioseparation. Subsequently, the separation of adenosine enantiomers was investigated under different operating conditions, including changes in the eluent’s ionic strength and the proportion of organic modifiers as well as column temperatures. It was demonstrated that, under various conditions of use and storage, the present CSP was stable over time.     

Adsorption and covalent coupling of ATP-binding DNA aptamers onto cellulose

With the long-term goal of developing paper surfaces that will detect pathogens, we have investigated physical adsorption and covalent coupling as strategies for treating cellulose surfaces with a DNA aptamer that binds ATP. Physical adsorption was reversible and the isotherms fitted the Langmuir equation with an adsorption maximum of 0.105 mg/m2 at high ionic strength (300 mM NaCl, 25 mM Tris-HCl) and only 0.024 mg/m2 in lower ionic strength buffer (25 mM Tris-HCl). Covalent coupling of amine-terminated aptamer with oxidized cellulose film (Schiff base + reduction) gave 25% coupling efficiency while maintaining the aptamer activity which was illustrated by using a known fluorescent aptamer that is capable of ATP detection. Therefore, covalent coupling, without spacer molecules, is a promising approach for supporting biosensing aptamers on cellulose.     

New extraction sorbent based on aptamers for the determination of ochratoxin A in red wine

A new solid phase extraction method based on aptamers, an oligosorbent, was developed and applied to the determination of ochratoxin A (OTA) from red wine. Two solid supports were chosen to immobilize OTA aptamer by covalent binding (cyanogen bromide-activated sepharose) or noncovalent binding (streptavidin-activated agarose). The resulting oligosorbents were evaluated in terms of retention, selectivity, and capacity. To assess the selectivity of the resulting oligosorbents, control supports made only of a solid support without immobilized aptamers were simultaneously studied. After optimization of the selective extraction procedure, extraction recoveries close to 100% were obtained on both materials. No retention was observed on the control supports. A similar capacity was also found for both oligosorbents. However, the immobilization by covalent bonding appeared more robust for the determination of OTA in the wine. A conventional sorbent and an immunoaffinity column were also applied to the determination of OTA in red wine to compare the potential of the various approaches for the treatment of such complex samples.     

Renewable and optically transparent electroactive indium tin oxide surfaces for chemoselective ligand immobilization and biospecific cell adhesion

In this report, we show the successful transfer of a sophisticated electroactive immobilization and release strategy to an indium tin oxide (ITO) surface to generate (1) optically transparent, robust, and renewable surfaces, (2) inert surfaces that resist nonspecific protein adsorption and cell attachment, and (3) tailored biospecific surfaces for live-cell high-resolution fluorescence microscopy of cell culture. By comparing the surface chemistry properties on both ITO and gold surfaces, we demonstrate the ITO surfaces are superior to gold as a renewable surface, in robustness (durability), and as an optically transparent material for live-cell fluorescence microscopy studies of cell behavior. These advantages will make ITO surfaces a desired platform for numerous biosensor and microarray applications and as model substrates for various cell biological studies.     

DNA aptamer folding on gold nanoparticles: from colloid chemistry to biosensors

We have investigated the effect of the folding of DNA aptamers on the colloidal stability of gold nanoparticles (AuNPs) to which an aptamer is tethered. On the basis of the studies of two different aptamers (adenosine aptamer and K+ aptamer), we discovered a unique colloidal stabilization effect associated with aptamer folding: AuNPs to which folded aptamer structures are attached are more stable toward salt-induced aggregation than those tethered to unfolded aptamers. This colloidal stabilization effect is more significant when a DNA spacer was incorporated between AuNP and the aptamer or when lower aptamer surface graft densities were used. The conformation that aptamers adopt on the surface appears to be a key factor that determines the relative stability of different AuNPs. Dynamic light scattering experiments revealed that the sizes of AuNPs modified with folded aptamers were larger than those of AuNPs modified with unfolded (but largely collapsed) aptamers in salt solution. From both the electrostatic and steric stabilization points of view, the folded aptamers that are more extended from the surface have a higher stabilization effect on AuNP than the unfolded aptamers. On the basis of this unique phenomenon, colorimetric biosensors have been developed for the detection of adenosine, K+, adenosine deaminase, and its inhibitors. Moreover, distinct AuNP aggregation and redispersion stages can be readily operated by controlling aptamer folding and unfolding states with the addition of adenosine and adenosine deaminase.     

FluMag-SELEX as an advantageous method for DNA aptamer selection

Aptamers are ssDNA or RNA oligonucleotides with very high affinity for their target. They bind to the target with high selectivity and specificity because of their specific three-dimensional shape. They are developed by the so-called Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. We have modified this method in two steps—use of fluorescent labels for DNA quantification and use of magnetic beads for target immobilization. Thus, radioactive labelling is avoided. Immobilization on magnetic beads enables easy handling, use of very small amounts of target for the aptamer selection, rapid and efficient separation of bound and unbound molecules, and stringent washing steps. We have called this modified SELEX technology FluMag-SELEX. With FluMag-SELEX we have provided a methodological background for our objective of being able to select DNA aptamers for targets with very different properties and size. These aptamers will be applied as new biosensor receptors. In this work selection of streptavidin-specific aptamers by FluMag-SELEX is described. The streptavidin-specific aptamers will be used to check the surface occupancy of streptavidin-coated magnetic beads with biotinylated molecules after immobilization procedures.     

基于聚多巴胺磁性纳米微球的洛美沙星适配体筛选研究

基于纳米材料与单链核苷酸可能存在的氢键作用、π-π结合、电荷转移等非共价结合方式,可快速区分对目标靶分子有特异性结合的单链核酸适配体候选分子,从而缩短适配体筛选周期、提高筛选的成功率.本研究采用聚多巴胺磁性纳米微球(MNPs@PDAs)为分离载体,以洛美沙星(LMX)为靶标分子,利用磁分离技术建立了一种小分子的适配体筛选新方法.经过7轮筛选,获得了对洛美沙星分子具有高亲和性(KD=(17.57±0.5)nmol/L)的核酸适配体AF-3,且AF-3对于结构相似分子培氟沙星(PEFX)、氧氟沙星(OFLX)、诺氟沙星(NFLX)不具有亲和性.基于MNPs@PDAs的筛选方法有望于应用于其它重要靶分子的高效适配体探针获取.     

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.     

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.     

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.