Label-free aptamer-based electrochemical impedance biosensor for 17β-estradiol

A novel aptamer-based label-free electrochemical impedance spectroscopy biosensor for 17β-estradiol has been fabricated. The aptamers were firstly immobilized on the gold electrode through Au-S interaction; the aptamer probe was then bound with the addition of 17β-estradiol to form the estradiol/aptamer complex on the electrode surface. This leads to a significantly larger interfacial electron transfer resistance than that without the addition of 17β-estradiol. The change in the resistance had a linear relationship with 17β-estradiol concentration in the range of 1.0 × 10(-8) to 1.0 × 10(-11) mol L(-1), with a detection limit of 2.0 × 10(-12) mol L(-1). The biosensor showed high selectivity to 17β-estradiol and good stability. The designed biosensor has been applied to detect 17β-estradiol in human urine with satisfactory results.     

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.     

Selection of bovine catalase aptamers using non-SELEX

In this research, we used the non-SELEX method to successfully select an aptamer that binds to the protein target (bovine catalase) with a K(D) value in the low micro molar range. The time window was determined for the target and aptamer library by optimizing the buffer conditions using 3 × Tris-glycine-potassium phosphate (TGK) buffer as the run buffer and 1× TGK as the selection buffer to give the biggest complex peak. Fractions were collected by multistep nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM)-based partitioning for three rounds of selection. The fractions from each round were enriched using PCR and the progress of selection was monitored using bulk affinity analysis. Fraction 2 was determined to have the optimal bulk affinity (0.75 μM) and this enriched library was cloned and sequenced giving four aptamer sequences. These sequences were verified using affinity capillary electrophoresis (CAT 1 0.237 μM) and fluorescence intensity measurements (CAT 1 0.430 μM). The specificity of the aptamer was also determined by fluorescence intensity measurements. The results showed that the aptamer with the highest binding affinity showed at least a 100-fold decrease in affinity toward four other proteins (i.e. lysozyme, trypsinogen, chymotrypsinogen A, and myoglobin) tested and this confirmed that the aptamer exhibited a distinct specificity toward bovine catalase. This aptamer will be useful in biosensing, Western blot, and biomarker identification.     

Electrochemical immunoassay at a 17beta-estradiol self-assembled monolayer electrode using a redox marker

A simple electrochemical immunoassay was demonstrated using a 17beta-estradiol modified electrode. 17beta-estradiol was immobilized on the gold electrode surface with a self-assembly technique. The specific binding between estradiol antibody and 17beta-estradiol on the electrode surface was evaluated by monitoring the change in the electrode response with three hydrophilic redox markers. The decrease in the electrode response for the redox marker was observed, when the antibody was bound to the estradiol self-assembled monolayer (SAM) electrode surface. The change in the electrode response of the redox marker is attributed to the steric hindrance between the antibody on the electrode surface and the redox marker. The relative standard deviation at 30 microg ml(-1) estradiol antibody was 4.1% (n = 3). The competitive reaction between the antigen in the solution and 17beta-estradiol immobilized on the electrode surface for the limited binding sites on the antibody produced an increase in the electrode response with hydroquinone as the marker. The binding affinity of three antigens including 17beta-estradiol to the estradiol antibody was evaluated. Furthermore, the result obtained from this method was compared with the previously reported enzyme binding assay using the biotinylated estradiol and the biotin-immobilized microtiter plate.     

Kinetic capillary electrophoresis-based affinity screening of aptamer clones

DNA aptamers are single stranded DNA (ssDNA) molecules artificially selected from random-sequence DNA libraries for their specific binding to a certain target. DNA aptamers have a number of advantages over antibodies and promise to replace them in both diagnostic and therapeutic applications. The development of DNA aptamers involves three major stages: library enrichment, obtaining individual DNA clones, and the affinity screening of the clones. The purpose of the screening is to obtain the nucleotide sequences of aptamers and the binding parameters of their interaction with the target. Highly efficient approaches have been recently developed for the first two stages, while the third stage remained the rate-limiting one. Here, we introduce a new method for affinity screening of individual DNA aptamer clones. The proposed method amalgamates: (i) aptamer amplification by asymmetric PCR (PCR with a primer ratio different from unity), (ii) analysis of aptamer-target interaction, combining in-capillary mixing of reactants by transverse diffusion of laminar flow profiles (TDLFP) and affinity analysis using kinetic capillary electrophoresis (KCE), and (iii) sequencing of only aptamers with satisfying binding parameters. For the first time we showed that aptamer clones can be directly used in TDLFP/KCE-based affinity analysis without an additional purification step after asymmetric PCR amplification. We also demonstrated that mathematical modeling of TDLFP-based mixing allows for the determination of K_d values for the in-capillary reaction of an aptamer and a target and that the obtained K_d values can be used for the accurate affinity ranking of aptamers. The proposed method does not require the knowledge of aptamer sequences before screening, avoids lengthy (3-5 h) purification steps of aptamer clones, and minimizes reagent consumption to nanoliters.     

Evaluation of a molecularly imprinted polymer for the isolation/enrichment of β-estradiol

The selective preconcentration of estradiol was explored using the recognition ability of a molecularly imprinted polymer (MIP) in the solid phase extraction (SPE) format. Polymeric particles were imprinted with 17β-estradiol using methacrylic acid as functional monomer and divinylbenzene as crosslinker. Binding studies of these polymeric particles towards 17β-estradiol showed selectivity over non-imprinted polymers, using acetonitrile as solvent. The imprinted polymer showed a recovery of 88% for β-estradiol in deionized water and 81% in surface water. The selectivity of the MIP over the non-imprinted polymer was relatively low, only 10% higher recovery. The results indicate that the MIP imprinted with 17β-estradiol does not appear to provide a viable approach to be used in a sample clean-up or enrichment step for the determination of estradiol in aqueous systems.     

Nonequilibrium capillary electrophoresis of equilibrium mixtures: a universal tool for development of aptamers

Aptamers are DNA (or RNA) ligands selected from large libraries of random DNA sequences and capable of binding different classes of targets with high affinity and selectivity. Both the chances for the aptamer to be selected and the quality of the selected aptamer are largely dependent on the method of selection. Here we introduce selection of aptamers by nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). The new method has a number of advantages over conventional approaches. First, NECEEM-based selection has exceptionally high efficiency, which allows aptamer development with fewer rounds of selection. Second, NECEEM can be equally used for selecting aptamers and finding their binding parameters. Finally, due to its comprehensive kinetic capabilities, the new method can potentially facilitate selection of aptamers with predefined K(d), k(off), and k(on) of the aptamer-target interaction. In this proof-of-principle work, we describe the theoretical bases of the method and demonstrate its application to a one-step selection of DNA aptamers with nanomolar affinity for protein farnesyltransferase.     

Aptamer stationary phase for protein capture in affinity capillary chromatography

The thrombin-binding DNA aptamer was used with thrombin as a model system to investigate protein capture using aptamer stationary phases in affinity capillary chromatography. The aptamer was covalently attached to the inner surface of a bare fused-silica glass capillary to serve as the stationary phase. Proteins were loaded onto the capillary via an applied pressure. The capillary was then washed to remove unbound and non-specifically associated proteins. Finally, the bound protein was released and eluted using 20 mM Tris buffer containing 8 M urea, pH 7.3, at 50 degrees C. Eluate was collected after each step (load, wash and elute) and relative amounts of protein each were compared using fluorescence spectroscopy. The identity of the protein in the collections was confirmed using matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. The experiment was repeated for thrombin on a bare (unmodified) capillary and a capillary coated with a scrambled-sequence, non-G-quartet forming oligonucleotide that does not bind with thrombin. The results show that the aptamer stationary phase captures approximately three times as much thrombin as the control columns. The experiment was also repeated using human serum albumin (HSA) alone and in an equimolar mixture with thrombin. HSA was not retained on the aptamer capillary, nor did it affect the capture of thrombin from the mixture.     

Evaluation of Relative Aptamer Binding to Campylobacter jejuni Bacteria Using Affinity Probe Capillary Electrophoresis

Abstract

A qualitative capillary electrophoresis immunoassay was developed for the first-time to evaluate aptamer binding to bacterial cells. Binding affinity of aptamers developed against a Campylobacter jejuni bacterial cell target, relative to other common food-borne pathogens was investigated and specific binding affinity was evidenced by pronounced mobility shift and peak broadening with increasing bacteria concentration for both aptamers. Little to no mobility shift was observed for food-borne pathogens, Salmonella typhirium and Escherichia coli, even when increasing concentrations 10-fold over target. These results suggest that affinity probe capillary electrophoresis could be useful for qualitative screening of aptamer candidates for bacterial cell targets.     

Evaluation of a fluorescein-labeled estradiol derivative for use in affinity capillary electrophoresis

A fluorescein-labeled estradiol derivative was assessed for use in affinity capillary electrophoresis (ACE) in a competitive immunoassay format, in which the fluorescently labeled estradiol competed with unlabeled estradiol for a mouse anti-estradiol antibody. The preparation of the labeled estradiol produced a mixture of fluorescein-containing compounds that led to multiple peaks in the electropherogram and to which the antibody responded differently. Two of the components of the mixture, towards which the mouse antibody showed most affinity, were isolated using fraction collection via capillary electrophoresis (CE). The two fractions of the labeled estradiol products isolated by CE were characterized using mass spectrometric methods. The two active fluorescein-conjugated products differed in the carboxylate on the fluorescein moiety, one having a methyl group instead of the acidic hydrogen for the other. The estradiol antibody showed a stronger binding for the conjugate containing the methyl group, as determined from the estimated binding constants using Scatchard analysis. The isolated fractions of labeled estradiol were shown to be applicable to the ACE immunoassay method.     

Novel application of fluorescence coupled capillary electrophoresis to resolve the interaction between the G‐quadruplex aptamer and thrombin

The dynamic binding status between the thrombin and its G‐quadruplex aptamers and the stability of its interaction partners were probed using our previously established fluorescence‐coupled capillary electrophoresis method. A 29‐nucleic acid thrombin binding aptamer was chosen as a model to study its binding affinity with the thrombin ligand. First, the effects of the cations on the formation of G‐quadruplex from unstructured 29‐nucleic acid thrombin binding aptamer were examined. Second, the rapid binding kinetics between the thrombin and 6‐carboxyfluorescein labeled G‐quadruplex aptamer was measured. Third, the stability of G‐quadruplex aptamer–thrombin complex was also examined in the presence of the interfering species. Remarkably, it was found that the complementary strand of 29‐nucleic acid thrombin binding aptamer could compete with G‐quadruplex aptamer and thus disassociated the G‐quadruplex structure into an unstructured aptamer. These data suggest that our in‐house established fluorescence‐coupled capillary electrophoresis assay could be applied to binding studies of the G‐quadruplex aptamers, thrombin, and their ligands, while overcoming the complicated and costly approaches currently available.     

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.     

New fluorometric enzyme immunoassay for 17beta-estradiol by homogeneous reaction using biotinylated estradiol

A new fluorometric enzyme immunoassay for 17β-estradiol (E2) using biotinylated estradiol (BE) as a probe ligand, is described. In this method, E2 is detected indirectly by a solid-phase avidin-biotin binding assay, in which the biotin is immobilized on a microtiter plate (biotin-plate). After the competitive reaction between E2 and BE for the anti-E2 antibody in solution, the free E2 and BE are separated from the bound forms by means of ultrafiltration. The concentration of BE in the solution is determined from the reaction between the biotin immobilized on the plate and the free BE for the limited biotin binding sites of avidin conjugated with horseradish peroxidase (avidin-HRP), which is added to the solution. The enzymatic reaction of HRP was measured by a fluorometric analysis with the QuantaBlu™ Fluorogenic Peroxidase Substrate (QFPS) in order to detect of the avidin-biotin binding with a high degree of sensitivity. The detection limit and linear range for the determination of E2 were 0.12 nM and from 0.12 to 25 nM, respectively. The relative standard deviations (R.S.D.) for the E2 assay were between 2.2 and 9.1% (n = 3). The cross-reactivity for several other estrogens was also evaluated.     

A selective adenosine sensor derived from a triplex DNA aptamer

The aim of this study is to develop a selective adenosine aptamer sensor using a rational approach. Unlike traditional RNA aptamers developed from SELEX, duplex DNA containing an abasic site can function as a general scaffold to rationally design aptamers for small aromatic molecules. We discovered that abasic site-containing triplex DNA can also function as an aptamer and provide better affinity than duplex DNA aptamers. A novel adenosine aptamer sensor was designed using such a triplex. The aptamer is modified with furano-dU in the binding site to sense the binding. The sensor bound adenosine has a dissociation constant of 400 nM, more than tenfold stronger than the adenosine aptamer developed from SELEX. The binding quenched furano-dU fluorescence by 40%. It was also demonstrated in this study that this sensor is selective for adenosine over uridine, cytidine, guanosine, ATP, and AMP. The detection limit of this sensor is about 50 nM. The sensor can be used to quantify adenosine concentrations between 50 nM and 2 μM.     

Oligodeoxynucleotide-modified capillary for electrophoretic separation of single-stranded DNAs with a single-base difference.

We describe here a method of affinity capillary electrophoresis in which oligodeoxynucleotide (ODN) was immobilized onto the inner surface of the capillary. The immobilized ODN functioned successfully as an affinity ligand for sequence-based DNA separation. Six- or 12-mer ODN with a sequence complementary to one of the c-K-ras gene was used as an immobilized ligand. When the 12-mer ODN was used, the detection peak for the complementary ODN disappeared selectively, while the single-base mutant was detected as usual. In contrast, when the 6-mer ODN was used as the affinity ligand with a mixture of the complementary ODN and its single-base mutant, it was possible to detect both as completely separate peaks. That is, the separation mode was dependent on the base number of the immobilized ODN used as an affinity ligand.     

Capillary gel electrophoresis analysis of G-quartet forming oligonucleotides used in DNA-protein interaction studies

DNA-protein binding is among the most frequently studied biomolecular interactions with high importance in modern systems biology research. One interesting aspect of this rapidly developing field is the affinity capture of proteins by G-quartet forming oligonucleotides also referred to as aptamers. G-quartets are structural motifs formed by guanine-rich sequences commonly occurring in the human genome. In this paper, we describe a capillary gel electrophoresis based method to validate G-quartet formation of in-house designed oligonucleotides and discuss the effect of monovalent cation concentration on the development of this structure. The relevant aptamer was then bound to magnetic beads to form an affinity capture surface for target proteins, which were then analyzed by matrix-assisted laser desorption/ionization mass spectrometry.     

Selection of fluorescent aptamer beacons that light up in the presence of zinc

In order to generate nucleic acid biosensors that could undergo a reversible conformation change in the presence of the metal zinc, a random sequence pool of single-stranded DNA was immobilized on an oligonucleotide affinity column. In the presence of zinc, those species that underwent a conformational change were released from the column, collected, and amplified. A series of negative and positive selections refined the metal specificity of the selected aptamer beacons. Since the aptamer beacons contained a fluorophore, while the bound oligonucleotide contained a quencher, zinc binding also resulted in an increase in fluorescence. One of the selected beacons, Zn-6m2, bound zinc in the low micromolar range, gave a dose-dependent fluorescence signal, and showed an approximately sixfold increase in fluorescence on zinc binding. While some cross-reactivity with cadmium was observed, it should nonetheless prove possible to use the novel selection method to generate and tune the specificity of a variety of reversible metal biosensors. Such biosensors could potentially be used for continuous monitoring of metals in environmental samples.     

Combination of capillary electrophoresis and molecular modeling to study the enantiomer affinity pattern between β‐blockers and anionic cyclodextrin derivatives in a methanolic and water background electrolyte

In order to have deep insights into the mechanisms of enantiomer affinity pattern in both aqueous and non‐aqueous systems, an approach combining capillary electrophoresis and molecular modeling was undertaken. A chiral β‐blocker; acebutolol, was enantioseparated in aqueous capillary electrophoresis and non‐aqueous capillary electrophoresis using two anionic β‐cyclodextrin derivatives. The enantiomer affinity pattern of acebutolol was found to be opposite when an aqueous background electrolyte was replaced with non‐aqueous background electrolyte in the presence of heptakis(2,3‐di‐O‐acetyl‐6‐sulfo)‐β‐cyclodextrin but remained the same in the presence of heptakis(2,3‐di‐O‐methyl‐6‐sulfo)‐β‐cyclodextrin. Molecular docking of acebutolol into two β‐cyclodextrin derivatives indicated two distinct binding modes called ‘up’ and ‘down’ conformations. After structure optimization by molecular dynamics and energy minimization, both enantiomers of acebutolol were preferred to the ‘up’ conformation with heptakis(2,3‐di‐O‐methyl‐6‐sulfo)‐β‐cyclodextrin while ‘down’ conformation with heptakis(2,3‐di‐O‐acetyl‐6‐sulfo)‐β‐cyclodextrin. The further calculation of the complex energy with solvent effect indicated that heptakis(2,3‐di‐O‐acetyl‐6‐sulfo)‐β‐cyclodextrin had higher affinity to S‐acebutolol than R‐acebutolol in non‐aqueous capillary electrophoresis while it showed better binding to R‐acebutolol in aqueous capillary electrophoresis. However, the heptakis(2,3‐di‐O‐methyl‐6‐sulfo)‐β‐cyclodextrin bound better to R‐acebutolol in both aqueous and non‐aqueous capillary electrophoresis, implying that the binding mode played more important role in chiral separation of heptakis(2,3‐di‐O‐methyl‐6‐sulfo)‐β‐cyclodextrin while the solvent effect had prevailing impact on heptakis(2,3‐di‐O‐acetyl‐6‐sulfo)‐β‐cyclodextrin.     

毛细管电泳法高效筛选8-氧代鸟嘌呤DNA糖基化酶的核酸适配体

8-氧代鸟嘌呤DNA糖基化酶(OGG1)是人体中重要的功能蛋白,在修复DNA氧化性损伤过程中起关键作用。氧化应激等引起的氧化损伤易导致炎症反应的发生,对OGG1的抑制可以一定程度上起到缓解作用;对癌细胞OGG1的抑制有望作为癌症治疗的新方法。目前的研究多集中于小分子对OGG1功能的影响和调控,而OGG1的适配体筛选尚未见报道。作为功能配体,适配体具有合成简单、高亲和力及高特异性等优点。该文筛选了OGG1的核酸适配体,结合毛细管电泳高效快速的优点建立了两种基于毛细管电泳-指数富集进化(CE-SELEX)技术的筛选方法:同步竞争法和多轮筛选法。同步竞争法利用单链结合蛋白(SSB)与核酸库中单链核酸的强结合能力,与目标蛋白OGG1组成竞争体系,并通过增加SSB浓度来增加竞争筛选压力,以去除与OGG1弱结合的核酸序列,一步筛选即可获得与OGG1强结合的核酸序列。多轮筛选法在相同孵育条件和电泳条件下,经3轮筛选获得OGG1的核酸适配体。比较两种筛选方法的筛选结果,筛选结果中频次最高的3条候选核酸适配体序列一致,其解离常数(K_D)值在1.71~2.64 μmol/L之间。分子对接分析结果表明候选适配体1(Apt 1)可能与OGG1中具有修复氧化性损伤功能的活性口袋结合。通过对两种筛选方法的对比,证明同步竞争法更加快速高效,对其他蛋白核酸适配体筛选方法的选择具有一定的指导意义。得到的适配体有望用于OGG1功能调控,以抑制其修复功能。     

Designing highly specific biosensing surfaces using aptamer monolayers on gold

To build highly specific surfaces using aptamer affinity reagents, the effects of linker and coadsorbents were investigated for maximizing target binding and specificity for aptamer-based self-assembled monolayers (SAMs) supported on gold. An aptamer that binds the protein thrombin was utilized as a model system to compare different mixed monolayer systems toward maximizing binding and selectivity to the immobilized aptamer. Important factors used to optimize binding characteristics of thrombin to the aptamer-based monolayer films include changes in design elements of the linker and different coadsorbent thiols. Binding events measured by surface plasmon resonance (SPR) and ellipsometry showed that the binding performance of the aptamer SAMs depends principally on the linker and to a lesser extent on the coadsorbent. SAMs formed with HS-(CH2)6-OP(O)2O-(CH2CH2O)6-TTTTT-aptamer exhibited a 4-fold increase in binding capacity versus SAMs made using HS-(CH2)6-TTTTT-aptamer. Furthermore, SAMs made using HS-(CH2)6-OP(O)2O-(CH2CH2O)6-TTTTT-aptamer showed nearly complete specificity for thrombin versus bovine serum albumin (BSA, less than 2% bound), while a SAM incorporating a random DNA fragment (HS-(CH2)6-OP(O)2O-(CH2CH2O)6-TTTTT-RANDOM) showed little binding of thrombin. Irrespective of the aptamer-linker system, use of HS-(CH2)11(OCH2CH2)3OH, referred to as EG(3), as a coadsorbent enhanced binding of thrombin by approximately 2.5-fold compared to that of HS-(CH2)6-OH (mercaptohexanol, MCH).