Aptamer-based colorimetric biosensing of Ochratoxin A using unmodified gold nanoparticles indicator

This work presents an aptasensor for Ochratoxin A (OTA) using unmodified gold nanoparticles (AuNPs) indicator. The assay method is based on the conformation change of OTA's aptamer in phosphate buffered saline (PBS) containing Mg(2+) and OTA, and the phenomenon of salt-induced AuNPs aggregation. A single measurement took only five minutes. Circular dichroism spectroscopic experiments revealed for the first time that upon the addition of OTA, the conformation of OTA's aptamer in PBS buffer changed from random coil structure to compact rigid antiparallel G-quadruplex structure. This compact rigid G-quadruplex structure could not protect AuNPs against salt-induced aggregation, and thus the color change from red to blue could be observed by the naked eye. The linear range of the colorimetric aptasensor covered a large variation of OTA concentration from 20 to 625 nM and the detection limit of 20 nM (3σ) was obtained.     

Colorimetric aptasensor for progesterone detection based on surfactant-induced aggregation of gold nanoparticles

This paper proposes an aptasensor for progesterone (P4) detection in human serum and urine based on the aggregating behavior of gold nanoparticles (AuNPs) controlled by the interactions among P4-binding aptamer, target P4 and cationic surfactant hexadecyltrimethylammonium bromide (CTAB). The aptamer can form an aptamer-P4 complex with P4, leaving CTAB free to aggregate AuNPs in this aptasensor. Thus, the sensing solution will turn from red (520 nm) to blue (650 nm) in the presence of P4 because P4 aptamers are used up firstly owing to the formation of an aptamer-P4 complex, leaving CTAB free to aggregate AuNPs. However, in the absence of P4, CTAB combines with aptamers so that AuNPs still remain dispersed. Therefore, this assay makes it possible to detect P4 not only by absorbance measurement but also through naked eyes. By monitoring the variation of absorbance and color, a CTAB-induced colorimetric assay for P4 detection was established with a detection limit of 0.89 nM. Besides, the absorbance ratio A650/A520 has a linear correlation with the P4 concentration of 0.89–500 nM. Due to the excellent recoveries in serum and urine, this biosensor has great potential with respect to the visual and instrumental detection of P4 in biological fluids.     

Amplified electrochemical aptasensor taking AuNPs based sandwich sensing platform as a model

Here, we report a sensitive amplified electrochemical impedimetric aptasensor for thrombin, a kind of serine protease that plays important role in thrombosis and haemostasis. For improving detection sensitivity, a sandwich sensing platform is fabricated, in which the thiolated aptamers are firstly immobilized on a gold substrate to capture the thrombin molecules, and then the aptamer functionalized Au nanoparticles (AuNPs) are used to amplify the impedimetric signals. Such designed aptamer/thrombin/AuNPs sensing system could not only improve the detection sensitivity compared to the reported impedimetric aptasensors but also provide a promising signal amplified model for aptamer-based protein detection. In this paper, we realize a sensitive detection limit of 0.02 nM, with a linear range of 0.05-18 nM. Meanwhile, the effect of 6-mercaptohexanol (MCH) and 2-mercaptoethanol (MCE) on the modification of the electrode is investigated.     

The affinity ratio--its pivotal role in gold nanoparticle-based competitive colorimetric aptasensor

We present an important role of the ratio of affinities in unmodified gold nanoparticles-based colorimetric aptasensor reactions. An affinity ratio, representing the competitive interactions among aptamers, targets, and unmodified gold nanoparticles (umAuNPs), was found to be an important factor for the sensitivity (the performance), where the affinity ratio is the affinity of the aptamer to targets divided by the affinity to umAuNPs (K(dAuNP)/K(dTarget)). In this study, the five different aptamers having different affinity ratios to both umAuNPs and targets are used, and the degree of color change is well correlated with its affinity ratio. This result is verified by using a tetracycline binding aptamer (TBA) showing different affinities to its three derivatives, tetracycline, oxytetracycline and doxycycline. Based on this model, the sensitivity of umAuNPs based colorimetric detection for ibuprofen can be enhanced simply through reducing the ibuprofen binding aptamer's affinity to umAuNP by using bis (p-sulfonatophenyl) phenylphosphine as an AuNP-capping ligand, instead of using the citrate. As a result, a clear color change is observed even at a 20-fold less amount of ibuprofen. This study presents that the performance (detection sensitivity) of umAuNPs-based colorimetric aptasensors could be improved by simply adjusting the affinity ratio of the aptamers to targets and umAuNPs, without knowing the conformational changes of aptamers upon the target binding or needing any modification of aptamer sequences.     

Determination of glucose and uric acid with bienzyme colorimetry on microfluidic paper-based analysis devices

An aptamer is an artificial functional oligonucleic acid, which can interact with its target molecule with high affinity and specificity. Enzyme linked aptamer assay (ELAA) is developed to detect cocaine using aptamer fragment/cocaine configuration based on the affinity interaction between aptamer fragments with cocaine. The aptasensor was constructed by cleaving anticocaine aptamer into two fragments: one was assembled on a gold electrode surface, while the other was modified with biotin at 3'-end, which could be further labelled with streptavidin-horseradish peroxidase (SA-HRP). Upon binding with cocaine, the HRP-labelled aptamer fragment/cocaine complex formed on the electrode would increase the reduction current of hydroquinone (HQ) in the presence of H(2)O(2). The sensitivity and the specificity of the proposed electrochemical aptasensor were investigated by differential pulse voltammetry (DPV). The results indicated that the DPV signal change could be used to sensitively detect cocaine with the dynamic range from 0.1 μM to 50 μM and the detection limit down to 20 nM (S/N=3). The proposed aptasensor has the advantages of high sensitivity and low background current. Furthermore, a new configuration for ELAA requiring only a single aptamer sequence is constructed, which can be generalized for detecting different kinds of targets by cleaving the aptamers into two suitable segments.     

Selection of aptamers for aflatoxin M1 and their characterization

In the present work, aptamers against aflatoxin M1 and aflatoxin B1 were generated and tested for creating proof of principle of recognition of aflatoxin M1 by generated aptamers. The aptamers were selected through the process referred as systematic evolution of ligands by exponential enrichment. A total of 41 different aptamer (36 aptamers for aflatoxin M1 and 5 for aflatoxin B1) sequences were obtained. The determination of dissociation constant (K_d) values revealed that aptamers generated against aflatoxin M1 exhibited K_d values in the range of 35–1515 nM. Selected aptamers were grouped on the basis of the presence of common motifs or G‐quadruplex. We find it interesting that one aptamer with no conserved motif or G‐quadruplex had lowest K_d value (K_d = 35 nM). This structural motif is very distinct from motifs present in other aptamers. The K_d values of selected aptamers for aflatoxin B1 were in the range of 96–221 nM. One aptamer from each group was further tested for its ability to be used in aptasensor. The aptamer recognized aflatoxin M1 as indicated by color change (red to purple or blue) of aptamer‐coated gold nanoparticles in the presence of 250–500 nM aflatoxin M1. The aptamers can be used in developing methods for detection/estimation/separation of aflatoxin or antidote for aflatoxin toxicity. Copyright © 2014 John Wiley & Sons, Ltd.     

Aptabodies - new type of artificial receptors for detection proteins

We report on a new type of artificial receptor formed by hybridization of two DNA aptamers for human thrombin (aptabody). This aptasensor based on multiwalled carbon nanotubes allowed us to detect thrombin with detection limit of 0.3 nM, which was 3 times better in comparison with conventional aptamer.     

A colorimetric detection method of pesticide acetamiprid by fine-tuning aptamer length

This work investigates the effect of shortening aptamer sequences on the colorimetric detection of acetamiprid using aptamer-wrapped gold nanoparticles (AuNPs). Truncated 37-mer and 25-mer aptamers were generated by deleting excess flanking nucleotides from parental 49-mer acetamiprid-target aptamer. In comparing the responses of the three sequences, truncated aptamers did not improve the ability to discriminate against other tested pesticides. However, comparison between 49-mer and other shorter aptamers showed that shortening aptamer sequences through removing excess flanking nucleotides outsides of binding region improved colorimetric sensitivity for acetamiprid by 3.3 fold. Due to excess bases, the target-bound aptamer might still adhere to AuNPs, resulting in incomplete dissociation of aptamer from AuNPs and therefore the suppression of aggregation responses. This work provides further insight to the effects of aptamer structure on detection of the target, as well as a method by fine-tuning aptamer length for rapid detection of pesticide residues in environments or food.     

Probing the limits of aptamer affinity with a microfluidic SELEX platform

Nucleic acid-based aptamers offer many potential advantages relative to antibodies and other protein-based affinity reagents, including facile chemical synthesis, reversible folding, improved thermal stability and lower cost. However, their selection requires significant time and resources and selections often fail to yield molecules with affinities sufficient for molecular diagnostics or therapeutics. Toward a selection technique that can efficiently and reproducibly generate high performance aptamers, we have developed a microfluidic selection process (M-SELEX) that can be used to obtain high affinity aptamers against diverse protein targets. Here, we isolated DNA aptamers against three protein targets with different isoelectric points (pI) using a common protocol. After only three rounds of selection, we discovered novel aptamer sequences that bind to platelet derived growth factor B (PDGF-BB; pI = 9.3) and thrombin (pI = 8.3) with respective dissociation constants (K_d) of 0.028 nM and 0.33 nM, which are both superior to previously reported aptamers against these targets. In parallel, we discovered a new aptamer that binds to apolipoprotein E3 (ApoE; pI = 5.3) with a K_d of 3.1 nM. Furthermore, we observe that the net protein charge may exert influence on the affinity of the selected aptamers. To further explore this relationship, we performed selections against PDGF-BB under different pH conditions using the same selection protocol, and report an inverse correlation between protein charge and aptamer K_d.     

Development of a biosensor for electrochemical detection of tumor-associated proteins in blood plasma of cancer patients by aptamers

A molecular biosensor based on DNA aptamers (aptasensor) for the diagnosis of lung cancer in blood plasma samples was designed. To create the aptasensor, the aptamer 17_80, obtained in the study of postoperative material, was used. The affinity and binding selectivity of the aptamer 17_80 to the lung tumor tissue was confirmed on histological sections of postmortem samples of lung tissue. Using affinity enrichment and mass spectrometry, a possible target molecule of the aptamer 17_80, vimentin, was found.     

Sensitive label-free electrochemical analysis of human IgE using an aptasensor with cDNA amplification

In this study, we developed an ultrasensitive label-free aptamer-based electrochemical biosensor, featuring a highly specific anti-human immunoglobulin E (IgE) aptamer as a capture probe, for human IgE detection. Construction of the aptasensor began with the electrodeposition of gold nanoparticles (AuNPs) onto a graphite-based screen-printed electrode (SPE). After immobilizing the thiol-capped anti-human IgE aptamer onto the AuNPs through self-assembly, we treated the electrode with mercaptohexanol (MCH) to ensure that the remaining unoccupied surfaces of the AuNPs would not undergo nonspecific binding. We employed a designed complementary DNA featuring a guanine-rich section in its sequence (cDNA G1) as a detection probe to bind with the unbound anti-human IgE aptamer. We measured the redox current of methylene blue (MB) to determine the concentration of human IgE in the sample. When the aptamer captured human IgE, the binding of cDNA G1 to the aptamer was inhibited. Using cDNA G1 in the assay greatly amplified the redox signal of MB bound to the detection probe. Accordingly, this approach allowed the linear range (coefficient of determination: 0.996) for the analysis of human IgE to extend from 1 to 100,000pM; the limit of detection was 0.16pM. The fabricated aptasensor exhibited good selectivity toward human IgE even when human IgG, thrombin, and human serum albumin were present at 100-fold concentrations. This method should be readily applicable to the detection of other analytes, merely by replacing the anti-human IgE aptamer/cDNA G1 pair with a suitable anti-target molecule aptamer and cDNA.     

A novel impedimetric aptasensor,based on functionalized carbon nanotubes and prussian blue as labels

A simple and feasible electrochemical sensing protocol was developed for the detection of bisphenol A (BPA) by employing the gold nanoparticles (AuNPs), prussian blue (PB) and functionalized carbon nanotubes (AuNPs/PB/CNTs-COOH). An aminated complementary DNA as a capture probe and specific aptamer against BPA as a detection probe was immobilized on the surface of a modified glassy carbon (GC) electrode via the formation of covalent amide bond and hybridization, respectively. The proposed nanoaptasensor combined the advantages of the in situ formation of PB as a label, the deposition of neatly arranged AuNPs, and the covalent attachment of the capture probe to the surface of the modified electrode. Upon addition of target BPA, the analyte reacted with the aptamer and caused the steric/conformational restrictions on the sensing interface. The formation of BPA–aptamer complex at the electrode surface retarded the interfacial electron transfer reaction of the PB as a probe. Sensitive quantitative detection of BPA was carried out based on the variation of electron transfer resistance which relevant to the formation of BPA– aptamer complex at the modified electrode surface. Under the optimized conditions, the proposed aptasensor exhibited a high sensitivity, wide linearity to BPA and low detection limit. This aptasensor also displayed a satisfying electrochemical performance with good stability, selectivity and reproducibility.     

An ultrasensitive fluorescent aptasensor for adenosine detection based on exonuclease III assisted signal amplification

We report here a graphene oxide (GO)-based fluorescent aptasensor for adenosine detection by employing exonuclease III (Exo III) as a signal amplifying element. In the absence of adenosine, the adenosine aptamers hybridized with the complementary DNA (cDNA), and the Exo III could not cleave the single-strand signal probes labeled with carboxylfluorescein (FAM) at its 5' ends. When the graphene oxide was finally added, it could strongly adsorb the single-strand signal probes and quenched the fluorophore effectively. In the presence of adenosine, the aptamers associated with the targets, which led to the formation of duplex DNAs between the cDNAs and the signal probes. The Exo III thereafter could digest the duplex DNAs from 3' blunt terminus of signal probes, liberating the fluorophore. Upon adding the GO, the fluorophore could not be adsorbed and quenched. By coupling cyclic enzymatic cleavage, a remarkable fluorescent increase was obtained. Due to the specific recognition ability of the aptamer for the target and the powerful quenching property of GO for signal probe, this proposed approach has a good selectivity and high sensitivity for adenosine. In the optimum conditions described, >100% signal enhancement was achieved and a limit of detection as low as 1 nM was obtained, which is lower than those of commonly used fluorescent aptamer sensors. Moreover, the biosensor exhibited an ultrahigh sensitivity and held a versatile platform for clinical diagnostics, molecular biology and drug developments.     

Amplified surface plasmon resonance immunosensor for interferon-gamma based on a streptavidin-incorporated aptamer

Interferon-gamma (IFN-γ) is associated with susceptibility to tuberculosis, which is a major public health problem worldwide. Although significant progress has been made with regard to the design of enzyme immunoassays for IFN-γ, this assay is still labor-intensive and time-consuming. We therefore designed a DNA aptamer hairpin structure for the detection of IFN-γ with high sensitivity and selectivity. A streptavidin DNA aptamer was incorporated into the IFN-γ binding aptamer probe for the amplified detection of the target molecules. Initially, the probe remained in the inactive configuration. The addition of IFN-γ induced the rearrangement of the aptamer structure, allowing the self-assembly of the active streptavidin aptamer conformation for the streptavidin molecular recognition. Under optimized conditions, the detection limit was determined to be 33 pM, with a dynamic range from 0.3 to 333 nM, both of which were superior to those of corresponding optical sensors. Because combined aptamers are composed of nucleic acids, this optical aptasensor provided the advantages of high sensitivity, simplicity, reusability, and no further labeling or sample pre-treatment.     

Label-free and sensitive faradic impedance aptasensor for the determination of lysozyme based on target-induced aptamer displacement

A label-free and sensitive faradic impedance spectroscopy (FIS) aptasensor based on target-induced aptamer displacement was developed for the determination of lysozyme as a model system. The aptasensor was fabricated by self-assembling the partial complementary single strand DNA (pcDNA)–lysozyme binding aptamer (LBA) duplex on the surface of a gold electrode. To measure lysozyme, the change in interfacial electron transfer resistance of the aptasensor using a redox couple of [Fe(CN)₆]^3−/4− as the probe was monitored. The introduction of target lysozyme induced the displacement of the LBA from the pcDNA–LBA duplex on the electrode into the solution, decreasing the electron transfer resistance of the aptasensor. The decrease in the FIS signal is linear with the concentration of lysozyme in the range from 0.2 nM to 4.0 nM, with a detection limit of 0.07 nM. The fabricated aptasensor shows a high sensitivity, good selectivity and satisfactory regeneration. This work demonstrates that a high sensitivity of the fabricated aptasensor can be obtained using a relatively short pcDNA. This work also demonstrates that the target-induced aptamer displacement strategy is promising in the design of an electrochemical aptasensor for the determination of lysozyme with good selectivity and high sensitivity.     

A coordination polymer nanobelt (CPNB)-based aptasensor for sulfadimethoxine

A polymer-based aptasensor, which consisted of fluorescein amidite (FAM)-modified aptamers and coordination polymer nanobelts (CPNBs), was developed utilizing the fluorescence quenching effect to detect sulfadimethoxine residue in food products. A single-stranded DNA (ssDNA) aptamer, which was a specific bio-probe for sulfadimethoxine (Su13; 5'-GAGGGCAACGAGTGTTTATAGA-3'), was discovered by a magnetic bead-based systematic evolution of ligands by exponential enrichment (SELEX) technique, and the fluorescent quenchers CPNBs were produced by mixing AgNO(3) and 4,4'-bipyridine. This aptasensor easily and sensitively detected sulfadimethoxine in solution with a limit of detection (LOD) of 10ng/mL. Furthermore, the antibiotic dissolved in milk was also effectively detected with the same LOD value. In addition, this aptamer probe offered high specificity for sulfadimethoxine compared to other antibiotics. These valuable results provide ample evidence that the CPNB-based aptasensor can be used to quantify sulfadimethoxine residue in food products.     

Aptamer–nanoparticle-based chemiluminescence for p53 protein

A simple colorimetric biosensing technique based on the interaction of gold nanoparticles (AuNPs) with the aptamer was developed for detection of p53, a tumor suppressor protein, in the current study. Aggregation of AuNPs was induced by desorption of the p53 binding RNA aptamer from the surface of AuNPs as a result of the aptamer target interaction leading to the color change of AuNPs from red to purple. The detection limit of p53 protein by the colorimetric approach was 0.1 ng/ml after successful optimization of the amount of aptamer, AuNPs, salts, and incubation time. Furthermore, the catalytic activity of the aggregated AuNPs was greatly enhanced by chemiluminescence (CL) reaction, where the detection limit was enhanced to 10 pg/ml with a regression coefficient of R² = 0.9907. Here the sensitivity was increased by 10-fold compared with the AuNP-based colorimetric method. Hence, the sensitivity of detection was increased by employing CL, by using the catalytic activity of aggregated AuNPs, on the luminol–hydrogen peroxide reaction. Thus, the combination of colorimetric and CL-based aptasensor can be of great advantage in increasing the sensitivity of detection for any target analyte.     

Electrochemical aptasensor for tetracycline detection

An electrochemical aptasensor was developed for the detection of tetracycline using ssDNA aptamer that selectively binds to tetracycline as recognition element. The aptamer was highly selective for tetracycline which distinguishes minor structural changes on other tetracycline derivatives. The biotinylated ssDNA aptamer was immobilized on a streptavidin-modified screen-printed gold electrode, and the binding of tetracycline to aptamer was analyzed by cyclic voltammetry and square wave voltammetry. Our results showed that the minimum detection limit of this sensor was 10 nM to micromolar range. The aptasensor showed high selectivity for tetracycline over the other structurally related tetracycline derivatives (oxytetracycline and doxycycline) in a mixture. The aptasensor developed in this study can potentially be used for detection of tetracycline in pharmaceutical preparations, contaminated food products, and drinking water.     

Rapid real-time electrical detection of proteins using single conducting polymer nanowire-based microfluidic aptasensor

Single polypyrrole (PPy) nanowire-based microfluidic aptasensors were fabricated using a one-step electrochemical deposition method. The successful incorporation of the aptamers into the PPy nanowire was confirmed by fluorescence microscopy image. The microfluidic aptasensor showed responses to IgE protein solutions in the range from 0.01 nM to 100 nM, and demonstrated excellent specificity and sensitivity with faster response and rapid stabilization times (~20 s). At the lowest examined IgE concentration of 0.01 nM, the microfluidic aptasensor still exhibited ~0.32% change in the conductance. The functionality of this aptasensor was able to be regenerated using an acid treatment with no major change in sensitivity. In addition, the detection of cancer biomarker MUC1 was performed using another microfluidic aptasensor, which showed a very low detection limit of 2.66 nM MUC1 compared to commercially available MUC1 diagnosis assay (800 nM).     

Simultaneous generation of aptamers to multiple gamma-carboxyglutamic acid proteins from a focused aptamer library using DeSELEX and convergent selection

By using the in vitro selection method SELEX against the complex mixture of GLA proteins and utilizing methods to deconvolute the resulting ligands, we were able to successfully generate 2'-ribo purine, 2'-fluoro pyrimidine aptamers to various individual targets in the GLA protein proteome that ranged in concentration from 10 nM to 1.4 microM in plasma. Perhaps not unexpectedly, the majority of the aptamers isolated following SELEX bind the most abundant protein in the mixture, prothrombin (FII), with high affinity. We show that by deselecting the dominant prothrombin aptamer the selection can be redirected. By using this DeSELEX approach, we were able to shift the selection toward other sequences and to less abundant protein targets and obtained an aptamer to Factor IX (FIX). We also demonstrate that by using an RNA library that is focused around a proteome, purified protein targets can then be used to rapidly generate aptamers to the protein targets that are rare in the initial mixture such as Factor VII (FVII) and Factor X (FX). Moreover, for all four proteins targeted (FII, FVII, FIX, and FX), aptamers were identified that could inhibit the individual protein's activitity in coagulation assays. Thus, by applying the concepts of DeSELEX and focused library selection, aptamers specific for any protein in a particular proteome can theoretically be generated, even when the proteins in the mixture are present at very different concentrations.